Patent Application
20240026407
The present application generally relates to saponins, in particular methods for the enzymatic modification of saponins, products made thereby, uses of said products and also to other associated aspects. The present application further relates to glucosidases and rhamnosidases, in particular mutated glucosidases and rhamnosidases which may be of use in methods for the enzymatic modification of saponins. The saponins may be obtainable from Quillaja species, including extracts obtainable from Quillaja species, such as extracts of Quillaja saponaria Molina.
Saponins are steroid or terpenoid glycosides. They have a broad range of uses from fire extinguisher foams to food additives and immunostimulants (Reichert, 2019).
Quillaja extract (E 999) is currently approved by the European Food Safety Authority under EU Regulation 1129/2011 as a food additive in flavoured drinks (14.1.4), cider and perry (14.2.3). Quillaja extract (E 999) is described as being obtained by aqueous extraction of the milled inner bark or wood of Quillaja saponaria, or other Quillaja species. It is also described as containing a number of triterpenoid saponins consisting of glycosides of quillaic acid. Sugars—including glucose, galactose, arabinose, xylose, and rhamnose—are also said to be present, along with tannin, calcium oxalate and other minor components. (EFSA Journal 2019 17(3):5622)
Saponins have been of interest as immunostimulants for many decades (see, for example, Hyslop, 1969). Quil A is a saponin fraction derived from an aqueous extract from the bark of Quillaja saponaria (Dalsgaard, 1974). Quil A itself contains a plurality of components with the four most predominant Quil A fractions purified by reverse phase chromatography, namely QS-7, QS-17, QS-18 and QS-21, all having immunostimulatory activity although varying in haemolytic activity and toxicity (Kensil, 1991; Kensil, 1995). The main saponin fraction, QS-18, was found to be highly toxic in mice but saponin fractions QS-7 and QS-21 were far less toxic. QS-21, being more abundant than QS-7, has been the most widely studied saponin adjuvant (Ragupathi, 2011).
Liquid chromatography/mass spectrometry analysis of Quillaja saponaria bark water/methanol extracts has revealed over 100 saponins, many of which have been assigned structures (Nyberg, 2000; Nyberg, 2003; Kite, 2004).
Quillaja brasiliensis (A St.-Hil & Tul) Mart. extracts have been described, with the identity of various components therein determined by mass spectrometry. Many saponin components in Quillaja brasiliensis extracts correspond to saponins found in Quillaja saponaria extracts (Wallace, 2017; Wallace, 2019) and Quillaja brasiliensis extracts have also been shown to have immunostimulant effects (Cibulski, 2018; Yendo, 2017).
The Adjuvant System 01 (AS01) is a liposome-based adjuvant which contains two immunostimulants, 3-O-desacyl-4′-monophosphoryl lipid A (3D-MPL) and QS-21 (Garcon, 2011; Didierlaurent, 2017). 3D-MPL is a non-toxic derivative of the lipopolysaccharide from Salmonella minnesota. AS01 is included in vaccines for malaria (RTS, S-Mosquirix™) and Herpes zoster (HZ/su—Shingrix™), and in multiple candidate vaccines. AS01 injection results in rapid and transient activation of innate immunity in animal models. Neutrophils and monocytes are rapidly recruited to the draining lymph node (dLN) upon immunization. Moreover, AS01 induces recruitment and activation of MHCIIhigh dendritic cells (DC), which are necessary for T cell activation (Didierlaurent, 2014). Some data are also available on the mechanism of action of the components of AS01. 3D-MPL signals via TLR4, stimulating NF-κB transcriptional activity and cytokine production and directly activates antigen-presenting cells (APCs) both in humans and in mice (De Becker, 2000; Ismaili, 2002; Martin, 2003; Mata-Haro, 2007). QS-21 promotes high antigen-specific antibody responses and CD8+ T-cell responses in mice (Kensil, 1998; Newman, 1992; Soltysik, 1995) and antigen-specific antibody responses in humans (Livingston, 1994). Because of its physical properties, it is thought that QS-21 might act as a danger signal in vivo (Lambrecht, 2009; Li, 2008). Although QS-21 has been shown to activate ASC-NLRP3 inflammasome and subsequent IL-1β/IL-18 release (Marty-Roix, 2016), the exact molecular pathways involved in the adjuvant effect of saponins have yet to be clearly defined.
Extracts of Quillaja saponaria are commercially available, including fractions thereof with differing degrees of purity such as Quil A, Fraction A, Fraction B, Fraction C, QS-7, QS-17, QS-18 and QS-21.
The enzymatic hydrolysis of Quil-A by Rapidase® Revelation Aroma has been described during the development of a quality control method to provide a degraded reference sample (Lecas, 2021).
Availability of saponins is constrained, particularly those obtained from rarer plants or where saponins of interest are present in relatively low amounts. Furthermore, separation of certain saponins from other components, particularly other saponin components which may have similar structures, can be burdensome. Consequently, there remains a need for new methods which may improve the yield of saponins of interest and/or facilitate removal of undesired saponin components.
Modestobacter marinus glucosidase (Uniparc reference UPI000260A2FA, Uniprot reference I4EYD5) is a naturally occurring glucosidase. There remains a need for further glucosidases which may have improved properties.
Kribbella flavida rhamnosidase (Uniparc reference UPI00019BDB13, Uniprot reference D2PMT) is a naturally occurring rhamnosidase. There remains a need for further rhamnosidases which may have improved properties.
The present invention provides a method for making a product saponin, said method comprising the step of enzymatically converting a starting saponin to the product saponin. Suitably the method uses a polypeptide of the invention, such as an engineered glucosidase polypeptide or an engineered rhamnosidase polypeptide.
Also provided is a method for increasing the amount of a product saponin in a composition, said method comprising the step of enzymatically converting a starting saponin to the product saponin. Suitably the method uses a polypeptide of the invention, such as an engineered glucosidase polypeptide or an engineered rhamnosidase polypeptide.
Further provided is a method for reducing the amount of a starting saponin in a composition, said method comprising the step of enzymatically converting the starting saponin to a product saponin. Suitably the method uses a polypeptide of the invention, such as an engineered glucosidase polypeptide or an engineered rhamnosidase polypeptide.
The use of a glycosidase for enzymatically converting a starting saponin to a product saponin is also provided by the invention. Suitably the glycosidase is a polypeptide of the invention, such as an engineered glucosidase polypeptide or an engineered rhamnosidase polypeptide.
Additionally provided is a method for identifying a candidate enzyme having beta exo glucosidase activity, comprising selecting an enzyme comprising, such as consisting of: (i) an amino acid sequence according to SEQ ID No. 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325, 338, 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 or 809 or functional variants thereof; or (ii) an amino acid sequence according to SEQ ID No. 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325, 338, 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 or 809 or functional variants thereof
Also provided is a method for identifying a candidate enzyme having alpha exo rhamnosidase activity, comprising selecting an enzyme comprising, such as consisting of, an amino acid sequence according to SEQ ID No. 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993, 1077, 1046, 1015, 1063, 1054, 1074, 1067 or 1033, or functional variants thereof.
Also provided are engineered glucosidase and rhamnosidase polypeptides as further detailed below (referred to as polypeptides of the invention).
The present invention provides an engineered glucosidase polypeptide comprising, such as consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes at least one residue substitution from:
The present invention provides an engineered rhamnosidase polypeptide comprising, such as consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 1017, or a functional fragment thereof, wherein the engineered rhamnosidase polypeptide includes at least one residue substitution from:
The invention also provides a saponin prepared by the methods herein, a saponin containing composition comprising a product saponin prepared by the methods herein, adjuvant compositions comprising said saponins or saponin containing compositions, and adjuvant compositions prepared using said saponins or saponin containing compositions. Use of a saponin or saponin containing composition of the invention in the manufacture of an adjuvant composition is also provided.
Further provided are immunogenic compositions comprising a saponin or saponin containing composition according to the invention and an antigen or a polynucleotide encoding an antigen. Kits comprising (i) a saponin or saponin containing composition according to the invention and (ii) an antigen or a polynucleotide encoding an antigen are also provided.
As mentioned previously, saponins are steroid or terpenoid glycosides which have a broad range of uses. Current approaches to obtaining certain saponins in suitable quantities and of suitable purities are limiting. The present inventors have surprisingly found that enzymatic modification of saponins can facilitate improved availability of saponins of interest and/or facilitate removal of undesired saponin components. The present invention therefore provides methods for the enzymatic modification of saponins, products made by such methods, uses of said products and associated aspects. In methods of the invention a starting saponin (i.e. a saponin to be modified by an enzyme) is converted into a product saponin (i.e. the saponin resulting from enzymatic modification of the starting saponin).
Engineered glucosidase polypeptides of the present invention may be used in methods for the enzymatic modification of saponins.
Engineered rhamnosidase polypeptides of the present invention may be used in methods for the enzymatic modification of saponins.
The present invention can be applied to achieve a plurality of objectives, such as: (i) improving the yield of saponins of interest obtainable from a given starting material; (ii) broadening the range of starting materials suitable for obtaining saponins of interest; and/or (iii) convenient removal of undesired saponins from saponins of interest.
Where supply is constrained for a starting material from which a saponin of interest is isolated, achieving the maximum yield of the saponin of interest is clearly important. Independent of the efficiency of extraction and separation processes which would generally be adapted for optimal isolation of existing saponin of interest, the present invention may be applied to increase the amount of a saponin of interest which may be obtained from a given starting material. Enzymatic modification of other saponins present in the starting material to form a saponin of interest can increase the amount of the saponin of interest which may be obtained.
Saponins may be obtained from a broad range of starting materials. The presence of specific saponins and their levels in plant material may depend on a range of factors such as a plant species, tissue, age, season, environmental conditions and the like. Variation may be observed between plants (such as trees) of the same species (see, for example, WO2018057031). The burden associated with extraction and/or isolation of a saponin of interest may mean that certain potential sources of the saponin of interest are not commercially viable, due to the saponin of interest being present at relatively low levels. Enzymatic modification of other saponins present in the starting material to form a saponin of interest can expand the range of viable starting materials for obtaining the saponin of interest.
It is well understood that different saponins may have different activity profiles—both positive/desired activities and negative/undesired activities. Some uses of saponins require a high degree of purification and separating a saponin of interest from other saponins, particularly those of similar structure or physical properties, can be burdensome. Enzymatic modification of such other saponins may alter their physical properties and may thereby facilitate separation from a saponin of interest. Other uses of saponins may not require a high degree of purity per se, nevertheless it may still be desirable to remove or reduce the amount of a particular saponin component (or components) within a saponin mixture without burdensome chromatographic methods. Enzymatic modification can facilitate removal or reduction in the level of a particular saponin component within a saponin mixture without the need for chromatographic means.
The methods of the present invention require a starting saponin (i.e. a saponin which is intended to be enzymatically modified). The starting saponin may be a naturally occurring saponin (i.e. a steroid or terpenoid glycoside found in nature) or an artificially created saponin (i.e. a steroid or terpenoid glycoside not found in nature).
In some embodiments the starting saponin is a steroid glycoside, in other embodiments the starting saponin is a terpenoid glycoside, especially a triterpenoid glycoside.
Naturally occurring starting saponins may be obtained by extraction or may be prepared synthetically (fully or semi-synthetically).
Naturally occurring starting saponins include those obtainable from, such as obtained from, plants of the genera Gypsophilia, Saponaria or Quillaja (Bomford, 1992). Especially of interest are starting saponins obtainable from plants of Quillaja species. Particular starting saponins of interest include those obtainable from Quillaja brasiliensis or Quillaja saponaria. In one embodiment the starting saponin is obtainable from Quillaja saponaria, such as obtained from Quillaja saponaria. In one embodiment the starting saponin is obtainable from Quillaja brasiliensis, such as obtained from Quillaja brasiliensis.
In certain embodiments the starting saponin is a quillaic acid glycoside. In certain embodiments the starting saponin is a phytolaccinic acid glycoside. In certain embodiments the starting saponin is an echinocystic acid glycoside. In certain embodiments the starting saponin is a 22-beta-hydroxylated quillaic acid glycoside. In certain embodiments the starting saponin is an gypsogenin glycoside.
Analysis of water/methanol extracts of Quillaja saponaria bark by liquid chromatography/mass spectrometry has revealed over 100 saponins (Nyberg, 2000; Nyberg, 2003; Kite, 2004). Quillaja brasiliensis extracts have also been described, with many saponin components in Quillaja brasiliensis extracts corresponding to saponins found in Quillaja saponaria extracts. (Wallace, 2017; Wallace, 2019).
The following text describes particular quillaic acid derived starting and product saponins which are grouped by ‘family’. Each family has one or more common structural features which characterise the family relative to other families. Individual components within each family also display certain structural features which characterise the component relative to other components of the family, including: xylose or rhamnose chemotype—the presence of a xylose or rhamnose residue in the C3 saccharide; A or B isomers—A having the acyl chain linked through the 4-position of the D-fucose, B at having the acyl chain linked through the 3-position of the D-fucose; V1 and V2—the presence of a terminal apiose or xylose residue in the C28 saccharide (in other components of a family this terminal residue may also be absent). The text focuses on components which typically have a significant presence in Quillaja saponaria aqueous extracts, but it will be appreciated that (i) other components of a family also exist and (ii) the proportions of different components of a family may vary both between families and between different saponin sources (Kite, 2004). The specific extraction method used may also influence the proportions of different components obtained.
A and B isomers may be separable using chromatographic techniques. However, under suitable solvent conditions these isomers will revert to equilibrium proportions (see e.g. Cleland, 1996). Xylose and rhamnose chemotypes typically elute closely, depending on chromatographic technique the rhamnose chemotype may form a minor peak closely preceding or overlapping with the main peak for the family.
Those skilled in the art will also recognise that the structures described contain ionisable groups and under appropriate circumstances may exist in dissociated forms or as salts. Structures are generally shown with the glucuronate moiety in ionised form and the indicated molecular weight is calculated directly from the ion shown (corresponding to the monoisotopic m/z observed with negative mode electrospray mass spectrometry), however, all non-dissociated, dissociated and salt forms are intended to be encompassed by the recited definitions. Salts are desirably pharmaceutically acceptable, although non-pharmaceutically acceptable salts can nevertheless be useful during manufacture of pharmaceuticals or for non-pharmaceutical uses.
Starting saponins obtainable from Quillaja saponaria include:
Starting saponins of direct relevance to the engineered glucosidase polypeptides are those having cleavable glucose residues, nevertheless, the engineered glucosidase polypeptides may be utilised in conjunction with additional enzymes capable of cleaving other sugar residues. Particular starting saponins of relevance to the engineered glucosidase polypeptides include:
Starting saponins of direct relevance to the engineered rhamnosidase polypeptides are those having cleavable rhamnose residues, nevertheless, the engineered rhamnosidase polypeptides may be utilised in conjunction with additional enzymes capable to cleaving other sugar residues. Particular starting saponins of relevance to the engineered rhamnosidase polypeptides include:
The methods of the present invention enzymatically modify a starting saponin to provide a product saponin. The product saponin may be a naturally occurring saponin (i.e. a steroid or terpenoid glycoside found in nature, though the product saponin is itself obtained by the methods of the invention) or an artificially created saponin (i.e. a steroid or terpenoid glycoside not found in nature).
In some embodiments the product saponin is a steroid glycoside, in other embodiments the product saponin is a terpenoid glycoside, especially a triterpenoid glycoside.
Naturally occurring product saponins include those obtainable from plants of the genera Gypsophilia, Saponaria or Quillaja (Bomford, 1992). Especially of interest are product saponins obtainable from plants of Quillaja species. Particular product saponins of interest include those obtainable from Quillaja brasiliensis or Quillaja saponaria. In one embodiment the product saponin is obtainable from Quillaja saponaria. In one embodiment the product saponin is obtainable from Quillaja brasiliensis.
In certain embodiments the product saponin is a quillaic acid glycoside.
Product saponins obtainable from Quillaja saponaria include:
Product saponins of direct relevance to the engineered glucosidase polypeptides are those where a glucose residue has been cleaved relative to a starting saponin. Nevertheless, the engineered glucosidase polypeptides may be utilised in conjunction with additional enzymes capable to cleaving other sugar residues. Particular product saponins of relevance to the engineered glucosidase polypeptides include:
Product saponins of direct relevance to the engineered rhamnosidase polypeptides are those where a rhamnose residue has been cleaved relative to a starting saponin. Nevertheless, the engineered rhamnosidase polypeptides may be utilised in conjunction with additional enzymes capable to cleaving other sugar residues. Particular product saponins of relevance to the engineered rhamnosidase polypeptides include:
The term QS-18 family components as used herein means the xylose chemotype QS-18 2150 component (A and B isomers, and apiose and xylose isomers: QS-18 2150 A V1, QS-18 2150 A V2, QS-18 2150 B V1 and QS-18 2150 B V2), the xylose chemotype QS-18 2018 component (A and B isomers: QS-18 2018 A and QS-18 2018 B), the rhamnose chemotype QS-18 2164 component (A and B isomers, and apiose and xylose isomers: QS-18 2164 A V1, QS-18 2164 A V2, QS-18 2164 B V1 and QS-18 2164 B V2).
