Patent Application
20060286657
Due to recent advances in genetic and cell engineering technologies, peptides and proteins known to exhibit various pharmacological actions in vivo can be produced in quantities useful for pharmaceutical applications. A limitation to the development of these therapeutics is the preparation of stable pharmaceutical compositions of the proteins.
Therapeutic proteins are typically administered by frequent injection over time because the proteins often have a short in vivo half-life and negligible oral bioavailability. In some cases, frequent injection regimens pose a physical burden on the patient and are associated with increased administrative costs, as compared to costs associated with administering small molecules. As such, there is currently a great deal of interest in developing and evaluating longer lasting or sustained-release compositions and formulations.
Effective sustained-release compositions and formulations can provide a means of controlling blood levels of the active ingredient, and also provide greater efficacy, safety, patient convenience and patient compliance.
To date, two of the most widely used approaches to obtain sustained-action of a protein therapeutic include: 1) modifying the protein to increase the circulating half-life of the protein, for example by increasing the molecular weight and reducing immunogenicity; and 2) encapsulating the protein, for example, in polymer microspheres.
In connection with the first mechanism, i.e., protein modification, conjugation of biologically active molecules with biocompatible polymers is one way to improve formulation properties and in vivo performance of such molecules. Polyethylene glycol (PEG) is one of the most useful polymers often employed for this purpose. The properties that are attainable by PEG attachment to various low and high molecular weight drugs, and the corresponding applications of the resulting macromolecular conjugates have been extensively documented (for a review, see Zalipsky, Bioconjugate Chem., 6:150-165, 1995 and Adv. Drug Delivery Rev., 16:157-182, 1995). In particular, proteins are often conjugated with PEG, usually methoxy-PEG (mPEG), to gain longer in vivo circulation, reduced immunogenicity, and improved solubility and resistance to proteolytic enzymes.
PCT publication WO 02/049673 (Burg et al.) “refers to conjugates of erythropoietin with poly(ethylene glycol) comprising an erythropoietin glycoprotein having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells and selected from the group consisting of human erythropoietin and analogs thereof which have sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; said glycoprotein being covalently linked to one poly(ethylene glycol) group of the formula —CO—(CH2)x)—(OCH2CH2)m—OR with the —CO of the poly(ethylene glycol) group forming an amide bond with amino groups; wherein R is lower alkyl; x is 2 or 3; and m is from about 450 to about 1350.”
PCT publication WO 02/32957 (Nakamura et al.) describes PEG-modified EPO prepared by “chemically modifying the lysine residue at the 52-position of natural erythropoietin (natural EPO) with polyethylene glycol,” which PEG-modified EPO is stated to show a “ . . . long-lasting drug effect.”
PCT publication WO 97/24440 (De Sauvage et al.) describes “OB protein-immunoglobulin chimeras and polyethylene glycol (PEG)-OB derivatives” stated to have “extended half-life as compared to the corresponding native OB proteins.”
PCT publication WO 94/28024 (Chyi et al.) describes “[b]iologically active conjugates of glycoproteins having erythropoietic activity and having at least one oxidized carbohydrate moiety covalently linked to a non-antigenic polymer.”
PCT publication WO 90/13540 (S. Zalipsky) describes poly(ethylene glycol)-N-succinimide carbonate and its preparation.
Many methods are available for linking mPEG to proteins, usually to their amino groups of lysine residues or N-terminal of the polypeptide sequence (Zalipsky & Lee, 1992, supra, “Use of Functionalized Polyethylene Glycols for Modification of Polypeptides,” in Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., Plenum, New York N.Y., pp. 347-370). Urethane (carbamate) attachment of PEG to a protein is a convenient way to form PEG-protein conjugates. Carbamate linkages are more resistant to hydrolysis than amide linkages, which are also often utilized for protein PEGylation. Thus the urethane-linked conjugates are very stable in a variety of physiological conditions. There are a few known PEG reagents that are used to make urethane linked PEG-proteins (Zalipsky & Lee, 1992; Veronese et al., 1985, Appl. Biochem. Biotechnol., 11:141-152). These include slow-reacting imidazolyl formate, trichlorophenyl carbonate, and nitrophenyl carbonate (NPC) derivatives. A more reactive reagent, mPEG-succinimidyl carbonate (mPEG-SC), is often utilized (e.g., U.S. Pat. Nos. 5,122,614, 5,324,844, 5,612,460 and 5,808,096 to Zalipsky). As a rule, in comparison to less reactive reagents, a more reactive reagent allows faster, more efficient reaction under milder conditions. On the other hand, the less reactive reagents have better storage stability, and usually better selectivity.
PEGylation of proteins with slow-reacting reagents such as PEG-NPC proceeds more efficiently at a pH range of 8-10, as most amino groups of proteins are deprotonated and are highly reactive in this pH range. Many proteins are either not soluble or not stable at this basic pH range. Under these conditions, multiple amino groups react randomly with low selectivity. On the other hand, PEG-NPC is not very reactive at pH<8, thus these reactions proceed very slowly and not efficiently. This reagent is essentially unreactive at neutral and acidic pH (about 5-7).
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and upon study of the drawings.
The following aspects and embodiments thereof described and illustrated below are meant to be exemplary and illustrative, not limiting in scope.
In one aspect, an alternative method of generating PEGylated peptides and proteins is provided. In a preferred embodiment, the method allows for efficient modification of proteins at a pH at or below about 7 or about 6. The method allows for efficient formation of urethane-linked PEG proteins using mild PEG reagents and mild reaction conditions. The process results in the formation of moderately PEGylated proteins. In one embodiment, the method is advantageous for the modification of proteins that are not stable or not soluble at or above a neutral pH range. In another embodiment, the method is useful to PEGylate a protein that is insoluble or unstable at a pH higher than about 7.0 or 8.0.
In another aspect, a method useful for optimizing the yield of 1:1 PEG-protein in a heterogeneous population of PEG-protein molecules is described. This method comprises combining a PEG derivatized acylating agent and a protein in the presence of an activating agent at a pH of less than about 7.0 or neutral pH. In embodiments, the PEG derivatized acylating agent is mPEG-NPC. In another embodiment, the activating agent is selected from the group consisting of HOSu, HOBt, and HOAt.
In yet another aspect, the method is useful to maximize the yield of minimally conjugated proteins, i.e. 1:1, 1:2, and 1:3 protein:PEG.
In a further aspect, a method useful for PEGylating a protein that is insoluble or unstable at a pH higher than about 7.0 or neutral pH is described. The method comprises reacting the protein with a PEG derivatized acylating agent and an activating agent at a pH lower than about 7.0. In embodiments, The PEG derivatized acylating agent is mPEG-NPC. In another embodiment, the activating agent is selected from the group consisting of HOSu, HOBt, and HOAt.
In an additional aspect, a method to PEGylate a protein to form predominately 1:1 PEG-protein at a pH of less than about 8 and greater than the pKa of the additive to boost the rate of the reaction is described. The method comprises reacting the protein with a PEG derivatized acylating agent and an activating agent at a pH lower than about 8.0. In an embodiment, The PEG derivatized acylating agent is mPEG-NPC. In another embodiment, the activating agent is selected from the group consisting of HOSu, HOBt, and HOAt.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.
I. Definitions
The terms below have the following meanings unless indicated otherwise.
“Peptide” as used herein refers to any of the various amides that are derived from two or more α-amino acids by combination of the amino group of one acid with the carboxyl group of another. Peptides may be obtained by partial hydrolysis of proteins. “Polypeptide” as used herein refers to a chain of peptides. “Protein” as used herein refers to any of the numerous naturally occurring, usually extremely complex, substances that consist of amino-acid residues joined by peptide bonds. Proteins may further contain carbon, hydrogen, nitrogen, oxygen, usually sulfur, and occasionally other elements (such as phosphorus or iron). Proteins are generally characterized by a biological function including, for example, enzymes, hormones, or immunoglobulins. Unless specifically stated or recognizable by context, these terms are used interchangeably herein.
“Hydrophilic polymer” as used herein refers to a polymer having moieties soluble in water, which lend to the polymer some degree of water solubility at room temperature. Exemplary hydrophilic polymers include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylyoxazoline, polyhydroxypropyl-methacrylamide, polymethacrylamide, polydimethyl-acrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, polyaspartamide, copolymers of the above-recited polymers, and polyethyleneoxide-polypropylene oxide copolymers. Properties and reactions with many of these polymers are described in U.S. Pat. Nos. 5,395,619 and 5,631,018.
“PEGylation” refers to the attachment of one or more polyethylene glycol (PEG) substituent or derivatives to a biologically active protein.
Acylating agent” refers to an agent capable of connecting an acyl group to another chemical compound, whereby the acylating agent provides the acyl group. Exemplary acylating reagents include nitrophenyl carbonate, trichlorophenyl carbonate, pentachlorophenyl carbonate, and carbonyl imidazole as well as various active esters, e.g. nitrophenyl ester, pentafluoroethyl ester, trichlorophenyl ester.
“PEG derivatized acylating reagent” refers to an acylating agent that is derivatized to include polyethylene glycol.
Abbreviations: PEG: polyethylene glycol; mPEG: methoxy polyethylene glycol; HOSu: N-hydroxysuccinimide; HOBt: N-Hydroxybenzotriazole; NPC: nitrophenyl carbonate; DTB: dithiobenzyl; SC: succinimidyl carbonate.
II. Method of Forming Conjugates
The solubility of proteins in aqueous solutions varies enormously based on the protein structure and composition. While some proteins are very soluble, many proteins, especially structural proteins are essentially insoluble under physiological conditions and exist normally as solids. (Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, N.Y. (1993)). The solubility of proteins is typically lowest near pl≈pH.
As noted above, however, PEGylation of proteins with typical acylating reagents such as PEG-NPC proceeds quickly at a pH greater than 7.5, typically greater than 8, as PEG-NPC is highly reactive in this pH range, but is not very reactive at neutral or lower pH values where the reaction proceeds very slowly and is not efficient.
The present methods take advantage of some of the benefits of low- and high-reactivity PEG reagents. Specifically, less reactive reagents, such as mPEG-NPC, have superior shelf life and selectivity characteristics as compared with more reactive reagents, yet their sluggish reactivity, particularly under neutral conditions, makes them undesirable for use. In contrast, the more reactive mPEG-SC and similar compounds suffer the disadvantages of being less stable and less selective than their less reactive counterparts, and can undergo undesirable side reactions (Zalipsky, Chem Com, 1:69-70, 1998).