The term desglucosyl-QS-17 family components as used herein means the xylose chemotype desglucosyl-QS-17 2134 component (A and B isomers, and apiose and xylose isomers: desglucosyl-QS-17 2134 A V1, desglucosyl-QS-17 2134 A V2, desglucosyl-QS-17 2134 B V1 and desglucosyl-QS-17 2134 B V2), the xylose chemotype desglucosyl-QS-17 2002 component (A and B isomers: desglucosyl-QS-17 2002 A and desglucosyl-QS-17 2002 B), the rhamnose chemotype desglucosyl-QS-17 2148 component (A and B isomers, and apiose and xylose isomers: desglucosyl-QS-17 2148 A V1, desglucosyl-QS-17 2148 A V2, desglucosyl-QS-17 2148 B V1 and desglucosyl-QS-17 2148 B V2).
The term QS-17 family components as used herein means the xylose chemotype QS-17 2296 component (A and B isomers, and apiose and xylose isomers: QS-17 2296 A V1, QS-17 2296 A V2, QS-17 2296 B V1 and QS-17 2296 B V2), the xylose chemotype QS-17 2164 component (A and B isomers: QS-17 2164 A and QS-17 2164 B), the rhamnose chemotype QS-17 2310 component (A and B isomers, and apiose and xylose isomers: QS-17 2310 A V1, QS-17 2310 A V2, QS-17 2310 B V1 and QS-17 2310 B V2).
The term QS-21 family components as used herein means the xylose chemotype QS-21 1988 component (A and B isomers, and apiose and xylose isomers: QS-21 1988 A V1, QS-21 1988 A V2, QS-21 1988 B V1 and QS-21 1988 B V2), the xylose chemotype QS-21 1856 component (A and B isomers: QS-21 1856 A and QS-21 1856 B), the rhamnose chemotype QS-21 2002 component (A and B isomers, and apiose and xylose isomers: QS-21 2002 A V1, QS-21 2002 A V2, QS-21 2002 B V1 and QS-21 2002 B V2).
The term desarabinofuranosyl-QS-18 family components as used herein means the xylose chemotype desarabinofuranosyl-QS-18 2018 component (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl-QS-18 2018 A V1, desarabinofuranosyl-QS-18 2018 A V2, desarabinofuranosyl-QS-18 2018 B V1 and desarabinofuranosyl-QS-18 2018 B V2), the xylose chemotype desarabinofuranosyl-QS-18 1886 component (A and B isomers: desarabinofuranosyl-QS-18 1886 A and desarabinofuranosyl-QS-18 1886 B), the rhamnose chemotype desarabinofuranosyl-QS-18 2032 component (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl-QS-18 2032 A V1, desarabinofuranosyl-QS-18 2032 A V2, desarabinofuranosyl-QS-18 2032 B V1 and desarabinofuranosyl-QS-18 2032 B V2).
The term acetylated desglucosyl-QS-17 family components as used herein means xylose chemotype acetylated desglucosyl-QS-17 2176 component (apiose and xylose isomers: acetylated desglucosyl-QS-17 2176 A V1 and acetylated desglucosyl-QS-17 2176 A V2), the xylose chemotype acetylated desglucosyl-QS-17 2044 A component, the rhamnose chemotype acetylated desglucosyl-QS-17 2190 component (apiose and xylose isomers: acetylated desglucosyl-QS-17 2190 A V1 and acetylated desglucosyl-QS-17 2190 A V2).
The term desarabinofuranosyl-QS-21 family components as used herein means xylose chemotype desarabinofuranosyl-QS-21 1856 component (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl-QS-21 1856 A V1, desarabinofuranosyl-QS-21 1856 A V2, desarabinofuranosyl-QS-21 1856 B V1 and desarabinofuranosyl-QS-21 1856 B V2), the xylose chemotype desarabinofuranosyl-QS-21 1712 component (A and B isomers: desarabinofuranosyl-QS-21 1712 A and desarabinofuranosyl-QS-21 1712 B), the rhamnose chemotype desarabinofuranosyl-QS-21 1870 component (A and B isomers, and apiose and xylose isomers: desarabinofuranosyl-QS-21 1870 A V1, desarabinofuranosyl-QS-21 1870 A V2, desarabinofuranosyl-QS-21 1870 B V1 and desarabinofuranosyl-QS-21 1870 B V2).
The term acetylated QS-21 family components as used herein means xylose chemotype acetylated QS-21 2030 component (apiose and xylose isomers: acetylated QS-21 2030 A V1 and acetylated QS-21 2030 A V2), the xylose chemotype acetylated QS-21 1898 A component, the rhamnose chemotype acetylated QS-21 2044 component (apiose and xylose isomers: acetylated QS-21 2044 A V1 and acetylated QS-21 2044 A V2).
Suitably a starting saponin is obtained by extraction from a starting material. The starting material may be plant material obtained from plants of the genera Gypsophilia, Saponaria or Quillaja (Bomford, 1992), such as plant material obtained from plants of Quillaja species. Particular plant material includes that obtained from Quillaja brasiliensis or Quillaja saponaria. In one embodiment the plant material is obtained from Quillaja saponaria. In one embodiment the plant material is obtained from Quillaja brasiliensis.
Extraction may be from complete plants. Alternatively, extraction may be from selected plant tissues. Extraction from selected plant tissues may be from plant material including wood or bark, such as from plant material which is wood or bark. In some embodiments, extraction is from plant material including bark, such as from plant material which is bark.
Extraction may be from plant material obtained from an adult plant. Alternatively, extraction may be from plant material obtained from a young plant, such as plants of less than 5 years old, such as less than 3 years old. (Schlotterbeck, 2015; WO2018057031)
Extraction may be performed using water or lower alcohols (e.g. methanol or ethanol) as solvents, including mixtures thereof. In one embodiment the starting saponin is obtained by aqueous extraction (e.g. using solvent comprising at least 80% v/v water, especially at least 90% v/v water, such as at least 95% v/v water). In one embodiment the starting saponin is obtained by methanol extraction (e.g. using solvent comprising at least 80% v/v methanol, especially at least 90% v/v methanol, such as at least 95% v/v methanol). In one embodiment the starting saponin is obtained by ethanol extraction (e.g. using solvent comprising at least 80% v/v ethanol, especially at least 90% v/v ethanol, such as at least 95% v/v ethanol). In one embodiment the starting saponin is obtained by methanol/ethanol extraction (e.g. using solvent comprising at least 20% v/v methanol, especially at least 30% v/v methanol, such as at least 40% v/v methanol and at least 20% ethanol, especially at least 30% v/v ethanol, such as at least 40% v/v ethanol). In one embodiment the starting saponin is obtained by water/ethanol extraction (e.g. using solvent comprising at least 20% v/v water, especially at least 30% v/v water, such as at least 40% v/v water and at least 20% ethanol, especially at least 30% v/v ethanol, such as at least 40% v/v ethanol. In one embodiment the starting saponin is obtained by water/methanol extraction (e.g. using solvent comprising at least 20% v/v water, especially at least 30% v/v water, such as at least 40% v/v water and at least 20% methanol, especially at least 30% v/v methanol, such as at least 40% v/v methanol).
Methods of the invention may be applied to starting saponin in a range of contexts. A starting saponin may be in the form of a minor component in a saponin containing composition (ignoring solvents, if any), such as a minor component of a plant material extract. A starting saponin may be in the form of a major component in a saponin containing composition, such as a major component in a plant material extract. A starting saponin may be in the form of a minor component in a processed, such as partially purified, plant material extract. A starting saponin may be in the form of a major component in a processed, such as partially purified, plant material extract. In some embodiments the starting saponin is substantially purified at the time of enzymatic modification.
Purification refers to the isolation of a component from other components. Partial purification therefore means the isolation of a components, to some degree, from other components. Substantial purification means the substantial isolation of a component from other components, such as wherein the component comprises at least 50% w/w, especially as at least 70%, particularly at least 80%, for example at least 90% of the component content (50%, 70%, 80% and 90% purity, respectively). Partial purification, in relation to an extract, means the isolation of the starting saponin, to some degree, from other extracted components. Substantially purified, in relation to an extract, means the substantial isolation of the starting saponin from other extracted components, such as wherein the starting saponin comprises at least 50% w/w, especially as at least 70%, particularly at least 80%, for example at least 90% of the extracted component content. Partial or substantial purification can be undertaken through various means including chromatography, filtration over semi-permeable membranes, treatment with selective adsorbants such as polyvinylpolypyrrolidone (PVPP) and the like.
Although a starting saponin may be a specific chemical entity, in many circumstances involving saponins obtained by extraction a plurality of starting saponins may be present, these being enzymatically modified to provide their corresponding product saponins. As mentioned above for individual saponins, the invention may be applied to a plurality of starting saponins in a range of contexts mutatis mutandis. A plurality of starting saponins comprising related starting saponins may undergo equivalent enzymatic modification concurrently. A plurality of starting saponins comprising distinguishable starting saponins may undergo different enzymatic modifications concurrently (in the presence of more than one enzyme) or in series (sequential treatment with separate enzymes). A plurality of starting saponins may contain both related and distinguishable starting saponins.
Methods of the invention may be applied to a starting saponin in the form of a component of:
Methods of the invention may be applied to a starting saponin in a composition comprising:
As with other QS families, the QS-7 family components contains a plurality of related structures including xylose and rhamnose chemotypes, xylose and apiose isomers, A and B isomers:
Certain QS-7 family compounds may lack glucose, or the rhamnose attached to the beta-D-fuc.
The present invention provides the enzymatic modification of saponins. Enzymatic modifications envisaged in the present invention include the conversion of a starting saponin into a product saponin by the removal of one or more sugar residues from the starting saponin. Suitably the enzymatic modifications envisaged in the present invention are the conversion of a starting saponin into a product saponin by the removal of one or more sugar residues from the starting saponin.
In certain embodiments the enzymatic modification involves the removal of a single sugar residue i.e. removal of a terminal sugar residue (‘exo’ action) from a starting saponin. In other embodiments enzymatic conversion involves the removal of a plurality of sugar residues from a starting saponin i.e. cleavage at a saccharide linkage other than in a terminal location (‘endo’ action), resulting in removal of a plurality of sugar residues (such as 2, 3 or 4 sugar residues) attached through said saccharide linkage.
Particular sugar residues which may be removed comprise (such as consist of):
Particular single sugar enzymatic conversions of interest include:
Enzymatic conversions may be applied to a single starting saponin or a plurality of starting saponins in parallel. It will be appreciated that a process may comprise or consist of the conversions specified above, depending on the composition of the starting material and the enzymes used. Furthermore, while a process may be limited to the use of a single enzyme intended to remove a particular sugar residue or group of sugar residues from (i) a single starting saponin, (ii) a family of starting saponins, or (iii) from a plurality of families of starting saponins; processes may also use a plurality of enzymes intended to remove a plurality of sugar residues from (i) a single starting saponin, (ii) a family of starting saponins, or (iii) from a plurality of families of starting saponins. Processes involving multiple enzymes may be undertaken in series (i.e. a single enzyme is applied to saponin material at any time) or in parallel (i.e. more than one enzyme is applied to saponin material at any time, such as two or three enzymes, in particular two enzymes), or combinations thereof.
Processes involving the removal of multiple sugar residues may involve the removal of single (but different) sugar residues from multiple starting saponins and/or the removal of multiple sugar residues from particular starting saponins (such as 2, 3 or 4 residues, in particular 2 or 3, especially 2 residues). Removal of multiple sugar residues from particular starting saponins may involve any combination of removal of single residues and/or removal of a plurality of residues in a single cleavage.
Exemplary processes may comprise (such as consist of) the removal of glucose and rhamnose, in particular an alpha-rhamnose residue and a beta-glucose residue, such as the alpha-L-rhamnose residue and the beta-D-glucose residue from quillaic acid glycosides:
Particular multi-sugar enzymatic conversions of interest include:
Extracts may contain complex mixtures of saponin components and consequently may experience a plurality of conversions when multiple enzymes are present. For example, a starting mixture containing QS-17, QS-18 and desglucosyl-QS-17 components which is treated with an appropriate beta-glucosidase and alpha-rhamnosidase in parallel may undergo conversions including:
Extensive protein or DNA databases of natural and artificial glycosidases are available. Candidate enzymes may be selected and screened to assess suitability for achieving a particular conversion under particular reaction conditions. Suitability of an enzyme will depend on a number of factors including:
Additional factors which facilitate effective conversions include:
Those skilled in the art will appreciate that the level and type of specificity required of an enzyme will depend on the objective to be achieved and the general circumstances.
Conversion of QS-18 family components to QS-21 family components requires an enzyme demonstrating beta exo glucosidase activity.
Conversion of QS-17 family components to desglucosyl-QS-17 family components requires an enzyme demonstrating beta exo glucosidase activity.
Conversion of desglucosyl-QS-17 family components to QS-21 family components requires an enzyme demonstrating alpha exo rhamnosidase activity.
Conversion of QS-17 family components to QS-18 family components requires an enzyme demonstrating alpha exo rhamnosidase activity.
It may be noted that many Quillaja saponaria starting saponins of interest contain only one glucose residue. Many Quillaja saponaria starting saponins of interest contain a plurality of rhamnose residues, therefore selectivity for specific rhamnose residues is of greater importance practically. For example, conversion of desglucosyl-QS-17 family components to QS-21 components or QS-17 family components to QS-18 family components requires specificity for exo-rhamnosidase action over endo-rhamnosidase action. Furthermore, rhamnosidase specificity for the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety over other terminal rhamnose residues (e.g. in the rhamnose chemotype components) may also be desirable. In certain embodiments it may be desirable to remove the terminal rhamnose from rhamnose chemotype components (alone or in conjunction with any alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety), to better facilitate their chromatographic separation from xylose chemotype components.
In one embodiment saponin starting material is subjected to enzymatic modification by a single enzyme. The single enzyme may be a glucosidase, in particular a beta exo glucosidase. A single enzyme glucosidase may be an engineered glucosidase polypeptide of the present invention. Alternatively, the single enzyme is a rhamnosidase, in particular an alpha exo rhamnosidase. A single enzyme rhamnosidase may an engineered rhamnosidase polypeptide of the present invention.
Preferred enzymes are those which efficiently enzymatically convert a starting saponin(s) to the desired product saponin(s) while demonstrating limited or no undesired conversion(s) of other saponin components present.
In one embodiment saponin starting material is subjected to enzymatic modification by more than one enzyme, such as by two or three enzymes, especially by two enzymes. Enzymatic modification by more than one enzyme may involve sequential/series enzymatic modification. Alternatively, enzymatic modification by more than one enzyme may involve concurrent/parallel enzymatic modification. Enzymatic modification by at least three enzymes may involve a combination of sequential/series (modification by one enzyme) and concurrent/parallel (modification by at least two other enzymes) enzymatic modification, in any order. Where a plurality of enzymes are provided, these may be as distinct proteins or may be in the form of one or more fusion proteins.
An enzyme of interest is a glucosidase, such as a beta exo glucosidase. A glucosidase may be an engineered glucosidase polypeptide of the present invention. Another enzyme of interest is a rhamnosidase, such as an alpha exo rhamnosidase. A rhamnosidase may an engineered rhamnosidase polypeptide of the present invention. Enzyme combinations of interest include those comprising, such as consisting of, a glucosidase and a rhamnosidase, in particular a beta exo glucosidase and an alpha exo rhamnosidase. Enzymatic modification involving a glucosidase and a rhamnosidase, in particular a beta exo glucosidase and an alpha exo rhamnosidase, may be undertaken: sequentially with glucosidase (e.g. beta exo glucosidase) followed by rhamnosidase (e.g. alpha exo rhamnosidase), sequentially with rhamnosidase (e.g. alpha exo rhamnosidase) followed by glucosidase (e.g. beta exo glucosidase) or, conveniently, concurrently with both glucosidase (e.g. beta exo glucosidase) and rhamnosidase (e.g. alpha exo rhamnosidase). Particular enzyme combinations of interest are those comprising, such as consisting of, an engineered glucosidase of the present invention and an engineered rhamnosidase polypeptide of the present invention.
Enzymes utilised will typically be of external origin to saponin material i.e. not naturally found within the source of saponins obtained by extraction.
Enzymes may be native, i.e. naturally occurring glycosidases, or alternatively may be non-naturally occurring glycosidases. In one embodiment a glucosidase enzyme is a naturally occurring glucosidase (e.g. exo glucosidase, such as beta exo glucosidase). In a second embodiment a glucosidase enzyme is a non-naturally occurring glucosidase (e.g. exo glucosidase, such as beta exo glucosidase). In one embodiment a rhamnosidase enzyme is a naturally occurring rhamnosidase (e.g. exo rhamnosidase, such as alpha exo rhamnosidase). In a second embodiment a rhamnosidase enzyme is a non-naturally occurring rhamnosidase (e.g. exo rhamnosidase, such as alpha exo rhamnosidase).