As seen in
It will be appreciated that many water-soluble, non-carboxylic, Brönstead acids of moderate acidity having the propensity to donate N- or O-linked protons to the PEGylation reagent are suitable for use as the activating agent in the present methods. General examples include acidic alcohols, phenols, imidazols, triazols and tetrazols, among others. Examples of acidic acids suitable for use in this aspect of the invention include, but are not limited to, N-hydroxydicarboxyimides, N-hydroxyphthalimides particularly with nitro and other electron withdrawing substituents on the aromatic ring, N-hydroxy tetrahydrophthalimide, N-hydroxyglutarimide, N-hydroxy-5-norbornene-2,3-dicarboxyimide, and N-hydroxy-7-oxabicyclo[2.21]hept-5-ene-2,3-dicarboxyimide. 1-N-hydroxybenzotriazol and derivatives with electron withdrawing groups on the aromatic ring, e.g. nitro, chloro, 3-hydroxy-1,2,3-benzotriazin-4(3H)-one. N-hydroxysulfosuccinimide sodium salt is very soluble in water, which means that it can be used at even higher concentration in aqueous buffers than HOSu. Exemplary hydroxy amine derivatives include N-hydroxysuccinimide (HOSu), sulfonate derivatives of HOSu, 1-hydroxybenzotriazole (HOBt), and hydroxyl-7-azabenzotriazole (HOAt). These coupling agents can act as an efficient buffer component in a pH range of about 4 to about 7.5, based on their pKa. For example, HOSu, being a weak acid of pKa=6, acts as an efficient buffer component in a pH range of about 5 to about 7. The coupling reagent may be added to a buffer, or may comprise the buffer with or without other salts. Further, as HOSu is quite soluble in an aqueous solution, it can be added to buffers at relatively high concentration to further boost the PEGylation reaction.
Exemplary phenols include, but are not limited to, dinitrophenol, trinitrophenol, trifluorophenol, pentafluorophenol, and pentachlorophenol. It should be noted that water solubility is a factor for pentafluorophenol and pentachlorophenol. In addition, 4- or 2-hydroxypyridine and derivatives are also suitable for use in the present methods as exemplified by hydroxyl-2-nitropyridine.
Other exemplary compounds for use as the activating agent include compounds having an acidic N—H functionality, such as imidazol derivatives with electron withdrawing groups (imidazol, pKa=7), e.g. 4- or 2-nitroimidazol, triazol, tetrazol, and some derivatives, such as 2-nitro-1,2,4-triazole.
As mentioned above, in one exemplary embodiment, the invention relates to a method using acylating PEG reagents of low to medium reactivity, particularly reagents having low to medium reactivity at room temperature and/or at pH≦7.0. A particularly preferred acylating reagent is mPEG-NPC. Nitrophenyl carbonate derivatized polyethylene glycol is exemplified for use in this aspect of the present invention, however, as seen in Example 4, the method has also been applied to preparation of thiolytically cleavable dithiobenzyl (DTB) urethane-linked PEG-protein conjugates, utilizing a mPEG-DTB-NPC reagent. The reactivity and selectivity benefits were similar to those obtained using the non-cleavable mPEG-NPC reagent. Other low-reactivity PEG reagents suitable for use in the present invention include, but are not limited to, carbonyl imidazolyls, trichlorophenyl carbonates, and other nitrophenyl carbonates, which have been utilized to make urethane-linked PEG-proteins (see, e.g. Zalipsky & Lee, 1992, supra). Likewise, methoxy-PEG (mPEG) is exemplified herein, but is not the only modified PEG that can be used. Other modified PEGs, preferably rendered monofunctional by addition of an inert group to one end, are also suitable for use in the method. Examples include, but are not limited to, short alkoxy PEG derivatives (ethoxy, butoxy, and the like) or PEG modified with various protected functional groups, as would be understood by one of skill in the art.
It will further be appreciated that while a PEG reagent comprising 12,000 and 30,000 Da PEG is exemplified herein, other PEG lengths are also contemplated for use in the present method. A preferred size range of PEG is 1,000-50,000 Da. Preferably, the PEG length is 40,000 Da or less, more preferably 30,000 Da or less.
In one embodiment, the method preferably includes the step of combining the protein with an activating agent and mPEG-NPC in an aqueous medium at a pH of less than about 8.0.
In one preferred embodiment, the pH is about 5.0 or 6.0 to about 7.5. In other embodiments, the pH is less than about 7 or 8. It will be appreciated that neutral conditions are often more favorable for protein stability and/or solubility. Neutral conditions also generally favor modification of the most reactive and least basic amino groups on a protein. As the N-terminal amino group is usually a few orders of magnitude less basic (pKa=7.9) than the ε-amino group of lysine (pKa=10-11), lower pH tends to keep most of the latter groups fully protonated. Therefore, a lower pH is generally more favorable for selective modification of the N-terminal amino group. It will be appreciated that often proteins PEGylated predominantly on the N-terminal amino retain higher functional activity.
The protein, activating agent, and mPEG-NPC are combined for a period of time from about 0.5 hours to about 24 hours. In one embodiment, the time is from about 2 hours to about 6 hours. It will be appreciated that one of skill in the art can readily determine and/or vary the time to optimize the reaction.
The reaction temperature is generally about room temperature, or between 8° C. and 37° C., however, it will be appreciated that one of skill in the art can readily determine and/or vary the temperature to optimize the reaction.