Enzymes may be modified relative to a reference enzyme (‘engineered’). Point mutations, either singly or in combination, introduced by engineering may provide benefits such as increased activity, increased specificity, increased stability, increased expression or other the like. Assays to confirm the properties of the enzymes are well known to those skilled in the field. For example, activity may be quantified by methods such as those shown in the examples (see Examples 4 to 7) or by analogous methods.
Different enzymes may show different sensitivity to environmental conditions, such as pH, temperature, substrate concentration, product concentration, solvent composition, presence of contaminants and the like. Such parameters may be taken into consideration during screening of candidate enzymes for the desired activity.
Candidate enzymes having beta glucosidase activity include those in EC3.2.1.21.
Beta exo glucosidases of interest include those described in Table 7, especially SEQ ID Nos. 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324, 319, 9, 240, 325 and 338, and functional variants thereof. Particular beta exo glucosidases of interest include SEQ ID Nos. 262, 208, 63, 229, 250, 5, 101, 207, 169, 247, 302, 324 and 319, and functional variants thereof, such as SEQ ID Nos. 262, 208, 63, 229, 250, 5, 101 and 207, and functional variants thereof.
Another group of beta exo glucosidases of interest include those described in Table 9, especially SEQ ID Nos. 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890, 841, 832, 830, 845, 871, 837, 883 and 809, and functional variants thereof. Particular beta exo glucosidases of interest include SEQ ID Nos. 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810, 894, 849, 803, 890 and 841, and functional variants thereof, such as SEQ ID Nos. 850, 879, 868, 826, 804, 888, 881, 891, 816, 827, 857, 853, 842, 814, 886, 885, 838, 829, 808, 828, 870, 873, 844, 882, 874, 825, 824, 823, 810 and 894, and functional variants thereof.
SEQ ID No. 262, and functional variants thereof, are particularly desirable beta exo glucosidases. In one embodiment the beta exo glucosidase comprises, such as consists of: (i) SEQ ID. 262; or (ii) a functional variant thereof having at least 80% identity to SEQ ID. 262, especially at least 90%, in particular at least 95%, such as at least 96%, at least 97%, at least 98%, for example at least 99% identity; or (iii) a functional fragment of at least 100, especially at least 200, particularly at least 300, such as at least 400, for example at least 500 contiguous amino acids of SEQ ID. 262.
Candidate enzymes having alpha rhamnosidase activity include those in EC3.2.1.40.
Alpha exo rhamnosidases of interest include SEQ ID Nos. 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993, 1077, 1046, 1015, 1063, 1054, 1074, 1067 and 1033, and functional variants thereof. Particular alpha exo rhamnosidases of interest include SEQ ID Nos. 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041, 989, 1053, 1018, 1066, 1082, 1076, 993 and 1077, and functional variants thereof, such as SEQ ID Nos. 992, 1003, 1052, 1073, 1017, 1055, 1075, 1001, 1007, 1061, 1079, 1027, 1039, 1041 and 989, and functional variants thereof.
SEQ ID No. 1017, and functional variants thereof, are particularly desirable exo rhamnosidases. In one embodiment the alpha exo rhamnosidase comprises, such as consists of: (i) SEQ ID. 1017; or (ii) a functional variant thereof having at least 80% identity to SEQ ID. 1017, especially at least 90%, in particular at least 95%, such as at least 96%, at least 97%, at least 98%, for example at least 99% identity; or (iii) a functional fragment of at least 100, especially at least 200, particularly at least 300, such as at least 400, for example at least 500 contiguous amino acids of SEQ ID. 1017.
Functional variants of interest in the present application include those comprising, such as consisting of: (i) a sequence having at least 80% identity to the reference sequence, especially at least 90%, in particular at least 95%, such as at least 96%, at least 97%, at least 98%, for example at least 99% identity; or (ii) a fragment of at least 100, especially at least 200, particularly at least 300, such as at least 400, for example at least 500 contiguous amino acids of the reference sequence.
Certain desirable functional variants of interest include those comprising, such as consisting of, a sequence having 1 to 20 additions, deletions and/or substitutions relative to the reference sequence, especially 1 to 15 additions, deletions and/or substitutions, particularly 1 to additions, deletions and/or substitutions, such as 1 to 5 additions, deletions and/or substitutions.
The degree of sequence identity may be determined using by the homology alignment algorithm of Needleman and Wunsch, the ClustalW program or the BLASTP algorithm, using default settings. An algorithm using global alignment (Needleman and Wunsch) is preferred.
“Percentage of sequence identity,” “percent identity,” and “percent identical” are used herein to refer to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Determination of optimal alignment and percent sequence identity is performed using the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul, 1990; Altschul, 1997). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. Briefly, the BLAST analyses involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (N) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff, 1989).
Numerous other algorithms are available that function similarly to BLAST in providing percent identity for two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith, 1981, by the homology alignment algorithm of Needleman, 1970, by the search for similarity method of Pearson, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, 1995)). Additionally, determination of sequence alignment and percent sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided. The ClustalW program is also suitable for determining identity.
Modestobacter marinus glucosidase (Uniparc reference UPI000260A2FA, Uniprot reference I4EYD5-SEQ ID No. 262 herein) is a naturally occurring glucosidase demonstrating beta exo glucosidase activity and, for example, is capable of the conversion of QS-18 family components to QS-21 family components. Despite its potent activity, the present inventors have found that the properties of wild type Modestobacter marinus glucosidase may be altered by the introduction of one or more mutations.
The present invention provides an engineered glucosidase polypeptide comprising, such as consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes at least one residue substitution from:
The glucosidases will contain one to forty-two of the substitutions, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six to thirty or thirty-one to forty-three substitutions.
The present invention also provides an engineered glucosidase polypeptide comprising, such as consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes at least one residue substitution from:
The glucosidases will contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or all sixteen substitutions.
The engineered glucosidase polypeptide may comprise, such as consist of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes at least one residue substitution from: F44Y, V263L, A355W, R357M, T365N, L367C, Q396R, F442Q, L474C, I475F and F541I.
Suitably the engineered glucosidase polypeptide comprises, such as consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 262, or a functional fragment thereof, wherein the engineered glucosidase polypeptide includes the residue substitutions: F44Y, V263L, A355W, R357M, T365N, L367C, Q396R, F442Q, L474C, I475F and F541I.
The present invention provides a polypeptide comprising an amino acid sequence of sequence of SEQ ID No. 262 with one to twenty-five mutations selected from the list consisting of:
Variant glucosidases will contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four or all twenty-five mutations.
In some embodiments an engineered glucosidase is not a polypeptide comprising an amino acid sequence of sequence of SEQ ID No. 262 with one to twenty-five mutations selected from the list consisting of:
The above-mentioned engineered glucosidase polypeptides may also be referred to herein as examples of ‘variant glucosidases’.
A variant glucosidase may contain F44Y.
A variant glucosidase may contain V60L.
A variant glucosidase may contain G117A.
A variant glucosidase may contain F170N.
A variant glucosidase may contain V263G or V263L, in particular V263L.
A variant glucosidase may contain N351H or N351Q, in particular N351H.
A variant glucosidase may contain A355H, A355I, A355L, A355M, A355R, A355T or A355W. In some embodiments a variant glucosidase contains A355H. In some embodiments a variant glucosidase contains A355I. In some embodiments a variant glucosidase contains A355L. In some embodiments a variant glucosidase contains A355M. In some embodiments a variant glucosidase contains A355R. In some embodiments a variant glucosidase contains A355T. In some embodiments a variant glucosidase contains A355W.
A variant glucosidase may contain A356P.
A variant glucosidase may contain R357A, R357C, R357K, R357M or R357Q, in particular R357M.
A variant glucosidase may contain G362C.
A variant glucosidase may contain T365A, T365N or T365S, in particular T365N.
A variant glucosidase may contain L367C.
A variant glucosidase may contain V394R.
A variant glucosidase may contain V395Y.
A variant glucosidase may contain Q396E, Q396G, Q396N, Q396P, Q396R, Q396S or Q396Y, in particular Q396R.
A variant glucosidase may contain F430W.
A variant glucosidase may contain R435F.
A variant glucosidase may contain V438T.
A variant glucosidase may contain V440F.
A variant glucosidase may contain F442M or F442Q, in particular F442Q.
A variant glucosidase may contain G443D.
A variant glucosidase may contain G444T.
A variant glucosidase may contain A473F or A473R, in particular A473F.
A variant glucosidase may contain L474O, L474I or L474V, in particular L474C.
A variant glucosidase may contain I475F.
A variant glucosidase may contain L4920, L492G, L492H, L492I, L492N, L492Q, L492V, L492W or L492Y, in particular L492H, L492N, L492V. In some embodiments a variant glucosidase contains L492H. In some embodiments a variant glucosidase contains L492N. In some embodiments a variant glucosidase contains L492V.
A variant glucosidase may contain Q493F or Q493H.
A variant glucosidase may contain P494H or P494I, in particular P494I.
A variant glucosidase may contain S495I, S495K or S495Q.
A variant glucosidase may contain G496P or G496W, in particular G496P.
A variant glucosidase may contain D498A, D498E, D498F, D498I, D498K, D498L, D498N, D498P, D498R, D498S, D498T or D498V, in particular D498P.
A variant glucosidase may contain A502R.
A variant glucosidase may contain M504G or M504R, in particular M504R.
A variant glucosidase may contain L507A or L507R, in particular L507R.
A variant glucosidase may contain T508M.
A variant glucosidase may contain L529M.
A variant glucosidase may contain F535P.
A variant glucosidase may contain A536D or A536E.
A variant glucosidase may contain A537R.
A variant glucosidase may contain F541A, F541I, F541L, F541M or F541V, in particular F541I.
A variant glucosidase may contain L542I.
A variant glucosidase may contain Q543G or Q543L.
A variant glucosidase may contain E547L.
A variant glucosidase may contain Y585W.
A variant glucosidase may contain E588K.
Variant glucosidases may comprise R357M, T365N, A473F, L474O and I475F.
Variant glucosidases may comprise F44Y, R357M, T365N, F442Q, A473F, L474O and I475F.
Variant glucosidases may comprise F44Y, V263L, R357M, T365N, F442Q, A473F, L474C, I475F and F541I.
Variant glucosidases may comprise F44Y, V263L, A355W, R357M, T365N, L367C, Q396R, F442Q, L474O, I475F and F541I.
Variant glucosidases may comprise F44Y, V263L, R357M, T365N, F442Q, L474O, I475F, F541I and zero to seventeen mutations selected from the list consisting of:
A variant glucosidase may comprise a “tag,” a sequence of amino acids that allows for the isolation and/or identification of the polypeptide. For example, adding an affinity tag can be useful in purification. Exemplary affinity tags that can be used include histidine (HIS) tags (e.g., hexa histidine-tag, or 6×His-Tag), FLAG-TAG, and HA tags. Tags may be located N-terminally or C-terminally and may be directly connected or attached via a linking sequence. SEQ ID No. 1177 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be N-terminally attached. SEQ ID No. 1178 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be C-terminally attached.
In certain embodiments, the tags used herein are removable, e.g., removal by chemical agents or by enzymatic means, once they are no longer needed, e.g., after the polypeptide has been purified.
A variant glucosidase may comprise 1000 residues or fewer, especially 950 residues or fewer, in particular 900 residues or fewer, such as 850 residues or fewer.
A variant glucosidase may consist of an amino acid sequence of SEQ ID No. 262 with one to twenty-five mutations selected from the list consisting of:
Variant glucosidases desirably demonstrate a FIOP (Fold Improvement Over Parent) relative to SEQ ID No. 262 of at least 1.05, especially at least 2, in particular at least 10, such as at least 50. FIOP may be determined by the methods described in Example 4.
Kribbella flavida rhamnosidase (Uniparc reference UPI00019BDB13, Uniprot reference D2PMT5—SEQ ID No. 1017 herein) is a naturally occurring rhamnosidase demonstrating alpha exo rhamnosidase activity and, for example, is capable of the conversion of desglucosyl-QS-17 family components to QS-21 family components. Despite its potent activity, the present inventors have found that the properties of wild type Kribbella flavida rhamnosidase may be altered by the introduction of one or more mutations.
The present invention provides an engineered rhamnosidase polypeptide comprising, such as consisting of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 1017, or a functional fragment thereof, wherein the engineered rhamnosidase polypeptide includes at least one residue substitution from:
Consequently, the present invention provides a polypeptide comprising an amino acid sequence of sequence of SEQ ID No. 1017 with one to twenty-four mutations selected from the list consisting of:
Such polypeptides may be referred to herein as ‘variant rhamnosidases’.
Variant rhamnosidases will contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three or all twenty-four mutations.
A variant rhamnosidase may contain A56C.
A variant rhamnosidase may contain A143P.
A variant rhamnosidase may contain Q181H, Q181R or Q181S. In some embodiments a variant rhamnosidase contains Q181H. In some embodiments a variant rhamnosidase contains Q181R. In some embodiments a variant rhamnosidase contains Q181S.
A variant rhamnosidase may contain L214M.
A variant rhamnosidase may contain G215S.
A variant rhamnosidase may contain F216M.
A variant rhamnosidase may contain G218D or G218N. In some embodiments a variant rhamnosidase contains G218D. In some embodiments a variant rhamnosidase contains G218N.
A variant rhamnosidase may contain K219G.
A variant rhamnosidase may contain A238M.
A variant rhamnosidase may contain T252Y.
A variant rhamnosidase may contain T311W.
A variant rhamnosidase may contain V326C.
A variant rhamnosidase may contain G357C.
A variant rhamnosidase may contain S369C, S369I, S369K or S369M. In some embodiments a variant rhamnosidase contains S369C. In some embodiments a variant rhamnosidase contains S369I. In some embodiments a variant rhamnosidase contains S369K. In some embodiments a variant rhamnosidase contains S369M.
A variant rhamnosidase may contain I487M, I487Q or I487V. In some embodiments a variant rhamnosidase contains I487M. In some embodiments a variant rhamnosidase contains I487Q. In some embodiments a variant rhamnosidase contains I487V.
A variant rhamnosidase may contain K492N.
A variant rhamnosidase may contain V499T.
A variant rhamnosidase may contain G508S.
A variant rhamnosidase may contain R543C.
A variant rhamnosidase may contain L557Y.
A variant rhamnosidase may contain G634A.
A variant rhamnosidase may contain S635N.
A variant rhamnosidase may contain A690C.
A variant rhamnosidase may contain Q921H.
Variant rhamnosidases may comprise A143P, L214M, K219G and Q921H.
Variant rhamnosidases may comprise A143P, L214M, K219G, G357C and Q921H.
Variant rhamnosidases may comprise A143P, L214M, G215S, G218N, K219G, G357C, G508S, G634A and Q921H.
Variant rhamnosidases may comprise A143P, L214M, G215S, G218D, K219G, G357C, G508S, G634A, A690C and Q921H.
Variant rhamnosidases may comprise A143P, L214M, G215S, K219G, G357C, G508S, G634A and Q921H and one to sixteen mutations selected from the list consisting of:
Variant rhamnosidases may comprise A143P, L214M, G215S, K219G, G357C, G508S, G634A, Q921H, G218D or G218N, and one to fifteen mutations selected from the list consisting of:
A variant rhamnosidase may comprise a “tag,” a sequence of amino acids that allows for the isolation and/or identification of the polypeptide. For example, adding an affinity tag can be useful in purification. Exemplary affinity tags that can be used include histidine (HIS) tags (e.g., hexa histidine-tag, or 6×His-Tag), FLAG-TAG, and HA tags. Tags may be located N-terminally or C-terminally and may be directly connected or attached via a linking sequence. SEQ ID No. 1177 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be N-terminally attached. SEQ ID No. 1178 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be C-terminally attached.
In certain embodiments, the tags used herein are removable, e.g., removal by chemical agents or by enzymatic means, once they are no longer needed, e.g., after the polypeptide has been purified.
A variant rhamnosidase may comprise 1100 residues or fewer, especially 1050 residues or fewer, in particular 1000 residues or fewer, such as 950 residues or fewer.
A variant rhamnosidase may consist of an amino acid sequence of SEQ ID No. 1017 with one to twenty-four mutations selected from the list consisting of:
Variant rhamnosidases desirably demonstrate a FIOP relative to SEQ ID No. 1017 of at least 1.05, especially at least 2, in particular at least 10, such as at least 50. FIOP may be determined by the methods described in Example 4.
Function, in respect of functional variants, requires that the glycosidase activity is not notably reduced as a result of sequence variation, typically at least 50% of glycosidase activity, especially at least 75% activity, particularly at least 90%, such as at least 100% activity is maintained for at least one saponin modification reaction under at least one set of conditions (activity being determined by rate of modification of starting saponin to product saponin). Variants may be created with the intention of improving the glycosidase in some manner (e.g. conversion rate; specificity, which may be increased or reduced depending on needs; tolerance to environmental conditions, such as pH, substrate concentration, product concentration, other contaminants and the like; stability, thermal or chemical; production, such as facilitating expression or purification of the glycosidase either pre- or post-saponin modification). Variants need not be improved in all respects and may simply demonstrate a different balance of characteristics relative to the reference sequence.
Glycosidases will typically be 2000 amino acids or fewer, such as 1500 amino acids or fewer.
Suitably, glycosidases are soluble.
Glycosidases may be immobilised, such as by attachment to solid (e.g. polymer) particles. Immobilisation of glycosidases may facilitate separation from a reaction mixture, improve thermal stability and/or tolerance to environmental conditions.
Glycosidases may comprise a “tag,” a sequence of amino acids that allows for the isolation and/or identification of the polypeptide. For example, adding an affinity tag can be useful in purification. Exemplary affinity tags that can be used include histidine (HIS) tags (e.g., hexa histidine-tag, or 6×His-Tag), FLAG-TAG, and HA tags. Tags may be located N-terminally or C-terminally and may be directly connected or attached via a linking sequence. SEQ ID No. 1177 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be N-terminally attached. SEQ ID No. 1178 provides a sequence for an exemplary 6×His-Tag with linker sequence which may be C-terminally attached.
Any suitable reaction conditions may be used. Optimal conditions will depend on a range of factors including the identity of the starting saponin, product saponin, enzyme utilised and the like.
The reaction requires treatment of a starting saponin(s) with a glycosidase(s). Appropriate glycosidases may be added to a saponin containing composition in a range of forms such as solution (typically aqueous), suspension (typically aqueous) or solid. Glycosidases may be in a purified, partially purified (such as clarified cell lysate) or unpurified form (crude cell lysate or unlysed cells). The use of partially purified or unpurified forms may be of interest when source cells (e.g. recombinant host cells, such as E. coli) express the enzyme to an extent that desired activity sufficiently exceeds any deleterious impact arising from other host cell contaminants. Desirably the glycosidase(s) are added in the form of clarified lysates. Glycosidases may be freshly prepared (e.g. clarified lysate) or taken from storage, such as thawed frozen liquid (e.g. clarified lysate) or reconstituted dried material (e.g. freeze-dried clarified lysate). Where a plurality of glycosidases is used in parallel, these will typically be expressed in different host cells to ensure adequate process control. A plurality of glycosidases used in parallel may be added together or separately (in the same or different forms).
Glycosidases may be produced using a protein secretion system, such as Bacillus licheniformis.
The weight of a glycosidase present may be in the range of 0.0001 mg to 25 mg per ml, especially 0.0001 mg to 5 mg per ml, in particular 0.0001 mg to 1 mg per ml, such as 0.001 mg to 0.5 mg per ml. When provided in the form of dried clarified lysate, the weight of a glycosidase present may be in the range of 0.01 mg to 100 mg of lysate per ml, especially 0.01 mg to 30 mg per ml, in particular 0.01 mg to 5 mg per ml, such as 0.01 mg to 1 mg per ml.
Any appropriate pH may be used, though typically between pH 4 to 9, especially pH 5 to 8, and in particular pH 5.5 to 7.5 such as pH 5.5 to 6.5. Where a plurality of glycosidases is used in series, each enzymatic modification may be undertaken at a different pH though for convenience they may be undertaken at the same pH.
Buffers may be used to aid control of the pH. Suitable buffers and appropriate concentrations may be obtained from standard sources. Inorganic salt buffers are typically used, such as potassium phosphate, sodium phosphate, potassium acetate, sodium acetate, potassium citrate, sodium citrate and the like. A suitable buffer concentration may be 10 mM to 500 mM, especially 25 mM to 250 mM and in particular 50 mM to 100 mM. Buffer concentrations of about 50 mM, such as 50 mM or about 100 mM, such as 100 mM, may be used.
Any appropriate temperature may be used, though typically between 10 degC to 60 degC, especially 15 degC to 50 degC, in particular 15 degC to 45 degC, such as 20 degC to 42 degC.
An appropriate time such that the reaction proceeds sufficiently is usually up to 10 days, especially up to 5 days, in particular up to 3 days. Desirably the enzyme and reaction conditions are chosen such that the reaction proceeds sufficiently in a period of up to 2 days, especially up to 1 day, in particular up to 18 hrs, such as 12 hrs, for example up to 6 hrs.
The reaction will be undertaken in a suitable solvent, typically water or an aqueous solution with water miscible co-solvent(s) such as methanol, ethanol, n-propanol, i-propanol, tetrahydrofuran, ethylene glycol, glycerol,1,3-propanediol or acetonitrile. Any co-solvent(s) should be present in amounts which are not excessively deleterious to the reaction proceeding, such as 50% or less v/v, especially 20% or less, in particular 10% or less, such as 5% or less, for example 2% or less (in total).
The reaction may be homogeneous or heterogeneous, monophasic, bi-phasic or multiphasic with particulates, dispersed solids in suspension and/or colloidal micelles present. Desirably the reaction will be monophasic.
The starting saponins may be present at a concentration of 0.001 to 100 g per litre, especially 0.005 to 75 g per litre, in particular 0.01 to 50 g per litre, such as 0.1 to 25 g per litre, for example 1 to 10 g per litre.
The reaction may be carried out in various modes of operation such as batch mode, fed batch mode or continuous mode.
The reaction is typically performed at a scale which can provide commercial quantities of product saponin. A batch reaction volume may be at least 10 ml, especially at least 100 ml, in particular at least 1 L. A batch reaction volume may be 500 ml to 2000 L, especially 1 L to 1000 L, in particular 10 L to 500 L, such as 25 L to 200 L.
Enzymes are desirably adequately selective for the conversion of a starting saponin into a product saponin rather than other conversions of the starting saponin. As used herein, the term selectivity means at least 25% (mole basis) of converted starting saponin results in the intended product saponin, in particular at least 50%, especially at least 75%, such as at least 90% (e.g. at least 95%).
The concept of selectivity may also be applied in the context of the conversion of a plurality of starting saponins into a plurality of product saponins such that at least 25% of converted starting saponins (mole basis) result in the intended product saponins, in particular at least 50%, especially at least 75%, such as at least 90% (e.g. at least 95%).
Desirably conversion of a starting saponin into a product saponin is complete. However, rate of conversion, specificity of conversion (including rate of non-specific conversion(s)), product inhibition, starting saponin stability under reaction conditions, product saponin stability under reaction conditions and the like mean that conversions may not be complete or that it is desirable (e.g. for maximum yield or to obtain a balance between yield and process time) for a conversion to be stopped prior to completion.
At the point the reaction has progressed to the desired extent it may be stopped by denaturing or otherwise removing the enzyme. For example, the pH of reaction mixture may be adjusted to about pH to 3.5 to 4, especially pH 3.5 to 4, in particular pH 3.8 and/or the addition of sufficient quantities of anti-solvents or denaturing solvents such as acetonitrile. Precipitated enzyme may be removed by filtration.
By the term ‘Preceding peak’ is meant the peak immediately preceding the QS-21 main peak in the HPLC-UV methods described herein (see
By the term ‘m/z’ is meant the mass to charge ratio of the monoisotope peak. Unless otherwise specified, ‘m/z’ is determined by negative ion electrospray mass spectrometry.
By the term ‘ion abundance’ is meant the amount of a specified m/z measured in the sample, or in a given peak as required by the context. The mass chromatogram for the specified m/z may be extracted from the MS total ion chromatogram in the UPLC-UV/MS methods described herein. The mass chromatogram plots the signal intensity versus time. Ion abundance is measured as the area of the integrated peak. The area for a specified m/z/area for a relative reference m/z=relative abundance.
By the term ‘UV absorbance at 214 nm’ is meant the area of an integrated peak in the UV absorbance chromatogram. The (area for a specified peak)/(area of all integrated peaks in the chromatogram)×100=percentage area for the specified peak.
By the term ‘UV absorbance at 214 nm and relative ion abundance’ is meant an estimate for the percentage of a given m/z for co-eluting species. (Percentage area for given UV peak)×(relative ion abundance for m/z of interest in given peak)/(sum of all relative ion abundance for given peak)=percentage of m/z of interest in the given UV peak, assumes relative ion abundance included for all coeluting species.
By the term ‘wherein the monoisotope of the most abundant species is 1988 m/z’ is meant the monoisotope of the most abundant species, first peak in the isotopic group with highest response per m/z is m/z 1987.9. The most abundant species may be determined by creating a combined spectrum across the entire total ion chromatogram using the UPLC-UV/MS method (negative ion electrospray) as described herein.
By the term ‘dried’ is meant that substantially all solvent has been removed. A dried extract will typically contain less than 5% solvent w/w, especially less than 2.5% (such as less than 5% water w/w, especially less than 2.5%). Suitably the dried extract will contain 100 ppm or less acetonitrile (w/w).
Further, there is provided a method for the manufacture of a saponin composition comprising the steps of:
There is also provided a method for the manufacture of a saponin composition comprising the steps of:
Additionally provided a method for the manufacture of a saponin composition comprising the steps of:
Also provided is a method for the manufacture of a saponin composition comprising the steps of:
Typically, the crude aqueous extract is a bark extract. Suitably the QS-21 main peak content in an aqueous solution of crude aqueous extract of Quillaja saponaria is at least 1 g/L, such as at least 2 g/L, especially at least 2.5 g/L and in particular at least 2.8 g/L (e.g. as determined by UV absorbance relative to a control sample of known concentration).
The step of purifying the extract by polyvinylpolypyrrolidone adsorption involves treatment of the extract with polyvinylpolypyrrolidone adsorbant e.g. resin. Typically, the extract is agitated with the polyvinylpolypyrrolidone resin. The extract may subsequently be separated from the polyvinylpolypyrrolidone resin with adsorbed impurities by filtration. This step of the process generally removes polyphenolic impurities such as tannins.
The step of purifying the extract by reverse phase chromatography using a polystyrene resin typically uses acetonitrile and water as solvent, usually acidified with a suitable acid such as acetic acid. An example of a suitable resin is Amberchrom XT20. Chromatography may be undertaken using isocratic conditions, though is typically operated under a solvent gradient (continuous, such as linear, or stepped), such as those provided in the Examples. This step of the process generally removes non-saponin material and enriches the desired saponins. Each polystyrene chromatography run is typically at a scale of between 25-200 g of QS-21, such as between 50-150 g and in particular between 70-110 g (amounts being based on QS-21 main peak content in the material by UV).
Purifying the extract by reverse phase chromatography using a phenyl resin typically uses acetonitrile and water as solvent, usually acidified with a suitable acid such as acetic acid. Chromatography may be undertaken using a solvent gradient (continuous, such as linear, or stepped), though is typically operated under isocratic conditions. This step of the process provides the final purification of the desired saponins. Selected fractions may be pooled to maximise yield of material matching the required criteria. Each phenyl chromatography run is typically at a scale of between 4-40 g of QS-21, such as between 10-30 g and in particular between 13-21 g (amounts being based on QS-21 main peak content in the material by UV).
The method may comprise the further step of removing solvent to provide a dried saponin extract. Consequently, the invention provides a method for the manufacture of a saponin composition comprising the steps of:
The invention also provides a method for the manufacture of a saponin composition comprising the steps of:
Further provided is a method for the manufacture of a saponin composition comprising the steps of:
Additionally provided is a method for the manufacture of a saponin composition comprising the steps of:
In order to improve drying efficiency, it may be desirable to undertake further steps of concentrating the extract, such as by capture and release using an appropriate technique, for example reverse phase chromatography (e.g. using a C8 resin), and/or exchanging the solvent in advance of the drying step.
Also provided is a method for the manufacture of a saponin composition comprising the steps:
Further provided is a method for the manufacture of a saponin composition comprising the steps:
Additionally provided is a method for the manufacture of a saponin composition comprising the steps:
The invention provides a method for the manufacture of a saponin composition comprising the steps:
Also provided is a method for the manufacture of a saponin composition comprising the steps:
Further provided is a method for the manufacture of a saponin composition comprising the steps:
Additionally provided is a method for the manufacture of a saponin composition comprising the steps:
The invention provides a method for the manufacture of a saponin composition comprising the steps:
The step of purifying the extract by diafiltration, ultrafiltration or dialysis, is suitably purification by diafiltration. typically using tangential flow. An appropriate example of a membrane is a 30 kDa cut-off. This step of the process generally removes salts, sugars and other low molecular weight materials.
Concentration of the extract may be performed using any suitable technique. For example, concentration may be performed using a capture and release methodology, such as reverse phase chromatography, in particular using a C8 resin. The reverse phase chromatography typically uses acetonitrile and water as solvent, usually acidified with a suitable acid such as acetic acid. Chromatography is typically operated under a solvent gradient, with the saponin extract captured in low organic solvent and eluted in high organic solvent, in particular, a stepped solvent gradient.
Exchanging the solvent may be performed using any suitable technique, in particular diafiltration, ultrafiltration or dialysis, especially diafiltration. Solvent exchange may be useful, for example, in reducing the acetonitrile content such as described in WO2014016374. A suitable membrane may be selected to allow solvent exchange while retaining the saponin extract, such as a 1 kDa membrane.
Drying, by removing the solvent, may be undertaken by any suitable means, in particular by lyophilisation. During drying, degradation of the saponin extract can occur, leading to the formation of Iyo impurity. Consequently, it is desirable to dry under conditions which limit formation of Iyo impurity, such as by limiting the drying temperature and/or drying time. Suitably removal of solvent is undertaken by a single lyophilisation process. The extent of drying required will depend on the nature of the solvent, for example non-pharmaceutically acceptable solvents will desirably be removed to a high degree, whereas some pharmaceutically acceptable solvents (such as water) may be removed to a lesser degree.
Suitably the methods of the present invention are undertaken at a scale of between 25-1000 g of QS-21, such as between 50-500 g and in particular between 100-500 g (amounts being based on QS-21 main peak content in the material by UV).
Provided is a product saponin prepared according to the present invention. There is provided the use of a product saponin prepared according to the present invention in the manufacture of a medicament. Additionally, provided is a product saponin prepared according to the present invention for use as a medicament, in particular as an adjuvant. Also provided is an adjuvant composition comprising a product saponin prepared according to the present invention.
There is provided a crude extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase. There is also provided a crude extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a rhamnosidase. Also provided is a crude extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase and a rhamnosidase.
There is provided a crude bark extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase. There is also provided a crude bark extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a rhamnosidase. Also provided is a crude saponin extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase and a rhamnosidase.
There is provided a PVPP treated extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase. There is also provided a PVPP treated extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a rhamnosidase. Also provided is a PVPP treated extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase and a rhamnosidase.
There is provided a PVPP treated bark extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase. There is also provided a PVPP treated bark extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a rhamnosidase. Also provided is a PVPP treated saponin extract (such as from Quillaja species, especially Quillaja saponaria), such as water and/or lower alcohol extract, especially aqueous extract which has been treated by a glucosidase and a rhamnosidase.
Also provided is a saponin composition containing at least 93% QS-21 main peak and <0.25% 2018 component by UV absorbance at 214 nm. Suitably wherein the monoisotope of the most abundant species is 1987.9 m/z. Desirably, the saponin composition contains at least 98% QS-21 group by UV absorbance at 214 nm. Desirably, the extract contains 1% or less of Iyo impurity by UV absorbance at 214 nm. Desirably, the extract contains 1% or less of largest peak outside the QS-21 group by UV absorbance at 214 nm.
Also provided is a saponin composition containing at least 98% QS-21 group, at least 93% QS-21 main peak, <0.25% 2018 component, 1% or less of largest peak outside the QS-21 group by UV absorbance at 214 nm and wherein the monoisotope of the most abundant species is 1987.9 m/z. Suitably the saponin composition contains <0.23% 2018 component, especially <0.21% 2018 component, in particular <0.21% 2018 component, such as 0.2% or less 2018 component.
The saponin compositions desirably comprise at least 40%, such as at least 50%, suitably at least 60%, especially at least 70% and desirably at least 80%, for example at least 90% (as determined by UV absorbance at 214 nm and by relative ion abundance) QS-21 1988 A component, QS-21 1856 A component and/or QS-21 2002 A component. In certain embodiments, the saponin composition comprises at least 40%, such as at least 50%, in particular at least 60%, especially at least 65%, such as at least 70%, QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments the saponin composition contain 90% or less, such as 85% or less, or 80% or less, QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin composition contain from 40% to 90% QS-21 1988 A component, such as 50% to 85% QS-21 1988 A component, especially 70% to 80% QS-21 1988 A component as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin compositions contain 30% or less, such as 25% or less, QS-21 1856 A as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments the saponin composition contain at least 5%, such as at least 10% QS-21 1856 A by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin compositions contain from 5% to 30% QS-21 1856 A, such as 10% to 25% QS-21 1856 A as determined by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin composition contains 40% or less, such as 30% or less, in particular 20% or less, especially 10% or less QS-21 2002 A component by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin composition contain at least 0.5%, such as at least 1%, QS-21 2002 A component by UV absorbance at 214 nm and by relative ion abundance. In certain embodiments, the saponin composition contain from 0.5% to 40% QS-21 2002 A component, such as 1% to 10% QS-21 2002 A component as determined by UV absorbance at 214 nm and by relative ion abundance.