As described in Example 1, EPO was conjugated to mPEG30k-NPC using the HOSu in the in situ activation process. The reaction was efficient with 81% of the EPO being conjugated, leading to the formation of mainly mono- and di-PEGylated-EPO as seen in
Electrophoresis gels were run and are shown in
As detailed in Example 2, conjugates of mPEG30k-EPO were prepared in either MOPS buffer or in MOPS buffer with HOSu as an additive and reactivity boosting agent at a pH of 6.5, 7.0, or 8.0. In the reaction with the MOPS buffer alone, conjugation of the mPEG30k to EPO produced low yields of PEG-EPO conjugates, with the majority of the protein remaining unconjugated at all three pHs tested. As seen in
As further illustrated in Example 2, boosting the conjugation of mPEG-NPC at lower pH ranges may be especially beneficial for proteins that are not stable at higher pH ranges. Addition of the activating agent is effective to increase at least one of the rate of reaction, the extent of reaction, and/or the time to completion for the reaction. This data further illustrates that PEG-NPC in the absence of the reactivity boosting agent, HOSu, is rather inert at pH 7.
As also detailed in Example 2, conjugates of mPEG30k-EPO were prepared by reacting mPEG30k-SC with EPO in MOPS buffer at a pH of 6.5, 7.0, or 8.0 for comparison. Conjugation of the mPEG30k-SC to EPO reacted efficiently at all pH, however formation of di- and tri-PEGylated-EPO was more highly favored than found using conjugation of mPEG-NPC to EPO in the presence of HOSu at all pHs. As detailed in Table 2, at pH 8.0, 98% of the EPO formed a conjugate (24% as 1:1, 50% as 1:2, and 24% as 1:3 conjugates). At a pH 7.0, 96% of the EPO was conjugated with 29% as 1:1 conjugates, 48% as 2:1 conjugates, and 19% as 3:1 conjugates. At pH 6.5, 90% of the EPO was conjugated with 39% as 1:1 conjugates, 42% as 2:1 conjugates, and 10% as 3:1 conjugates.
Bone Morphogenic Protein 7 or Bone Morphogenetic Protein 7 (BMP7) is a member of a large, structurally-related subgroup of the TGF-β super family of proteins. BMP7 is not soluble at a pH>7, particularly in the presence of salts. Therefore, any PEGylation reaction must be carried out below pH 7, preferably between pH 6 and 7. As described in Example 5, conjugates of mPEG30k-BMP7 were prepared with HOSu as an activating and buffering agent. The BMP7 reacted with mPEG30k-NPC efficiently, resulting in a composition favoring the formation of mono- and di-PEGylated-BMP7.
Electrophoresis gels were run with the mPEG30k-BMP7 sample as shown in
Lysozyme is an enzyme found in egg whites, milk, tears, and other secretions. It acts as an antibiotic by breaking down the polysaccharide walls of many kinds of bacteria. As described in Example 6, conjugates of mPEG30k-lysozyme were prepared in either a MOPS buffer solution or a MOPS/HOSu buffer solution at a pH of 6.5, 7.0, or 8.0 for comparison. As seen in Table 4, at a pH 8.0, 41% of the lysozyme reacted with mPEG30k-NPC to form a conjugate (34% as 1:1 conjugate and 7% as 2:1 conjugate) in the MOPS buffer. At pH 7.0, only 10% of the lysozyme reacted with mPEG30k-NPC as 1:1 conjugate. At pH 6.5, only 6% of the lysozyme reacted with mPEG30k-NPC as 1:1 conjugate. In the MOPS/HOSu buffer, the lysozyme reacted with the mPEG 30k-NPC to form primarily mono- and di-PEGylated-lysozyme. At pH 8.0, 46% of the lysozyme reacted with mPEG30k-NPC to form a conjugate (38% as 1:1 conjugate, 8% as 2:1 conjugate, and 1% as 3:1 conjugate). At pH 7.0, 40% of the lysozyme reacted with mPEG30k-NPC (34% as 1:1 conjugate and 6% as 2:1 conjugate). At pH 6.5, 30% of the lysozyme reacted with mPEG30k-NPC (27% as 1:1 conjugate and 3% as 2:1 conjugate).
In a preferred embodiment, the total conjugation in the presence of the activating agent is 1 to 7 fold greater than conjugation without the activating agent. In a more preferred embodiment, conjugation with the activating agent is 2 to 5 fold greater than conjugation without the activating agent.
As detailed in Example 3, the rate of hydrolysis of mPEG30k-NPC was measured in MOPS buffer, with and without the presence of HOSu at pH 6.5, 7.0, or 8.0. Briefly, mPEG30k-NPC was added to vials containing either MOPS buffer or MOPS buffer with HOSu. The rate of formation of p-nitrophenol was measured at 400 nm.
The activating agent produces transesterification of the carbonate ester of the mPEG-NPC, which is then hydrolyzed to produce the p-nitrophenol, mPEG-OH, and CO2. In a preferred embodiment, addition of an activating agent results in at least a 100 fold increase in the rate of reaction. In other embodiments, use of an activating agent results in a 10, 100, or 150 or more fold increase in the rate of reactions.
It will be appreciated that each or any of the temperature, pH and time involved can be varied as needed to maximize yield and/or minimize time for the reaction.