By the term ‘Iyo impurity’ is meant the triterpenoid glycosides identified as ‘Lyophilization Peak’ in
The saponin compositions of the present invention (i.e. a composition comprising a product saponin prepared according to the present invention) may be combined with further adjuvants, such as a TLR4 agonist, in particular lipopolysaccharide TLR4 agonists, such as lipid A derivatives, especially a monophosphoryl lipid A e.g. 3-de-O-acylated monophosphoryl lipid A (3D-MPL). 3D-MPL is sold under the name ‘MPL’ by GlaxoSmithKline Biologicals N.A. and is referred throughout the document as 3D-MPL. See, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL can be produced according to the methods described in GB 2 220 211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
Other TLR4 agonists which may be of use in the present invention include Glucopyranosyl Lipid Adjuvant (GLA) such as described in WO2008/153541 or WO2009/143457 or the literature articles Coler R N et al. (2011) Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant. PLoS ONE 6(1): e16333. doi:10.1371/journal.pone.0016333 and Arias M A et al. (2012) Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promotes Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgp140. PLoS ONE 7(7): e41144. doi:10.1371/journal.pone.0041144. WO2008/153541 or WO2009/143457 are incorporated herein by reference for the purpose of defining TLR4 agonists which may be of use in the present invention.
A particular alkyl glucosaminide phosphate (AGP) of interest is set forth as follows:
TLR4 agonists of interest include:
3-deacyl monophosphoryl hexa-acyl lipid A.
Another TLR4 agonist of interest is:
3-deacyl monophosphoryl lipid A.
A TLR4 agonist of interest is dLOS (as described in Han, 2014):
A typical adult human dose of adjuvant will comprise a saponin composition, such as a Q-21 composition, at amounts between 1 and 100 ug per human dose. The saponin extract may be used at a level of about 50 ug. Examples of suitable ranges are 40-60 ug, suitably 45-ug or 49-51 ug, such as 50 ug. In a further embodiment, the human dose comprises saponin composition, such as a Q-21 composition, at a level of about 25 ug. Examples of lower ranges include 20-30 ug, suitably 22-28 ug or 24-26 ug, such as 25 ug. Human doses intended for children may be reduced compared to those intended for an adult (e.g. reduction by 50%).
The TLR4 agonists, such as a lipopolysaccharide, such as 3D-MPL, can be used at amounts between 1 and 100 ug per human dose. 3D-MPL may be used at a level of about 50 ug. Examples of suitable ranges are 40-60 ug, suitably 45-55 ug or 49-51 ug, such as 50 ug. In a further embodiment, the human dose comprises 3D-MPL at a level of about 25 ug. Examples of lower ranges include 20-30 ug, suitably 22-28 ug or 24-26 ug, such as 25 ug. Human doses intended for children may be reduced compared to those intended for an adult (e.g. reduction by 50%).
When both a TLR4 agonist and a saponin composition, such as a Q-21 composition, are present in the adjuvant, then the weight ratio of TLR4 agonist to saponin is suitably between 1:5 to 5:1, suitably 1:1. For example, where 3D-MPL is present at an amount of 50 ug or 25 ug, then suitably QS-21 may also be present at an amount of 50 ug or 25 ug per human dose.
Adjuvants may also comprise a suitable carrier, such as an emulsion (e.g. an oil in water emulsion, such as a squalene containing oil in water emulsion) or liposomes.
The present invention provides an adjuvant composition comprising a saponin composition according to the present invention. Suitably the adjuvant composition further comprises a TLR4 agonist.
The term ‘liposome’ is well known in the art and defines a general category of vesicles which comprise one or more lipid bilayers surrounding an aqueous space. Liposomes thus consist of one or more lipid and/or phospholipid bilayers and can contain other molecules, such as proteins or carbohydrates, in their structure. Because both lipid and aqueous phases are present, liposomes can encapsulate or entrap water-soluble material, lipid-soluble material, and/or amphiphilic compounds.
Liposome size may vary from 30 nm to several um depending on the phospholipid composition and the method used for their preparation.
The liposomes of use in the present invention suitably contain DOPC, or, consist essentially of DOPC and sterol (with saponin and optionally TLR4 agonist).
In the present invention, the liposome size will be in the range of 50 nm to 200 nm, especially 60 nm to 180 nm, such as 70-165 nm. Optimally, the liposomes should be stable and have a diameter of ˜100 nm to allow convenient sterilization by filtration.
Structural integrity of the liposomes may be assessed by methods such as dynamic light scattering (DLS) measuring the size (Z-average diameter, Zav) and polydispersity of the liposomes, or, by electron microscopy for analysis of the structure of the liposomes. In one embodiment the average particle size is between 95 and 120 nm, and/or, the polydispersity (Pdl) index is not more than 0.3 (such as not more than 0.2).
In a further embodiment, a buffer is added to an adjuvant composition. The pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the subject. Suitably, the pH of a liquid mixture is at least 4, at least 5, at least 5.5, at least 5.8, at least 6. The pH of the liquid mixture may be less than 9, less than 8, less than 7.5 or less than 7. In other embodiments, pH of the liquid mixture is between 4 and 9, between 5 and 8, such as between 5.5 and 8. Consequently, the pH will suitably be between 6-9, such as 6.5-8.5. In a particularly preferred embodiment the pH is between 5.8 and 6.4. An appropriate buffer may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. In one embodiment, the buffer is a phosphate buffer such as Na/Na2PO4, Na/K2PO4 or K/K2PO4.
The buffer can be present in the liquid mixture in an amount of at least 6 mM, at least 10 mM or at least 40 mM. The buffer can be present in the liquid mixture in an amount of less than 100 mM, less than 60 mM or less than 40 mM.
It is well known that for parenteral administration solutions should have a pharmaceutically acceptable osmolality to avoid cell distortion or lysis. A pharmaceutically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the compositions (when reconstituted, if presented in dried form) will have an osmolality in the range of 250 to 750 mOsm/kg, for example, the osmolality may be in the range of 250 to 550 mOsm/kg, such as in the range of 280 to 500 mOsm/kg. In a particularly preferred embodiment the osmolality may be in the range of 280 to 310 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).
An “isotonicity agent” is a compound that is physiologically tolerated and imparts a suitable tonicity to a formulation to prevent the net flow of water across cell membranes that are in contact with the formulation. In some embodiments, the isotonicity agent used for the composition is a salt (or mixtures of salts), conveniently the salt is sodium chloride, suitably at a concentration of approximately 150 nM. In other embodiments, however, the composition comprises a non-ionic isotonicity agent and the concentration of sodium chloride in the composition is less than 100 mM, such as less than 80 mM, e.g. less than 50 mM, such as less mM, less than 30 mM and especially less than 20 mM. The ionic strength in the composition may be less than 100 mM, such as less than 80 mM, e.g. less than 50 mM, such as less 40 mM or less than 30 mM.
In a particular embodiment, the non-ionic isotonicity agent is a polyol, such as sucrose and/or sorbitol. The concentration of sorbitol may e.g. between about 3% and about 15% (w/v), such as between about 4% and about 10% (w/v). Adjuvants comprising an immunologically active saponin fraction and a TLR4 agonist wherein the isotonicity agent is salt or a polyol have been described in WO2012/080369.
Suitably, a human dose volume of between 0.05 ml and 1 ml, such as between 0.1 and ml, in particular a dose volume of about 0.5 ml, or 0.7 ml. The volumes of the compositions used may depend on the delivery route and location, with smaller doses being given by the intradermal route. A unit dose container may contain an overage to allow for proper manipulation of materials during administration of the unit dose.
The ratio of saponin:DOPC will typically be in the order of 1:50 to 1:10 (w/w), suitably between 1:25 to 1:15 (w/w), and preferably 1:22 to 1:18 (w/w), such as 1:20 (w/w).
Suitably the saponin is presented in a less reactogenic composition where it is quenched with an exogenous sterol, such as cholesterol. Cholesterol is disclosed in the Merck Index, 13th Edn., page 381, as a naturally occurring sterol found in animal fat. Cholesterol has the formula (C27H46O) and is also known as (3β)-cholest-5-en-3-ol.
The ratio of saponin:sterol will typically be in the order of 1:100 to 1:1 (w/w), suitably between 1:10 to 1:1 (w/w), and preferably 1:5 to 1:1 (w/w). Suitably excess sterol is present, the ratio of saponin:sterol being at least 1:2 (w/w). In one embodiment, the ratio of saponin:sterol is 1:5 (w/w). In one embodiment, the sterol is cholesterol.
The amount of liposome (weight of lipid and sterol) will typically be in the range of 0.1 mg to 10 mg per human dose of a composition, in particular 0.5 mg to 2 mg per human dose of a composition.
In a particularly suitable embodiment, liposomes used in the invention comprise DOPC and a sterol, in particular cholesterol. Thus, in a particular embodiment, a composition used in the invention comprises saponin extract in the form of a liposome, wherein said liposome comprises DOPC and a sterol, in particular cholesterol.
A particular adjuvant of interest features liposomes comprising DOPC and cholesterol, with TLR4 agonist and a saponin prepared according to the present invention, especially 3D-MPL and a saponin prepared according to the present invention.
Another adjuvant of interest features liposomes comprising DOTAP and DMPC, with TLR4 agonist and a saponin prepared according to the present invention, especially dLOS and a saponin prepared according to the present invention.
The adjuvants prepared according to the present invention may be utilised in conjunction with an immunogen or antigen. In some embodiments a polynucleotide encoding the immunogen or antigen is provided.
The adjuvant may be administered to a subject separately from an immunogen or antigen, or the adjuvant may be combined, either during manufacturing or extemporaneously, with an immunogen or antigen to provide an immunogenic composition for combined administration.
As used herein, a subject is a mammalian animal, such as a rodent, non-human primate, or human.
Consequently, there is provided a method for the preparation of an immunogenic composition comprising an immunogen or antigen, or a polynucleotide encoding the immunogen or antigen, said method comprising the steps of:
There is also provided the use of an adjuvant comprising a saponin prepared according to the present invention in the manufacture of a medicament. Suitably the medicament comprises an immunogen or antigen, or a polynucleotide encoding the immunogen or antigen. Further provided is an adjuvant comprising a saponin prepared according to the present invention for use as a medicament. Suitably the medicament comprises an immunogen or antigen, or a polynucleotide encoding the immunogen or antigen.
By the term immunogen is meant a polypeptide which is capable of eliciting an immune response. Suitably the immunogen is an antigen which comprises at least one B or T cell epitope. The elicited immune response may be an antigen specific B cell response, which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFNgamma, TNFalpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFNgamma, TNFalpha and/or IL2.
The antigen may be derived (such as obtained from) from a human or non-human pathogen including, e.g., bacteria, fungi, parasitic microorganisms or multicellular parasites which infect human and non-human vertebrates, or from a cancer cell or tumor cell.
In one embodiment the antigen is a recombinant protein, such as a recombinant prokaryotic protein.
A plurality of antigens may be provided. For example, a plurality of antigens may be provided to strengthen the elicited immune response (e.g. to ensure strong protection), a plurality of antigens may be provided to broaden the immune response (e.g. to ensure protection against a range of pathogen strains or in a large proportion of a subject population) or a plurality of antigens may be provided to currently elicit immune responses in respect of a number of disorders (thereby simplifying administration protocols). Where a plurality of antigens are provided, these may be as distinct proteins or may be in the form of one or more fusion proteins.
Antigen may be provided in an amount of 0.1 to 100 ug per human dose. The present invention may be applied for use in the treatment or prophylaxis of a disease or disorder associated with one or more antigens described above. In one embodiment the disease or disorder is selected from malaria, tuberculosis, COPD, HIV and herpes.
The adjuvant may be administered separately from an immunogen or antigen, or may be combined, either during manufacturing or extemporaneously), with an immunogen or antigen to provide an immunogenic composition for combined administration.
For parenteral administration in particular, compositions should be sterile. Sterilisation can be performed by various methods although is conveniently undertaken by filtration through a sterile grade filter. Sterilisation may be performed a number of times during preparation of an adjuvant or immunogenic composition, but is typically performed at least at the end of manufacture.
By “sterile grade filter” it is meant a filter that produces a sterile effluent after being challenged by microorganisms at a challenge level of greater than or equal to 1×107/cm2 of effective filtration area. Sterile grade filters are well known to the person skilled in the art of the invention for the purpose of the present invention, sterile grade filters have a pore size between and 0.25 um, suitably 0.18-0.22 um, such as 0.2 or 0.22 um.
The membranes of the sterile grade filter can be made from any suitable material known to the skilled person, for example, but not limited to cellulose acetate, polyethersulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE). In a particular embodiment of the invention one or more or all of the filter membranes of the present invention comprise polyethersulfone (PES), in particular hydrophilic polyethersulfone. In a particular embodiment of the invention, the filters used in the processes described herein are a double layer filter, in particular a sterile filter with built-in prefilter having larger pore size than the pore size of the end filter. In one embodiment the sterilizing filter is a double layer filter wherein the pre-filter membrane layer has a pore size between 0.3 and 0.5 nm, such as 0.35 or 0.45 nm. According to further embodiments, filters comprise asymmetric filter membrane(s), such as asymmetric hydrophilic PES filter membrane(s). Alternatively, the sterilizing filter layer may be made of PVDF, e.g. in combination with an asymmetric hydrophilic PES pre-filter membrane layer. In light of the intended medical uses, materials should be of pharmaceutical grade (such as parenteral grade).
The invention is illustrated by the following clauses:
The teaching of all references in the present application, including patent applications and granted patents, are herein fully incorporated by reference to the fullest extent possible. A composition or method or process defined as “comprising” certain elements is understood to encompass a composition, method or process (respectively) consisting of those elements. As used herein, ‘consisting essentially of’ means additional components may be present provided they do not alter the overall properties or function.
In respect of numerical values, the terms ‘approximately’, ‘around’ or ‘about’ will typically mean a value within plus or minus 10 percent of the stated value, especially within plus or minus 5 percent of the stated value and in particular the stated value.
Throughout the specification, including the claims, where the context permits, the term “comprising” and variants thereof such as “comprises” are to be interpreted as including the stated element (e.g., integer) or elements (e.g., integers) without necessarily excluding any other elements (e.g., integers). Thus a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
As used herein, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
As used herein, ng refers to nanograms, ug or μg refers to micrograms, mg refers to milligrams, mL or ml refers to milliliter, and mM refers to millimolar. Similar terms, such as um, are to be construed accordingly.
Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
The invention will be further described by reference to the following, non-limiting, examples:
Crude bark extract was separated by reverse phase HPLC using a C4 column and gradient elution: mobile phase A—water/acetonitrile, 7/3 v/v with 0.15% trifluoroacetic acid; mobile phase B—acetonitrile with 0.15% trifluoroacetic acid. UV detection was at 214 nm.
Crude bark extract samples are diluted as necessary with purified water. Polyvinylpolypyrrolidone (PVPP; 60 mg/mL) was added, the mixture stirred for approximately 30 minutes, and then centrifuged to separate the PVPP resin from the supernatant.
The supernatant was then analysed to provide an HPLC UV chromatogram.
40 ul of sample is injected. UV detection is set at 214 nM.
Using a blank injection for reference, integration of peaks in the chromatogram provides a total absorbance. Peak of interest (e.g. QS-21 main peak) is compared to total absorbance to determine peak content as a percentage.
The HPLC-UV method is also conveniently used to determine QS-21 main peak content and Preceding peak to QS-21 main peak ratio.
Column temperature 28 degrees C. 10 ul of sample is injected. UV detection is set at 214 nM.
Using a blank injection for reference, integration of peaks in the chromatogram provides a total absorbance. Peak of interest (e.g. QS-21 main peak) is compared to total absorbance to determine peak content as a percentage.
The UPLC-UV method is also conveniently used to determine 2018/QS-21 Ratio.
Test sample is prepared in 0.2% acetic acid in water/acetonitrile (70:30 v/v). Column temperature 55 degrees C. 10 ul of sample is injected. UV detection is set at 214 nM.
The term ‘QS-21 group’ is meant the triterpenoid glycosides identified from the B-isomer to the peak preceding the Iyo impurity in the UPLC-UV/MS methods described herein. Although retention times vary slightly between runs, the QS-21 group is located at approximately 3.8 min (QS-21 B-isomer) to approximately 4.5 minutes (prior to Iyo impurity peak, containing desarabinofuranosyl-QS-21 1856 A component).
Using a blank injection for reference, integration of peaks in the chromatogram that elute after the solvent front between 0.5 and around 5.50 minutes and do not appear in the blank is undertaken.
The monoisotope of the most abundant species is identified by combining TIC over the entire chromatogram to create a combined spectrum.
Ratio of QS-21 2002 A component to QS-21 1988 A component is calculated by comparing the ion current associated with the QS-21 2002 A component with the ion current associated with the QS-21 1988 A component within the QS-21 main peak.