In one embodiment, the method results in formation of moderately PEGylated proteins, and is particularly advantageous for the preparations of PEG-protein comprising 1:1 PEGylated, 2:1 PEGylated, and/or 3:1 PEGylated proteins. Preferably, the reaction produces primarily 1:1 PEGylated protein. In a preferred embodiment, the method produces about 40-45% 1:1 PEGylated protein. In a more preferred embodiment, the method produces about 55-65% 1:1 PEGylated protein. In yet another embodiment, the method produces about 60% 1:1 PEGylated protein. In another embodiment, the method results in a population having about equal molar ratio of 1:1 PEGylated protein to 2:1 PEGylated protein. In other embodiments, the population comprises about 15-40% 1:1 PEGylated protein, about 30-50% 2:1 PEGylated protein, and about 15-40% 3:1 PEGylated protein. The resulting population may further include unmodified protein (nonPEGylated). In preferred embodiments, nonPEGylated protein is present in an amount less than about 20%, 10%, or 5% of the total protein. In another preferred embodiment, nonPEGylated protein is present in an amount of less than about 1% or less than about 0.1% of the total protein. In a preferred embodiment, at least 60-90% of the protein is PEGylated with the present method.
It will be appreciated that the composition of the resulting population may be analyzed according to any suitable method known in the art. In one embodiment, the composition of the population is analyzed by HPLC. Size exclusion chromatography is useful and readily separates the various PEGylated species from each other and from the nonPEGylated protein.
Another variable that can be utilized to maximize yield of PEGylated protein is the ratio of protein to mPEG-NPC and/or the activating agent. As described in Example 4, 0.2, 0.4, and 0.6 mM solutions of mPEG12k-DTB-NPC were used to prepare PEGylated EPO in the presence of HOSu or HOBt. The solutions had a molar ratio of 3, 6, or 9 PEG/EPO and a molar ratio of 100, 50, or 25 HOSu/NPC.
In another embodiment, the process described herein was used to attach a hydrophilic polymer poly(ethylene glycol) to a structure referred to in the art as a MIMETIBODY™ (see PCT Publication Nos. WO 04/002417; WO 04/002424; WO 05/081687; and WO 05/032460, all of which are incorporated by reference herein). A mimetibody can comprise at least one CH3 region directly linked with at least one CH2 region directly linked with at least one hinge region or fragment thereof directly linked with an optional linker sequence, directly linked to at least one therapeutic peptide, optionally further directly linked with at least a portion of at least one variable antibody sequence. In a preferred embodiment, the mimetibody comprises a pair of a CH3-CH2-hinge-linker-therapeutic peptide fusion polypeptides, the pair linked by association or covalent linkage, such as, but not limited to, a Cys-Cys disulfide bond. For example, an EPO mimetic CH1 deleted mimetibody mimics an antibody structure with its inherent properties and functions, while providing a therapeutic peptide and its inherent or acquired in vitro, in vivo or in situ properties or activities.
Mimetibodies provide at least one suitable property as compared to known proteins, such as, but not limited to, at least one of increased half-life, increased activity, more specific activity, a selected or more suitable subset of activities, less immunogenicity, increased quality or duration of at least one desired therapeutic effect, less side effects, and the like.
Human mimetibodies that are specific for at least one protein ligand or receptor thereof can be designed against an appropriate ligand, such as isolated and/or EPO protein receptor or ligand, or a portion thereof (including synthetic molecules, such as synthetic peptides). Preparation of such mimetibodies are performed using known techniques to identify and characterize ligand binding regions or sequences of at least one protein or portion thereof.
A mimetibody referred to in the art as CNTO528 was selected as a model biomolecule (mimetibody) for PEGylation according to the process described herein. CNTO528 is an Epo receptor agonist, described in PCT Publication WO 04/002417, incorporated by reference herein. Examples 7 and 8 describe reaction of CNTO528 and PEG according to the reaction method described herein.
It will be appreciated that the methods described herein are useful for PEGylating a variety of peptides and proteins that are not soluble and/or not stable at a pH higher than about 8.0 or about 7.0.
In another embodiment, at least one PEG is attached at one or more sites on the protein molecule. It will further be appreciated that conjugation of PEG at more than one site on the protein molecule may increase circulation time.
It will be appreciated that the rate of reactivity is highly dependent on the pH of the buffer in the PEGylation reaction and the accessibility of the individual primary amino groups.
The reaction may be stopped by mixing a free amino compound in the reaction medium. Such a free amino compound includes, but is not limited to, TRIS, lysine, glycine, or any amino with at least one free amino group. In a preferred embodiment, the free amino compound is glycine. In this embodiment, the glycine is preferably used at a concentration of between about 10-100 mM, about 1-50 mM, or about 50-100 mM.
The method may further include a purification step according to known methods in the art. In other embodiments, either the product of the combining step or the product of a purifying step can be concentrated. In one embodiment, the concentrating step can be performed using ultrafiltration or concentration with, for example, a nominal molecular weight limit (NMWL) cutoff filter. Filters for performing such concentrations are commercially available. Occasionally, optimized reactions may not require purification steps to remove excess PEG reagent. However, one of skill in the art will appreciate both purification and concentration steps can be selected among techniques known in the art.
Various patents and publications are cited throughout the specification. Each of these patents or publications is expressly incorporated by reference herein, in its entirety.
The following examples are provided for the purpose of illustrating various presently preferred embodiments of the invention. As such, they are intended to exemplify and clarify, but not to limit the understanding or description of the invention in its several aspects.
Materials and Methods
Erythropoietin (EPO) was obtained from Johnson & Johnson Pharmaceutical Research & Development (Raritan, N.J.), at a protein concentration of 3.56 mg/ml in 20 mM citrate, 100 mM NaCl, pH 6.9 buffer.