Crude aqueous extract of Quillaja saponaria material having a 2018 component to QS-21 main peak ratio of 0.064 or lower and a Preceding peak to QS-21 main peak ratio of 0.4 or lower, was treated with PVPP (1 kg PVPP per litre of crude aqueous extract). After adsorption the mixture was filtered to separate the PVPP and bound impurities from the liquor.
Filtered liquor was concentrated and further purified by ultrafiltration/diafiltration using water and a 30 kD Hellicon membrane.
Resulting permeate was purified by reverse phase chromatography using a polystyrene resin (Amberchrom XT20).
Fractions were pooled to provide polystyrene purified saponin extract with a composition:
% QS-21 main peak≥18% (by HPLC)
and
2018 component/QS-21 main peak ratio≤0.054 (by UPLC-UV).
The combined polystyrene purified fraction pool was further purified by reverse phase chromatography using a phenyl resin (EPDM).
QS-21 containing fractions were pooled to provide phenyl purified saponin extract with a composition:
% QS-21 group≥98.5%
QS-21 main peak≥94.5%
2018 component≤2.7%
Main peak outside of the QS-21 group≤1% (by UPLC-UV/MS).
The combined phenyl purified saponin extract was concentrated by capture and release with reverse phase chromatography using a C8 resin (Lichroprep RP8) and the following conditions:
The C8 concentrated saponin extract was subjected to solvent exchange using ultrafiltration/diafiltration and a Pellicon 1 kDa membrane to reduce acetonitrile content below 21%.
The resulting solvent exchanged saponin extract was then lyophilised in a single step to provide a final purified saponin extract product.
The use of the process as described in Example 3 can consistently provide a purified saponin extract of Quillaja saponaria having a defined content in terms of QS-21 main peak and 2018 component, presenting a chromatographic profile comparable to the chromatograms shown in
The enzyme family of hydrolases (E.C.3.2.1.-) that act on glycosidic bonds (‘glycoside hydrolases’ (GH) or ‘glycosidases’), contains at present approximately one million members having wide ranging activities across molecules containing glycans and polysaccharides. A typical QS-18 family molecule contains a number of such glycosidic bonds, with the presence of the 1-3 bond between the alpha-L-rhamnose on the linear tetrasaccharide and the branched terminal beta-D-glucose differentiating the QS-18 family from the QS-21 family. The specific hydrolysis of this bond by a beta-glucosidase, i.e. an enzyme with exo-beta-1,3-glucosidase activity (E.C. 3.2.1.21 and E.C. 3.2.1.58) will therefore convert QS-18 family components to QS-21 family components. Specific members of the glycoside hydrolase family having exo-beta-1,3-glucosidase activity were initially identified using the CAZy (Carbohydrate Active enZyme) database (www.cazy.com), with GH families 1, 3 and 5 purported to have enzyme members with the desired exo-beta-1,3 activity. All sequences annotated by CAZy from GH families 1,3 and 5 were obtained, and separate curated hidden Markov model profiles constructed for each which were then used to identify additional familial enzymes by searching the 209 million protein member Uniprot (www.uniprot.org) knowledgebase with the software HMMER (Eddy, 1998). In total, 22,594 sequences: 12,049, 9,278 and 1,267 representatives from GH families 1,3 and 5, respectively, were identified using this method. MMSeqs2 (Hauser, 2016) was then used to cluster each group of enzyme sequences using the default clustering workflow and parameters with a minimum sequence identity and coverage of 30% and 80%, respectively. In cases where the initial clustering yielded clusters with more than 1000 members, a second sub-clustering was performed at a higher 50% or 70% identity to ensure diverse exemplars from these larger clusters were represented more prominently. All clusters were then examined, and exemplars selected from each with preferences for annotation quality, known experimental activity, existing three dimensional structures from the Protein Data Bank (www.wwpdb.org) or known extremophile organisms as annotated by Uniprot. A final set of 400 diverse candidate enzymes was selected. Polynucleotide sequences encoding each selected enzyme linked to an N-terminal 6×His tag and Tev-cleavage site were prepared (amino acid sequence for His-tag linker, inserted N-terminally of normal start methionine, is provided in SEQ ID No. 1177) using a proprietary genetic-algorithm based codon optimization code.
Details of the candidate enzyme and polynucleotide sequences are summarised below in Table 6.
Cyberlindnera fabianii
Flavobacterium gilvum
Algibacter lectus
Microbacterium azadirachtae
Actinobacteria bacterium
Chloroflexi bacterium
Komagataeibacter rhaeticus
Bacteroides sp.
Streptomyces rubrolavendulae
Clostridium roseum
Firmicutes bacterium
Anthracocystis flocculosa
Bifidobacterium boum
Jejuia pallidilutea
Ceratocystis fimbriata
Actinobacteria bacterium
Rhodococcus sp.
Valsa mali
Eisenbergiella tayi
Streptomyces sp.
Firmicutes bacterium
Tenericutes bacterium
Gluconobacter oxydans
Bifidobacterium catenulatum
Bionectria ochroleuca
Parcubacteria sp.
Microbacterium ketosireducens
Roseburia faecis
Kwoniella dejecticola
Cyberlindnera jadinii
Bacteroidetes bacterium
Verrucomicrobia bacterium
Bacteroidetes bacterium
Lichtheimia ramosa
Bifidobacterium mongoliense
Vibrio ishigakensis
Phaeomoniella chlamydospora
Ardenticatena maritima
Coprococcus comes
Nocardioides dokdonensis
Acetobacterium wieringae
Tenericutes bacterium
Tenericutes bacterium
Parabacteroides distasonis
Bifidobacterium psychraerophilum
Hebeloma cylindrosporum
Brenneria goodwinii
Aspergillus calidoustus
Bacteroides finegoldii
Altererythrobacter dongtanensis
Candidatus firestone
Tenericutes bacterium
Lentisphaerae bacterium
Planctomycetes bacterium
Pseudallescheria apiosperma
Nonlabens sediminis
Gynuella sunshinyii
Verticillium longisporum
Cellulomonas sp.
Hungatella hathewayi
Mesorhizobium sp.
Clostridium sp.
Chlamydiales bacterium
Spirochaetes bacterium
bacterium
Thermotogae bacterium
Pseudallescheria apiosperma
Algibacter lectus
Paxillus involutus
Verticillium longisporum
Mucilaginibacter gotjawali
Bacteroides uniformis
Coprococcus sp.
Blautia sp.
Cellulomonas sp.
bacterium
Spirochaetes bacterium
Tenericutes bacterium
Flavobacterium gilvum
Algibacter lectus
Hydnomerulius pinastri
Nocardia farcinica
Bacteroides cellulosilyticus
Fonsecaea erecta
Bacteroides sp.
Tannerella forsythia
Microbacterium esteraromaticum
bacterium
Candidatus hydrogenedentes
Bacteroidetes bacterium
Penicillium solitum
Weissella soli
Acetatifactor muris
Corynespora cassiicola
Meira miltonrushii
Bacteroides fragilis
Malassezia restricta
Fusarium euwallaceae
Psathyrella aberdarensis
Aeromonas hydrophila
Saccharopolyspora erythraea
Streptomyces sviceus
Naematelia encephala
Hartmannibacter diazotrophicus
Pontimonas salivibrio
Cadophora sp.
Meira miltonrushii
Monilinia fructigena
Hortaea werneckii
Streptomyces netropsis
Aureobasidium pullulans
Aspergillus clavatus
Clavibacter michiganensis
Penicillium rubens
Lachnoclostridium sp.
Rhodobacteraceae bacterium
Bacteroides fragilis
Aspergillus indologenus
Acaromyces ingoldii
Monilinia fructigena
Paenibacillus xylanexedens
Actinomyces howellii
Friedmanniomyces endolithicus
Neosartorya fischeri
Pseudomonas aeruginosa
Talaromyces stipitatus
Aquimixticola soesokkakensis
Rhodobacterales bacterium
Methylorubrum extorquens
Clostridium perfringens
Acholeplasmatales bacterium
Bacteroidetes bacterium
Clostridium carnis
Mycolicibacterium flavescens
Streptococcus gallolyticus
Yersinia enterocolitica
Anaeromyxobacter sp.
Talaromyces stipitatus
Hortaea werneckii
Micavibrio sp.
Acidobacteriia bacterium
Corynebacterium jeikeium
Clostridiaceae bacterium
Anaerolineaceae bacterium
Gymnopilus dilepis
Kocuria rosea
Teredinibacter sp.
Aspergillus niger
Laccaria bicolor
Pedosphaera parvula
Megamonas hypermegale
Armillaria gallica
Micromonospora sp.
Klebsiella oxytoca
Candidatus ozemobacter
Coleophoma crateriformis
Apiotrichum porosum
Acholeplasma hippikon
Streptomyces spectabilis
Aspergillus niger
Xanthomonas campestris
Lactobacillus paracasei
Bifiguratus adelaidae
bacterium
Actinomadura parvosata
Melissococcus plutonius
Enterococcus durans
Malassezia restricta
Apiotrichum porosum
Streptomonospora sp.
Lactobacillus gasseri
Aspergillus niger
Neosartorya fumigata
Eubacterium eligens
Bifiguratus adelaidae
bacterium
Corynespora cassiicola
Pseudomicrostroma glucosiphilum
Staphylococcus saprophyticus
Malassezia restricta
Saitozyma podzolica
Tremella mesenterica
Arthrobacter sp.
Scheffersomyces stipitis
Leptothrix cholodnii
Thauera sp.
Kosmotoga olearia
Roseburia intestinalis
Streptococcus equinus
Streptococcus cristatus
Cellulosilyticum lentocellum
Streptococcus gallolyticus
Ketogulonicigenium vulgare
Spathaspora passalidarum
Niastella koreensis
Cellvibrio sp.
Flavobacterium sp.
Macrophomina phaseolina
Kosmotoga olearia
Blautia obeum
Bifidobacterium dentium
Anaerolinea thermophila
Coriobacterium glomerans
Microlunatus phosphovorus
Streptomyces sp.
Spathaspora passalidarum
Glarea lozoyensis
Glaciozyma antarctica
Arthrobacter sp.
Fusarium pseudograminearum
Hypocrea rufa
Ruminococcus torques
Bifidobacterium dentium
Bacteroides salanitronis
Bacteroides coprosuis
Marinomonas posidonica
Chaetomium thermophilum
Tetragenococcus halophilus
Paenibacillus sp.
Turneriella parva
Arthrobacter sp.
Agaricus bisporus
Nectria haematococca
Bacteroides xylanisolvens
Sediminispirochaeta smaragdinae
Deinococcus proteolyticus
Sphingobacterium sp.
Sphingobium chlorophenolicum
Caloramator australicus
Commensalibacter intestini
Paenibacillus sp.
Nitrolancea hollandica
Cryptococcus neoformans
Acidipropionibacterium acidipropionici
Prevotella sp.
Rhodobacter capsulatus
Stigmatella aurantiaca
Sphaerochaeta globosa
Sphaerochaeta coccoides
Novosphingobium sp.
Arthrobotrys oligospora
Lactococcus lactis
Phaeospirillum molischianum
Modestobacter marinus
Saccharothrix espanaensis
Cronobacter sakazakii
Verticillium alfalfae
Bacteroides xylanisolvens
Leadbetterella byssophila
Sphaerochaeta globosa
Sphaerochaeta coccoides
Haloplasma contractile
Nitrospirillum amazonense
Azospirillum brasilense
Phaeospirillum molischianum
Modestobacter marinus
Wickerhamomyces ciferrii
Gloeocapsa sp.
Sphaerobacter thermophilus
Bacteroides xylanisolvens
Prevotella buccae
Grosmannia clavigera
Melampsora larici-populina
Prevotella multisaccharivorax
Streptomyces zinciresistens
Granulicella mallensis
Gibberella zeae
Modestobacter marinus
Lactobacillus equicursoris
Colletotrichum fructicola
Streptosporangium roseum
Listeria grayi
Enterococcus italicus
Fluviicola taffensis
Shigella flexneri
Actinomyces sp.
Verticillium dahliae
Actinoplanes sp.
Gibberella zeae
Auricularia subglabra
Nitratireductor indicus
Thermoclostridium stercorarium
Geobacillus sp.
Burkholderia ambifaria
Aspergillus oryzae
Yersinia pseudotuberculosis
Wallemia ichthyophaga
Glarea lozoyensis
Moniliophthora roreri
Zhouia amylolytica
Fusarium oxysporum
Flavobacterium johnsoniae
Oryza sativa
Clostridium
saccharoperbutylacetonicum
Rhodococcus sp.
Burkholderia ambifaria
Xanthomonas campestris
Caulobacter vibrioides
Arcticibacter svalbardensis
Winogradskyella psychrotolerans
Methyloglobulus morosus
Pestalotiopsis fici
Capronia coronata
Aspergillus aculeatus
Thermotoga neapolitana
Clostridium saccharoperbutylacetonicum
Schizosaccharomyces pombe
Phaeosphaeria nodorum
Xylella fastidiosa Dixon
Schizosaccharomyces pombe
Arcticibacter svalbardensis
Colletotrichum gloeosporioides
Xanthomonas arboricola
Fusarium oxysporum
Paenibacillus polymyxa
Kluyveromyces marxianus
Ilumatobacter coccineus
Agrobacterium sp.
Cytophaga hutchinsonii
Thermobifida fusca
Botryotinia fuckeliana
Agarivorans albus
Enterococcus sp.
Salinispira pacifica
Bacteroides xylanisolvens
Fusarium oxysporum
Thermotoga maritima
Neotermes koshunensis
Thanatephorus cucumeris
Hungateiclostridium thermocellum
Koribacter versatilis
Neosartorya fumigata
Amycolatopsis vancoresmycina
Gibberella fujikuroi
Bifidobacterium longum
Bacteroides xylanisolvens
Fusarium oxysporum
Sorghum bicolor
Clostridium cellulovorans
Dacryopinax primogenitus
Rhizobium radiobacter
Phanerochaete chrysosporium
Enterobacter agglomerans
Candidatus microthrix
Chthonomonas calidirosea
Acholeplasma brassicae
Chania multitudinisentens
Gibberella moniliformis
Fusarium oxysporum
Phanerochaete chrysosporium
Secale cereale
Anoxybacillus gonensis
Thermotoga maritima
Rhodospirillum rubrum
Thermotoga neapolitana
Candidatus microthrix
Ruminiclostridium cellobioparum
Acholeplasma brassicae
Klebsiella pneumoniae
Rhizoctonia solani
Homo sapiens
Thermoanaerobacter thermohydrosulfuricus
Stigmatella aurantiaca
Burkholderia thailandensis
Yersinia pseudotuberculosis
Togninia minima
Ophiostoma piceae
Pyronema omphalodes
Ogataea parapolymorpha
Hymenobacter swuensis
Flavobacterium johnsoniae
Paenibacillus polymyxa
Oryza sativa
Oryza sativa
Nannochloris
Halothermothrix orenii
Neurospora crassa
Micrococcus antarcticus
Exiguobacterium antarcticum
Thermus thermophilus
Trichoderma harzianum
Hypocrea jecorina
Streptomyces sp.
Streptococcus pyogenes
Trifolium repens
Talaromyces emersonii
Hungateiclostridium thermocellum
Lactobacillus plantarum
Agrobacterium tumefaciens
Experiment 4-1—Screening of Glucosidases for Deglycosylation with Purified QS-18 (0.04 mg/ml) at pH 7.5 and Room Temperature
Nucleotide sequences were sub-cloned into pET24b+ for expression. 10 uL of E. coli cells (One Shot® BL21(DE3) chemically competent E. coli) were transferred to each well of a 96 well PCR plate (prechilled on ice). 10 uL autoclaved water was added to the DNA, resuspended by pipetting, and 1 uL of plasmid DNA (15-30 ng) was transferred to the competent cells. Immediately after addition, the resulting mixture was mixed by pipetting. Cells were heat shocked by placing the plate in a thermal cycler at 42° C. for 30 seconds then transferred directly to an ice bath for 2 min. 100 uL sterile Lysogeny Broth (LB) medium was added to each well containing transformed cells. The content of each plate was transferred into a 96-deep well plate pre-aliquoted with 400 uL LB and the plate was incubated at 37° C. with shaking and 85% humidity for 1 hour. After outgrowth, 500 uL of sterile LB containing 100 ug/mL kanamycin was added to the plates containing cells and plates incubated at 37° C. with shaking overnight (18 hours) with humidity control (85%).
1000 mLs of Overnight Express Media was supplemented with 1 mL kanamycin 50 mg/mL and 20 mL of 50% v/v glycerol added (50 ug/mL kanamycin final and 1% glycerol). 96-Well Assay Block 2 mL plates were aliquoted with 380 uL of media per well. Pre-inoculum (20 uL) from transformation plates was added. The plates were sealed appropriately and incubated at 37° C. at 300 with shaking for 2 h. After 2 h the temperature was lowered to 20° C. and incubation continued for 20 h.
The liquid cultures were centrifuged for 10 minutes at 4° C. The supernatant was discarded, plates blotted on an absorbent material to remove residue and the plates frozen at −80° C.