Recombinant human Bone Morphogenetic Protein-7 (BMP7) was obtained from CURIS (Cambridge, Mass.), as a lyophilized powder and kept at −70° C.
Nitrophenyl carbonate derivatized methoxy-polyethylene glycol 30,000 Daltons (mPEG30k-NPC) was purchased from NOF Corporation (Tokyo, Japan, #M35525).
Succinimidyl carbonate derivatized methoxy-polyethylene glycol 30,000 Da. (mPEG30k-SC) was prepared by reacting mPEG30k-NPC with N-hydroxysuccinimide in presence of di-isopropyl ethylamine, and purified by crystallization.
N-hydroxysuccinimide (HOSu), 1-hydroxybenzotriazole (HOBt), sodium phosphate (NaPO4), and 3-[N-Morpholino]propanesulfonic acid sodium salt (MOPS) were purchased from Sigma-Aldrich (St. Louis, Mo.).
A. Conjugation
A 10 mM stock solution of mPEG30k-NPC was prepared in acetonitrile. The conjugation buffer was prepared as a stock solution of 100 mM MOPS (3-[N-Morpholino]propanesulfonic acid sodium salt) and 100 mM HOSu (N-hydroxysuccinimide), and the pH was adjusted to 7.0±0.1 with 5 N NaOH. The reaction was initiated by first mixing 5.2 ml of EPO to 2.25 ml of MOPS/HOSu buffer, and 1.194 ml of distilled water. Afterward, 0.356 ml of mPEG30k-NPC was added drop by drop to the mixture, while gently vortexing. The reaction was allowed to proceed for 4 hours at room temperature (21-22° C.) on a rocking mixer, followed by an additional 18 hours at 4° C. The final reaction volume was 9 ml, containing 2 mg/ml (0.066 mM) of EPO, and 0.4 mM of mPEG30k-NPC, giving a molar ratio of 6 PEG/EPO, and 4% acetonitrile. The final HOSu concentration was 25 mM, which is approximately 62 molar excess over mPEG30k-NPC.
B. Analysis by HPLC Size Exclusion Column
The outcome of the conjugation reaction was analyzed by HPLC-SEC using Superose-6 10/300 GL, 1×30 cm column (Amersham Biosciences, Piscataway, N.J.), and 50 mM NaPO4, 150 mM NaCl, pH 6.5, mobile phase. The sample was diluted 1/20 in the mobile phase, and 50 μl were injected to the column. The flow rate was set at 0.5 ml/min, and elution from the column was monitored by a fluorescence detector set at an excitation wavelength of 295 nm, and emission wavelength of 360 nm (bandwidth 15 nm).
C. Purification
The conjugation reaction sample was purified by ion exchange preceded by dialysis.
1. Dialysis:
10 mM Tris buffer pH 7.5, using SPECTRA/POR 1 membrane tubing (Spectrum Medical Industries Inc., Los Angeles, Calif.), having a molecular weight cut-off of 6000-8000. The dialysis was carried out at 4° C. The first three buffer exchanges were each performed in 1 liter Tris buffer for 2 hours, the 4th exchange was performed overnight in 3 L Tris buffer, and the final exchange was done in 1 L buffer for 2 hours. At the end of dialysis, the sample was filtered through a 0.45 μm Acrodisc HT Tuffryn membrane syringe filter (PALL Life Sciences, Ann Arbor, Mich.).
2. Separation by Ion Exchange Chromatography:
A quaternary amine anion exchanger column, Source 15Q 4.6×100 mm (Amersham Biosciences, Piscataway, N.J.), 1.7 ml total volume, was equilibrated with 20 column volumes of 10 mM Tris pH 7.5 buffer. Next, 7.8 ml of the dialyzed conjugation sample were loaded on the column. Elution was performed by step gradient using mobile phase A containing 10 mM Tris pH 7.5, and mobile phase B containing 500 mM NaCl in 10 mM Tris pH 7.5, at a flow rate of 1.7 ml/min. The unbound material was washed from the column with 5 column volumes of mobile phase A. The elution started by increasing mobile phase B to 15% (75 mM NaCl) for 10 minutes, then to 20% B (100 mM NaCl) for 15 minutes, and finally to 70% B (350 mM NaCl) for 5 minutes. Fractions were collected at 0.85 ml/fraction through the entire separation.
D. Analysis by HPLC-SEC
To identify the content of the fractions collected throughout the ion exchange separation, aliquots from the fractions were analyzed by HPLC-SEC using TSKgel Super SW3000, 0.46×30 cm column (TOSOH Biosciences LLC, Montgomeryville, Pa.), 50 mM NaPO4, 150 mM NaCl, pH 6.5, mobile phase, and a flow rate of 0.25 ml/min.
E. Dialysis and Concentration
The pooled sample from the ion exchange separation was dialyzed in 20 mM sodium citrate, 100 mM sodium chloride, pH 6.9 buffer, using SPECTRA/POR 1 tubing described above. The dialysis was carried out at 4° C. The first buffer exchange was done in 2 L citrate/NaCl buffer for 2 days, the 2nd exchange was performed overnight in 2 L Tris buffer, and the final exchange was done in 1 L for 4 hours. The dialyzed sample was then concentrated, under nitrogen at 20 psi, in a 10 ml Amicon ultrafiltration stirred cell (Millipore Corp., Billerica, Mass.), using an OMEGA ultrafiltration membrane disc filter (PALL Life Sciences, Ann Arbor, Mich.), having a molecular weight cut-off of 3000. The sample volume was reduced from 18 ml to 4 ml final volume.