Lysis buffer was prepared according to the following protocol:
2 copies of each cell pellet plate were removed from −80 degC freezer and allowed to thaw. 200 uL of lysis buffer was added to each well of one copy of the cell pellet plates. Plates were shaken at room temperature for 10 mins. 190 uL of cell pellet/lysis buffer was transferred to a corresponding fresh cell pellet plate. These plates were incubated at room temperature with shaking for 2 hours. Lysate was clarified by centrifugation (10 min, 4 degC).
QS-18 was obtained by analogous methods to Example 3, collecting a QS-18 containing phenyl fraction following phenyl treatment (presence of m/z corresponding to key components was confirmed by MS and the phenyl fraction then used without further treatment). QS-18 solution was prepared by diluting aqueous QS-18 (ca 1 mg/mL) with 100 mM potassium phosphate pH 7.5 to 0.2 mg/ml. 40 uL clarified lysate was transferred into fresh 96 well PCR plates. 10 uL QS-18 solution added to each well of lysate to a final concentration of 0.04 mg/ml. Incubated at room temperature with shaking for 20 h. Quenched with 50 uL MeCN and shaken at room temperature for 10 mins. Samples were analysed by LC-MS/MS using a Waters Acquity H class coupled to a Waters Xevo Tandem Quadrupole (TQD) Mass Spectrometer.
Enzyme activity was calculated as:
The negative control reactions, which utilised a plasmid expressing an unrelated protein, had an average % conversion of 0.42% with a standard deviation (S.D.) of 0.10%. Candidate enzymes with % conversion >0.72%, i.e. >3 S.D. above negative control, were considered to be positive hits and are listed below in Table 7. Sample results are shown in
Experiment 4-2—Screening of Glucosidases for Deglycosylation with Purified QS-18 (1 mg/ml) at pH 6 and 30 Deg C.
Lysates were prepared in an identical manner to Experiment 4-1 above, except the lysis buffer was prepared in 100 mM potassium phosphate buffer pH 6. QS-18 solution was prepared by dissolving QS-18 in 100 mM potassium phosphate buffer pH 6 (2 mg/mL).
12 uL clarified lysate was transferred into fresh 96 well PCR plates. 12 uL QS-18 solution was added (1 mg/ml final concentration), plates sealed and incubated overnight (30° C.) for 18 hrs. After quenching with 25 uL MeCN and shaking for 10 mins (RT), samples were analysed using the LC-MS/MS protocol described in Experiment 4-1 and enzyme activity determined in an analogous manner.
The negative control reactions had an average % conversion of 0.38% with a standard deviation (S.D.) of 0.06%. Sequences with % conversion >0.56% i.e. >3 S.D. above negative control are listed in Table 7.
Experiment 4-3—Screening of Glucosidases for Deglycosylation with QS-18 in Crude Bark Extract (1 in 2000 Dilution) at pH 7 and 30 Deg C.
Lysates were prepared according to the following procedure.
50 uL of 50% v/v glycerol was transferred to each well of a flat bottom 96 well plate. 50 uL from each well of the overnight culture plate (in LB) from Experiment 4-1 was transferred and mixed by pipette aspiration. The plate was then covered with a foil seal and frozen at −80° C. as a glycerol stock of the transformants. Glycerol stock plates were removed from −80° C. freezer and allowed to thaw. Overnight cultures were prepared by pipetting 5 mL LB into 50 mL tubes with Kanamycin as a selection marker at a final concentration of 50 μg/mL. Cultures were inoculated with 10 μL of glycerol stock and incubated overnight at 37° C. with shaking.
Flask cultures were prepared by pipetting 25 mL Terrific Broth (TB) into 250 mL conical flasks with Kanamycin as selection marker at a final concentration of 50 μg/mL. Overnight cultures OD600 was measured using a spectrophotometer and initial inoculum volume calculated for a starting OD˜0.1. Cultures were inoculated and incubated at 37° C. with shaking up to OD˜0.6.
Cultures were induced with 1 mM IPTG and temperature was reduced to 20° C. with shaking. Cultures were then incubated overnight. Cultures were harvested in individual 1 mL aliquots (in 2 mL tubes). 1 mL aliquots were centrifuged at 13000 g for 3 min and supernatant discarded. Pellets were frozen at −20° C.
Lysis buffer was prepared according to the following protocol:
1 mL of Lysis buffer was added to a pellet from 1 mL culture aliquot. Lysed samples were incubated at room temperature with shaking for 2 hours. Lysate was clarified by centrifugation at 13000 g, 5 min, 4° C.
Crude bark extract (CBE) obtained by aqueous extraction of Quillaja saponaria and containing at least 2.80 mg/ml QS-21 (by HPLC-UV) was diluted 1 in 400 in 50 mM potassium phosphate buffer at pH 7. 100 ul diluted CBE was added to 400 ul of each lysate to give a final dilution of 1 in 2000.
As diluted CBE was added to the lysate, the solution was vortexed for ˜5 seconds and then a 80 ul sample taken and quenched with 160 ul methanol (MeOH). This was used as a time 0 sample. The reaction solutions and controls were then left to shake at 30° C. Samples were taken after 1 h in the same way as the time 0 sample. Samples were analysed by LCMS/MS using the protocol described in Experiment 4-1 and enzyme activity determined in an analogous manner.
Enzyme activity is calculated as the % conversion of the QS-18 present in the crude bark extract:
Lysates were prepared as in Experiment 4-2.
Crude bark extract (CBE) was adjusted to pH 6 by dropwise addition of 2M NaOH with stirring. 25 uL of clarified lysate was transferred to a reaction plate, 22.5 ul of 100 mM potassium phosphate buffer pH 6 was added, 2.5 ul CBE at pH 6 was added. Reaction plates were sealed, incubated at 25 degC with shaking for 18 hours, then quenched by addition of 50 uL acetonitrile (MeCN). Quenched reaction plates were re-sealed and incubated at 20 degC with shaking for 10 min. The reaction plates were centrifuged (10 min, 4 degC) and analysed by UV HPLC with the method below:
Three key peaks of interest are apparent using this chromatography: Left Peak (retention time approximately 2.30-2.35 min) comprising mainly QS-17 family components; Middle Peak (retention time approximately 2.37-2.42 min) comprising mainly QS-18 family components and desglucosyl-QS-17 family components; and Right Peak (retention time approximately 2.44-2.50 min) comprising mainly QS-21 family components. Peak identity was supported by MS/MS.
Enzyme activity is calculated as the % conversion of the Middle Peak present in the crude bark extract:
Based on detection of QS-18 2150 and QS-21 1988 components by LCMS/MS (Examples 4-1, 4-2 and 4-3) or UV HPLC quantification of Middle Peak (mainly QS-18 family and desglucosyl-QS-17 family) and Right Peak (mainly QS-21 family) (Example 4-4), Example 4 shows that a number of suitable glucosidases could be identified by screening a set of candidate enzymes (38 from 400, 9.5%). Glucosidases were capable of converting QS-18 family components to QS-21 family components at a range of pHs, concentrations of starting materials and purity of starting materials.
Although certain candidate enzymes did not demonstrate notable conversion under the conditions tested, this may be due to issues with enzyme expression, suitability of conditions (i.e. enzymes may function under other conditions) or a fundamental lack of required enzyme activity.
Additional candidate glucosidases were selected based on amino acid similarity to an active site model based on positive hits from Example 4.
A final set of 94 additional candidate enzymes was selected. Codon optimized polynucleotide sequences encoding each selected enzyme linked to an N-terminal 6×His tag were prepared. Details of the additional candidate enzyme and polynucleotide sequences are summarised below in Table 8.
Bifidobacterium actinocoloniiforme
Bifidobacterium psychraerophilum
Penicillium italicum
Microbacterium trichothecenolyticum
Bifiguratus adelaidae
Paenibacillus thiaminolyticus
Microbacterium lemovicicum
Rhodococcus erythropolis
Cutibacterium avidum
Clavibacter michiganensis
Microbacterium sp.
Bifidobacterium actinocoloniiforme
Bifidobacterium reuteri
Propionibacterium freudenreichii
Microbacterium hydrocarbonoxydans
Pseudonocardia sp.
Tuber aestivum
Propionibacterium australiense
Fusarium sp.
Nectria haematococca
Actinoplanes sp.
Streptomyces fulvissimus
Pestalotiopsis fici
Bifidobacterium bohemicum
Bifidobacterium saeculare
Bionectria ochroleuca
Bifidobacterium pseudocatenulatum
Pseudonocardia sp.
Coleophoma crateriformis
Arthrobotrys oligospora
Pyrenophora teres
Gordonia polyisoprenivorans
Stigmatella aurantiaca
Pestalotiopsis fici
Bifidobacterium magnum
Bifidobacterium stellenboschense
Bionectria ochroleuca
Hungatella hathewayi
Mycetocola reblochoni
Nonomuraea sp.
Coleophoma crateriformis
Paenarthrobacter aurescens
Kitasatospora setae
Nocardiopsis alba
Rhodococcus jostii
Bifidobacterium merycicum
Bifidobacterium scardovii
Fusarium oxysporum
Paraphaeosphaeria sporulosa
Clostridium oryzae
Corynespora cassiicola
Choiromyces venosus
Saccharopolyspora erythraea
Streptomyces venezuelae
Arthrobacter sp.
Thermobrachium celere
Drechslerella stenobrocha
Bifidobacterium minimum
Bifidobacterium thermacidophilum
Verruconis gallopava
Stagonospora sp.
Tuber borchii
Morchella conica
Dictyoglomus thermophilum
Microlunatus phosphovorus
Bifidobacterium asteroides
Dactylellina haptotyla
Fusarium oxysporum
Bifidobacterium longum
Bifidobacterium tsurumiense
Exophiala spinifera
Pyrenochaeta sp.
Cadophora sp.
Morchella conica
Bifidobacterium animalis
Treponema azotonutricium
Acidipropionibacterium acidipropionici
Salinispira pacifica
Bifidobacterium mongoliense
Bifidobacterium indicum
Brachyspira suanatina
Phialocephala scopiformis
Periconia macrospinosa
Arthrobacter ulcerisalmonis
Pseudarthrobacter chlorophenolicus
Haloplasma contractile
Cochliobolus heterostrophus
Microbacterium sp.
Experiment 5-1—Screening of Additional Glucosidases for Deglycosylation with Purified QS-18 (0.04 mg/ml) at pH 7.5 and 30 Deg C.
The 94 additional genes, together with positive control (DNA encoding SEQ ID No. 262) and negative control, were transformed, expressed, lysed and reacted in the same manner as described above for Experiment 4-1, except the reaction was incubated at 30 degrees C. for 18 hours.
Samples were analysed by LCMS/MS according to the procedure in Experiment 4-1. The results for all enzymes demonstrating a % conversion of at least 3 are shown in Table 9.
Experiment 5-2—Screening of Additional Glucosidases for Deglycosylation with QS-18 in Crude Bark Extract (80%) at pH 6 and 35 Deg C.
A plate was lysed at pH 6 as described in Experiment 4-2. 40 uL of clarified lysate was transferred to a reaction plate
The pH of CBE was adjusted to pH 6 by dropwise addition of 2M NaOH with stirring. 160 uL of pH 6 CBE was added to each well of the reaction plate. The reaction plate was sealed and incubated at 35 deg C. with shaking for 18 hours.
The reaction plate was quenched by adding 200 uL of MeCN (2% acetic acid (AcOH), 1 mg/mL hexanophenone) to each well of the plates. The quenched reaction plate was re-sealed and incubated at 20 deg C. with shaking for 10 min. The reaction plate was then centrifuged (10 min, 4 degC).
200 uL was transferred from each well of the quenched plate to the corresponding wells of a fresh 96 well plate and sealed. The plate was analysed by UV HPLC with the method of Experiment 4-4.
Experiment 5-3—Screening of Additional Glucosidases for Deglycosylation with QS-18 in Treated Bark Extract (80%) at pH 6 and 35 Deg C.
Experiment 5-1 was repeated, replacing CBE with Treated Bark Extract (TBE) at pH 6. TBE was prepared from CBE by PVPP treatment and concentration, to provide TBE with a QS-21 concentration of approximately 4 g/L. TBE was adjusted to pH 6 by dropwise addition of 2M NaOH with stirring.
Based on detection of QS-18 2150 and QS-21 1988 components by LCMS/MS (Example 5-1) or UV HPLC quantification of Middle Peak (mainly QS-18 family and desglucosyl-QS-17 family) and Right Peak (mainly QS-21 family) (Examples 5-2 and 5-3), Example 5 shows that a number of suitable glucosidases could be identified by screening a set of candidate enzymes, and also that candidate enzymes demonstrating similarity to previously identified suitable glucosidases were more likely to also be suitable glucosides (51 from 94, 54%). Glucosidases were capable of converting QS-18 family components to QS-21 family components at a range of pHs, concentrations of starting materials and purity of starting materials.
Again, although certain candidate enzymes did not demonstrate notable conversion under the conditions tested, this may be due to issues with enzyme expression, suitability of conditions (i.e. enzymes may function under other conditions) or a fundamental lack of required enzyme activity.
Conversion of QS-17 family components to QS-18 family components involves hydrolysis of the 1,2 glycosidic bond between the alpha-L-arabinofuranose and alpha-L-rhamnose found at the terminus of the acyl chain portion of the molecules. Glycoside hydrolases from families 78 and 106 exhibit the exo-alpha-1,2 rhamnosidase activity (E.C. 3.2.1.40) necessary to cleave this bond as annotated by the CAZy (Carbohydrate Active enZyme) database (www.cazy.com). All sequences annotated by CAZy from GH families 78 and 106 were obtained, and separate curated hidden Markov model profiles constructed for each which were then used to identify additional familial enzymes by searching the 209 million protein member Uniprot (www.uniprot.org) knowledgebase with the software HMMER (Eddy, 1998). In total, 11,749 sequences were identified: 10,653, and 1096 representatives from GH families 78 and 106, respectively. MMSeqs2 (Hauser, 2016) was then used to cluster each group of enzyme sequences using the default clustering workflow and parameters with a minimum sequence identity and coverage of 30% and 80%, respectively. In cases where the initial clustering yielded clusters with more than 1000 members, a second sub-clustering was performed at a higher 50% or 70% identity to ensure diverse exemplars from these larger clusters were represented more prominently. All clusters were then examined, and exemplars selected from each with preferences for annotation quality, known experimental activity, existing three dimensional structures from the Protein Data Bank (www.wwpdb.org) or known extremophile organisms as annotated by Uniprot. A final set of 94 diverse candidate enzymes was selected. Polynucleotide sequences encoding each selected enzyme linked to a C-terminal 6×His tag and Tev-cleavage site (amino acid sequence for linker His-tag, inserted N-terminally of stop codon, is provided in SEQ ID No. 1178) were prepared using a proprietary genetic-algorithm based codon optimization code.
Details of the candidate enzyme and polynucleotide sequences are summarised below in Table 10.
Rhodothermus marinus
Streptomyces bingchenggensis
Spirosoma linguale
Roseburia intestinalis
Draconibacterium orientale
Catenulispora acidiphila
Bacteroides thetaiotaomicron
Opitutus terrae
Lachnoclostridium phytofermentans
Rhodanobacter denitrificans
Prevotella ruminicola
Aspergillus terreus
Brachybacterium faecium
Flavobacterium johnsoniae
Rahnella aquatilis
Bifidobacterium moukalabense
Enterococcus casseliflavus
Geobacillus sp.
Modestobacter marinus
Pedobacter heparinus
Dyadobacter fermentans
Paenibacillus mucilaginosus
Paenibacillus sp.
Bacteroides thetaiotaomicron
Chloroflexus aurantiacus
Thermoclostridium stercorarium
Bifidobacterium moukalabense
Olsenella profusa
Kribbella flavida
Caulobacter vibrioides
Bacteroides thetaiotaomicron
Rhodonellum psychrophilum
Paenibacillus sp.
Catenovulum agarivorans
Zobellia galactanivorans
Bacteroides thetaiotaomicron
Bacteroides xylanisolvens
Pseudarthrobacter chlorophenolicus
Dictyoglomus thermophilum
Formosa agariphila
Rhodococcus jostii
Lactobacillus crispatus
Pedobacter heparinus
Spirosoma linguale
Pedobacter heparinus
Paenibacillus mucilaginosus
Caulobacter segnis
Bacteroides cellulosilyticus
Pedobacter heparinus
Formosa agariphila
Lactobacillus acidophilus
Rhodopirellula baltica
Frankia inefficax
Streptomyces scabiei
Flavobacterium johnsoniae
Streptomyces sp.
Acidobacterium capsulatum
Catenovulum agarivorans
Brachybacterium faecium
Klebsiella oxytoca
Chitinophaga pinensis
Streptomyces bottropensis
Subdoligranulum variabile
Microbacterium testaceum
Solibacter usitatus
Streptosporangium roseum
alpha proteobacterium
Solitalea canadensis
Parabacteroides goldsteinii
Cyclobacterium marinum
Solibacter usitatus
Lunatimonas lonarensis
Rhizobium leguminosarum
Streptosporangium roseum
Parabacteroides distasonis
Lachnospiraceae bacterium
Chitinophaga pinensis
Caulobacter segnis
Pedobacter heparinus
Pedobacter heparinus
Deltaproteobacteria bacterium
Thermobaculum terrenum
Opitutus terrae
Kribbella flavida
Streptomyces scabiei
Actinoplanes sp.