F. Sterile Filtration
The concentrated sample was sterile filtered through 0.22 μm Acrodisc HT Tuffryn membrane syringe filter, and sterilely filled into autoclaved glass vials. All vials were stored at 4° C.
The concentration of the mPEG30k-EPO preparations was determined to be 1.2 mg/ml based on the intrinsic fluorescence of EPO protein and calibrated with the parent EPO.
G. HPLC-SEC Analysis
The purified mPEG30k-EPO sample was analyzed by size exclusion chromatography using Superose-6 10/300 GL column (Amersham Biosciences, Piscataway, N.J.), and 50 mM NaPO4, 150 mM NaCl, pH 6.5, mobile phase. The sample was diluted 1/20 in the mobile phase, and 50 μl were injected to the column. The flow rate was set to 0.5 ml/min, and elution from the column was monitored by a fluorescence detector set at an excitation wavelength of 295 nm, and emission wavelength of 360 nm (bandwidth 15 nm). The results are shown in
H. SDS-PAGE Analysis
The mPEG30k-EPO sample was analyzed by gel electrophoresis under denaturing conditions, using NuPAGE® Bis-Tris 4-12% gradient gel and MOPS-SDS running buffer (Invitrogen Life Technology, Carlsbad, Calif.). Samples and controls were loaded on the gel at 10 μl/well containing 1.5 to 5 μg of protein. The gel was run at a constant voltage of 200 volts for 55 minutes. The gel was first stained in iodine for PEG detection, and subsequently in Coomassie Blue for protein detection. The electrophoresis gels are shown in
The composition of the purified mPEG30k-EPO sample determined by SDS-PAGE confirmed the HPLC-SEC results of
Three MOPS buffer solutions were prepared at 100 mM, and the pH values were adjusted to 6.5, 7.0, and 8.0 respectively, with 6 N HCl.
Three MOPS/HOSu buffer solutions were prepared at 100 mM MOPS/100 mM HOSu, and the pHs were adjusted to 6.5, 7.0, and 8.0 respectively, with 5 N NaOH.
A 10 mM (302.6 mg/ml) stock solution of mPEG30k-NPC was made in acetonitrile. A 10 mM (302 mg/ml) stock solution of mPEG30k-SC was made in acetonitrile. A stock solution of glycine was made with 500 mM in 10 mM Tris pH 7.5 buffer.
A total of 9 conjugation reactions were assembled. In the first set, EPO was reacted with mPEG30k-NPC in MOPS buffer at pH, 6.5, 7.0, or 8.0. In the second set, EPO was reacted with mPEG30k-NPC in MOPS/HOSu buffer at pH, 6.5, 7.0, or 8.0. In the third set, EPO was reacted with mPEG30k-SC in MOPS buffer at pH 6.5, 7.0, or 8.0. All the reactions were carried out at room temperature (21-22° C.) for 4 hours, while mixing, then transferred to 4° C. for an additional 18 hours.
The final concentrations in the reaction vials were 2 mg/ml (0.066 mM) for EPO, and 0.4 mM for mPEG30k, giving a molar ratio of 6 PEG/EPO. The final acetonitrile amount was 4%. In the reactions containing MOPS/HOSu buffer, the final HOSu concentration was 25 mM, which is equivalent to 62 molar excess over mPEG.
At the end of the incubation, glycine was added to all the reactions, to a final concentration of 25 mM, and allowed to react for 20 minutes at room temperature.
Samples from the conjugation reactions were diluted 1:25 in 50 mM NaPO4, 150 mM NaCl, pH 6.5 buffer, and 50 μl were injected to Superose-6 10/300 GL column (Amersham Biosciences, Piscataway, N.J.) to determine the amount of conjugation of the PEG and EPO. The mobile phase was 50 mM NaPO4, 150 mM NaCl, pH 6.5, run at 0.5 ml/min flow rate. A fluorescence detector was connected to the column outlet, and set to an excitation wavelength of 295 nm, and emission wavelength of 360 nm (bandwidth 15 nm). The chromatograms were analyzed by Star Chromatography Workstation 6.2 (Varian Inc., Walnut Creek, Calif.) and the results were expressed in percent of peak areas as shown in Table 1. The results for the HPLC-SEC analysis of the mPEG30k-EPO conjugates in the MOPS buffer alone are shown in
mPEG30k-NPC solution, MOPS buffer, MOPS/HOSu buffer were prepared as described in Example 2.
Forty microliters of the 10 mM mPEG30k-NPC were added to 3 vials, each containing 960 μl of 100 mM MOPS buffer solution at a pH of 6.5, 7.0, and 8.0, respectively. The mixture from each vial was immediately transferred to a 96-well microtiter plate in triplicates (300 μl/well). The plate optical density (OD) reading started at 400 nm and was collected for 2 hours in 2 minute reading intervals using a Vis range A400. Similar assays were repeated with the MOPS/HOSu buffer. The results are shown in
A. Reactions with HOSu in Phosphate Buffer
EPO was reacted at 2 mg/ml to 0.2, 0.4, and 0.6 mM mPEG12k-DTB-NPC, in conjugation buffer containing 100 mM sodium phosphate and 20 mM HOSu, at pH 7. The reaction was allowed to proceed for 5 hours at room temperature (22-24° C.) on a rocking mixer. During the reactions, the PEG/protein molar ratios were 3/1, 6/1, or 9/1, and HOSu/NPC molar ratios were 100/1, 50/1, and 25/1. At the end of incubation, the reactions were stopped with 9 mM glycine.