Asticcacaulis sp.
Kribbella flavida
Bacillus sp.
Flavobacterium johnsoniae
Lunatimonas lonarensis
Eisenbergiella massiliensis
Catenovulum agarivorans
Streptomyces avermitilis
Synthetic nucleotide sequences corresponding to SEQ ID 989 to 1082 were subcloned, transformed, expressed and lysed in an identical manner to Experiment 4-1 with the exception that a single cell pellet plate was lysed with 200 ul lysis buffer.
Treated bark extract (TBE) solution was prepared by diluting 1 volume with 9 volumes of 100 mM potassium phosphate pH 7.5. 40 uL clarified lysate was transferred into fresh 96 well PCR plates. 10 uL 10× diluted TBE solution added to each well of lysate to a final concentration of 2% ( 1/50). Plates were incubated at 30 degC with shaking for 18 h. Quenched with 50 uL MeCN+2% AcOH and shaken at room temperature for 10 mins prior to centrifugation (10 min, 4 degC) to remove particulates. Samples were analysed by LCMS/MS using method of Experiment 4-1. The following MS-MS transitions were monitored to observe loss of rhamnose from starting saponins to product derhamnosylated saponins.
Data is expressed as TIC peak area ratio percent (PAR %) for rhamnosylated starting saponin to derhamnosylated product:
Activity was measured for the removal of the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of QS-17 2296 component to produce QS-18 2150 component; desglucosyl-QS-17 2134 to produce QS-21 1988 component and QS-17 2310 component to produce QS-18 2164.
No activity was detected for the removal of the C3 saccharide rhamnose as demonstrated by no effect on QS-18 2164 component (QS-18 2164/QS-21 1988 ratio is unchanged). Additionally no endo cleavage of the C28 saccharide rhamnose attached to the C2 position of the fucose was observed.
A subset of rhamnosidases were expressed and lysed as in the method of experiment 6-1.
Treated bark extract (TBE) solution was adjusted to pH 7.4 by addition of NaOH (2M). 75 uL clarified lysate was transferred into fresh 96 well PCR plates. 25 uL TBE solution (pH 7.4) was added to each well of lysate to a final concentration of 25%. Plates were incubated at degC with shaking for 19.5 h.
Quenched with 100 uL MeCN+2% AcOH and shaken at room temperature for 10 mins prior to centrifugation (10 min, 4 degC) to remove particulates. Samples were analysed by UV HPLC using method of Experiment 4-4. Three key peaks of interest are apparent using this chromatography: Left Peak (retention time approximately 2.30-2.35 min) comprising mainly QS-17 family components; Middle Peak (retention time approximately 2.37-2.42 min) comprising mainly QS-18 family components and desglucosyl-QS-17 family components; and Right Peak (retention time approximately 2.44-2.50 min) comprising mainly QS-21 family components. Peak identity was supported by MS/MS.
Enzyme mediated hydrolysis of the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of QS-17 family components leads to a decrease in Left Peak and an increase in Middle Peak due to formation of QS-18 family components. Enzyme mediated hydrolysis of the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of desglucosyl-QS-17 family components leads to a decrease in Middle Peak and an increase in Right Peak due to formation of QS-21 family components.
The relative percentage of each peak was determined. A decrease in Left Peak and a concomitant increase in Middle Peak and Right Peak is observed for enzymes active under these conditions. Results for the tested subset of rhamnosidases are provided below in Table 12.
Example UV HPLC chromatograms are shown in
Selected variants were expressed and lysed as in the method of experiment 6-1.
Crude bark extract (CBE) solution was adjusted to pH 7.4 by addition of NaOH (2M). 20 uL clarified lysate was transferred into fresh 96 well PCR plates. 80 uL CBE solution (pH 7.4) was added to each well of lysate to a final concentration of 80%. Plates were incubated at 30 degC with shaking for 19.5 h.
Quenched with 100 uL MeCN+2% AcOH and shaken at room temperature for 10 mins prior to centrifugation (10 min, 4 degC) to remove particulates. Samples were analysed by UV HPLC using method of Experiment 4-4.
Three key peaks of interest are apparent using this chromatography: Left Peak (retention time approximately 2.30-2.35 min) comprising mainly QS-17 family components; Middle Peak (retention time approximately 2.37-2.42 min) comprising mainly QS-18 family components and desglucosyl-QS-17 family components; and Right Peak (retention time approximately 2.44-2.50 min) comprising mainly QS-21 family components. Peak identity was supported by MS/MS.
Enzyme mediated hydrolysis of the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of QS-17 family components leads to a decrease in Left Peak and an increase in Middle Peak due to formation of QS-18 family components. Enzyme mediated hydrolysis of the alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of desglucosyl-QS-17 family components leads to a decrease in Middle Peak and an increase in Right Peak due to formation of QS-21 family components.
The relative percentage of each peak was determined. A decrease in Left Peak and a concomitant increase in Middle Peak and Right Peak is observed for enzymes active under these conditions. Results for the tested subset of rhamnosidases are provided below in Table 13:
Example UV HPLC chromatograms are shown in
Based on detection of QS-17 2296, QS-17 2310, QS-18 2150, QS-18 2164, desglucosyl-QS-17 2134 and QS-21 1988 components by LCMS/MS (Example 6-1) or UV HPLC quantification of QS-17, QS-18 and QS-21 peaks (Examples 6-2 and 6-3), Example 6 shows that a number of rhamnosidases could be identified by screening a set of candidate enzymes (29 from 94, 31% achieving a QS-17 PAR % of 4.5 or less in Example 6-1). Rhamnosidases were capable of converting QS-17 family components to QS-18 family components and desglucosyl-QS-17 family components to QS-21 family components at a range of concentrations of starting materials and purity of starting materials.
Again, although certain candidate enzymes did not demonstrate notable conversion under the conditions tested, this may be due to issues with enzyme expression, suitability of conditions (i.e. enzymes may function under other conditions) or a fundamental lack of required enzyme activity.
E. coli cells expressing glucosidase SEQ ID No. 262 (as His-tagged enzyme, DNA SEQ ID No. 662) and separately rhamnosidase SEQ_ID No. 1017 (as His-tagged form, DNA SEQ ID No. 1111) were grown in a fermenter, isolated, lysed, clarified and the resulting lysate lyophilised to yield powder containing each of the expressed enzymes.
500 uL CBE was mixed with 500 uL volume sodium acetate buffer (50 mM, pH 6) containing 30 g/L lyophilised powder containing the glucosidase, and 3 g/L lyophilised powder containing the rhamnosidase, and incubated at 37 degC for 24 hours.
The reaction was quenched by the addition of an equal volume of MeOH and analysed by LC-MS/MS using the method of Experiment 4-1 monitoring the transitions in the table below
Components possessing alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety are reduced below the detection limit while components possessing a glucose moiety show >78% reduction after treatment for 24 h. The corresponding products of selective rhamnose and glucose hydrolysis show substantial increases.
Libraries of genetic variants encoding mutations in the wild type Modestobacter marinus glucosidase (SEQ ID No. 262) were prepared using molecular biology techniques, enzymes were prepared linked to an N-terminally located His-tag. Single monoclonal colonies were grown in 400 ul of expression medium and protein expressed. Cell pellets were lysed in 200 ul of the relevant buffer (Table 16)
Lysate was diluted appropriately in the relevant buffer to allow a lysate loading of the indicated % loading (1% loading corresponds to use of 2 ul original lysate in a 200 ul reaction). In some experiments a rhamnosidase was also present during the screening reaction (and also in controls, negating any impact on results).
Crude bark extract (CBE) obtained by aqueous extraction of Quillaja saponaria and containing at least 2.80 mg/ml QS-21 (by HPLC-UV). The pH of CBE was adjusted to pH 6 by dropwise addition of 2M NaOH with stirring. The relevant concentration of the relevant glucosidase was added. The appropriate relative volume of pH 6 CBE (160 ul (for 80%) or 150 ul (for 75%)) was added to each well of the reaction plate. The reaction plate was sealed and incubated at the relevant temperature with shaking overnight for between 18 and 22 hours.
The reaction plate was quenched by adding 200 uL of MeCN (2% AcOH, 1 mg/mL hexanophenone) to each well of the plates. The quenched reaction plate was re-sealed and incubated at 20 deg C. with shaking for 10 min. The reaction plate was then centrifuged (10 min, 4 degC).
200 uL was transferred from each well of the quenched plate to the corresponding wells of a fresh 96 well plate and sealed. The plate was analysed by UV HPLC with the method below:
UV Method EM2020N435545v2_2
A negative control (a lysate not expressing test enzymes) and a positive control (expressing the parent comparator—wild type or previous variant as appropriate). Fold improvement over parent (FIOP) for the glucosidase (shorter method) is calculated as follows:
% right peak=100*right peak area/(right peak area+left peak area)
Average % right peak area is calculated for negative controls (per plate) and subtracted from all wells to give the increase in % right peak for each well above average negative control Average increase in % right peak is calculated for positive controls per plate
FIOP=observed increase in % right peak divided by average positive control increase
20%
20%
10%
The following mutations were associated with enzymes demonstrating improved activity in at least one instance:
Libraries of genetic variants encoding mutations in the wild type Kribbella flavida rhamnosidase (SEQ ID No. 1017) were prepared using molecular biology techniques, enzymes were prepared linked to a C-terminally located His-tag. Single monoclonal colonies were grown in 400 ul of expression medium and protein expressed. Cell pellets were lysed in 200 ul of the relevant buffer (Table 17).
Lysate was diluted appropriately in the relevant buffer to allow a lysate loading of the indicated % loading (1% loading corresponds to use of 2 ul original lysate in a 200 ul reaction).
Crude bark extract (CBE) obtained by aqueous extraction of Quillaja saponaria and containing at least 2.80 mg/ml QS-21 (by HPLC-UV) was adjusted to pH 6 by dropwise addition of 2M NaOH with stirring. The relevant concentration of the relevant glucosidase was added. The appropriate relative volume of pH 6 CBE (160 ul (for 80%) or 150 ul (for 75%)) was added to each well of the reaction plate. The reaction plate was sealed and incubated at the relevant temperature and time.
The reaction plate was quenched by adding 200 uL of MeCN (2% AcOH, 1 mg/mL hexanophenone) to each well of the plates. The quenched reaction plate was re-sealed and incubated at 20 deg C. with shaking for 10 min. The reaction plate was then centrifuged (10 min, 4 degC).
200 uL was transferred from each well of the quenched plate to the corresponding wells of a fresh 96 well plate and sealed. The plate was analysed by UV HPLC with the method described in Example 4.
Three key peaks of interest are apparent using this chromatography: Left Peak (retention time approximately 2.30-2.35 min) comprising mainly QS-17 family components; Middle Peak (retention time approximately 2.37-2.42 min) comprising mainly QS-18 family components and desglucosyl-QS-17 family components; and Right Peak (retention time approximately 2.44-2.50 min) comprising mainly QS-21 family components. Peak identity was supported by MS/MS.
Enzyme activity is calculated as the % conversion of the Left Peak present in the crude bark extract:
The following mutations were associated with enzymes demonstrating improved activity in at least one instance:
Lyophilised powders from clarified cell lysate expressing glucosidases (from Example 8 WT glucosidase and engineered glucosidase polypeptides G1 to G5) and rhamnosidases (from Example 9 WT rhamnosidase and engineered rhamnosidase polypeptides R1 to R5) were dissolved in 200 mM sodium acetate aqueous solution at pH 5.8 to prepare the enzyme solutions at 4 fold the final reaction concentration as shown in Tables 19 and 20.
Each glucosidase solution was combined with an equal volume of 200 mM sodium acetate aqueous solution at pH 5.8 and separately with an equal volume of 200 mM sodium acetate aqueous solution at pH 5.8 containing 2 mg/ml rhamnosidase R5. This is a sufficient loading of rhamnosidase to effect complete hydrolysis of the relevant rhamnose moiety within 4 hours.
Each rhamnosidase solution was combined with an equal volume of 200 mM sodium acetate aqueous solution at pH 5.8 and separately with an equal volume of 200 mM sodium acetate aqueous solution at pH 5.8 containing 2 mg/ml glucosidase G5. This is a sufficient loading of glucosidase to effect complete hydrolysis of the relevant glucose moiety within 4 hours.
CBE was adjusted to pH of 6.0 to 6.2 with 2M sodium hydroxide and an equal volume added to the enzyme solution to prepare the reaction mix (i.e. 50% CBE concentration in reaction mix) and the concentration of glucosidase and/or rhamnosidase is shown in Tables 19 and 20. The reaction mix was heated to 35 degC for the time indicated in Tables 19 and 20, after which the reaction was quenched by addition of an equal volume of MeCN containing 2% acetic acid and shaken at room temperature for 10 mins prior to centrifugation (10 min, 4 degC) to remove particulates. Samples were analysed by UV HPLC using method of Experiment 4-4.
Results
The change in composition of the Left, Middle and Right peaks is shown in in Tables 19 and 20. The composition changes by the action of the enzymes depending on the presence or absence of the partner enzyme. The extent of reaction is proportional to the enzyme concentration and the reaction time under these conditions. The tables show data for enzyme concentrations and the reaction times providing for equivalent extents of reaction. The improvement resulting from the mutations introduced for each variant is equal to the fold change in enzyme concentration×time (i.e. fold improvement=(enzyme concentration×time) for preceding variant÷(enzyme concentration×time) for later variant). A cumulative fold improvement over the original enzyme variant is calculated by the product of the individual fold improvements. The glucosidase G5 shows approximately 800 fold improvement over WT glucosidase. The rhamnosidase R5 shows approximately 30 fold improvement over WT rhamnosidase.
Variants G5 and R5 were found to demonstrate activity across a range of reaction conditions from 25 degC to 40 degC, from pH 5 to 7 (maintaining >80% relative activity for pH 5.4 to 6.2, and >50% for pH 5.2 to 7), and with a range of CBE loadings to at least 150% (achieved by redissolving lyophilised CBE in a smaller volume).
Lyophilised powders from clarified cell lysate expressing engineered glucosidase polypeptide G3 from Example 8 and engineered rhamnosidase polypeptide R2 were dissolved in 200 mM sodium acetate aqueous solution at pH 5.8 to a concentration of 3 g/L (glucosidase) and 2 g/L (rhamnosidase). For a 1 L reaction, sodium acetate buffer 200 mM (700 mL) was charged to a stirred reactor. Under constant agitation, glucosidase enzyme powder (2.1 g) and rhamnosidase enzyme powder (1.4 g) were added and agitated for 30 mins until all the enzyme powder was suspended. The resulting enzyme solution (700 mL) was depth filtered (nom. 3-9 μm) and then sterile filtered (0.2 μm).
CBE (700 mL) containing 4.1 g/L QS-21, with a Preceding Peak ratio of 0.25 and a 2018/QS-21 ratio of 0.054 to 0.057 was depth filtered and then sterile filtered (0.2 μm).
Filtered CBE (500 mL) was charged to a stirred reactor, heated to 37 degC and the pH adjusted to pH of 6.0 to 6.2 with 2M sodium hydroxide. Enzyme solution (500 mL) was then charged to the reactor and the solution stirred at 37 degC for 5 hours.
After 5 hours glacial acetic acid was charged into the reaction mixture gradually under moderate agitation to adjust the pH to ˜pH3.8 (target range pH3.5 to 4.0).
The enzyme treated CBE was then purified analogously to the processes provided in Example 3.
The purified saponin extract was determined to contain at least 98% QS-21 group, at least 93% QS-21 main peak, 0.2% 2018 component, 1% or less of largest peak outside the QS-21 group by UV absorbance at 214 nm and wherein the monoisotope of the most abundant species was 1987.9 m/z.
The increase of QS-21 by mass, based on the QS-21 concentration and the sample volumes, shows a 2.6-3.0× increase in the enzyme treated CBE. The increase of % QS-21 (as % of saponins) showed a 3.0-3.1× increase.
Due to the improved saponin profile of the enzyme treated material a greater recovery yield is obtained while remaining within desired specifications (notably during polystyrene and phenyl resin chromatography where a greater proportion of QS-21 can be recovered). Overall dual enzyme treatment was found to result in approx. 5.2- to 5.3-fold increase in yield compared to a conventional (non-enzyme treated) process.
Cleland J. et al. Isomerization and Formulation Stability of the Vaccine Adjuvant QS-21 Journal of Pharmaceutical Sciences 1996 85(1):22-28
Lecas L. et al. Off-line two-dimensional liquid chromatography separation for the quality control of saponins samples from Quillaja saponaria Journal of Separation Science 2021 44:3070-3079
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/084813 | 12/8/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63123011 | Dec 2020 | US | |
| 63213382 | Jun 2021 | US | |
| 63213407 | Jun 2021 | US | |
| 63213340 | Jun 2021 | US |