The samples were analyzed by SDS-PAGE and the results are displayed as lanes 2, 3, and 4 in
B. Reactions with HOBt in Phosphate Buffer
Reactions were conducted as in section A, above except that the conjugation buffer was prepared as a stock solution of 100 mM sodium phosphate and 20 mM HOBt at pH 7. The samples were analyzed by SDS-PAGE as in section A, above, and the results are displayed as lanes 5, 6, and 7 in
C. Reactions in Phosphate Buffer
EPO was reacted at 2 mg/ml to mPEG12k-DTB-NPC at 0.4 mM, in conjugation buffer containing 100 mM sodium phosphate, at pH 7. The reaction was allowed to proceed for 5 hours at room temperature (22-24° C.) on a rocking mixer. During the reactions, the PEG/protein molar ratio was 6/1. At the end of incubation, the reactions were stopped with 9 mM glycine.
The samples were analyzed by gel electrophoresis as in section A, above. The results are displayed as lane 8 in
D. Reactions with HOSu in Phosphate Buffer at Various EPO Concentrations
EPO was reacted at 1 or 2 mg/ml to mPEG12k-DTB-NPC (at 0.1 or 0.2 mM), in conjugation buffer containing 100 mM sodium phosphate and 5 or 10 mM HOSu, at pH 7. The reaction was allowed to proceed for 5 hours at room temperature (22-24° C.) on a rocking mixer. During the reactions, the PEG/protein molar ratio was 3/1, and HOSu/NPC molar ratio was 50/1. At the end of incubation, the reactions were stopped with 9 mM glycine.
The samples were analyzed by gel electrophoresis as in section A, above, the results of which are displayed in lanes 10 and 11 in
A. Conjugation
A 1.4 mg/ml BMP7 stock solution was prepared in 25 mM HOSu (N-hydroxysuccinimide), pH 6. A 10 mM stock solution of mPEG30k-NPC was prepared in acetonitrile. A 12.86 ml of BMP7 (18 mg) was mixed with 4.24 ml of HOSu buffer, pH 6. Afterward, 0.9 ml of mPEG30k-NPC were added drop by drop to the mixture, while gently vortexing. The reaction was incubated for 16 hours at room temperature (21-22° C.) on a rocking mixer. The final reaction volume was 18 ml containing 1 mg/ml (0.028 mM) of BMP7, 0.5 mM of mPEG30k-NPC, 5% acetonitrile, and a molar ratio of 18 PEG/BMP7. The final HOSu concentration was 24 mM, which is approximately 48 molar excess over mPEG30k-NPC. The reaction was quenched with 10 mM glycine for 1 hour at room temperature.
B. Analysis by HPLC Size Exclusion Column
The outcome of the conjugation reaction was analyzed by HPLC-SEC using Superose-6 10/300 GL, 1×30 cm column (GE Healthcare, Piscataway, N.J.), and 25 mM Tris, 300 mM NaCl, 6 M Urea, pH 6.5, mobile phase. The sample was diluted 1/20 in the mobile phase, and 50 μl were injected to the column. The flow rate was set at 0.5 ml/min, and elution off the column was monitored by a fluorescence detector set at an excitation wavelength of 295 nm, and emission wavelength of 360 nm (bandwidth 15 nm) with the results shown in
C. Purification
1. Dialysis: 10 mM Na Acetate buffer pH 5, using SPECTRA/POR 1 membrane tubing (Spectrum Medical Industries Inc., Los Angeles, Calif.), having a molecular weight cut-off of 6000-8000. The dialysis was carried out at 4° C. At the end of dialysis, the sample was filtered through a 0.45 μm Acrodisc HT Tuffryn membrane syringe filter (PALL Life Sciences, Ann Arbor, Mich.).
2. Separation by Ion Exchange Chromatography:
A sulphopropyl cation exchanger column, Source 15S PE 4.6×100 mm (GE Healthcare, Piscataway, N.J.), 1.7 ml total volume, was equilibrated with 20 column volumes of 10 mM sodium acetate pH 5 buffer. Next, 20 ml of the dialyzed conjugation sample were loaded on the column. Elution was performed by gradient elution using mobile phase A containing 10 mM sodium acetate pH 5, mobile phase B1 containing 1 M NaCl in 10 mM sodium acetate pH 5, and mobile phase B2 containing 6 M urea, 1 M NaCl, 10 mM sodium acetate pH 5, at a flow rate of 1 ml/min. The unbound material to the column was washed out with 40 ml of mobile phase A. The gradient elution started by increasing mobile phase B1 from 10% to 60% in 50 minutes, then to 100% B2 (1 M NaCl, 6 M urea) for 10 minutes at 2 ml/min. Fractions were collected at 1 ml/fraction throughout the elution step with the results shown in
D. Analysis by HPLC-SEC
In order to identify the content of the fractions collected throughout the ion exchange separation, aliquots from the fractions were analyzed by HPLC-SEC using Superose-6 10/300 GL column described above and the results are depicted in
E. Dialysis and Concentration
Fractions B2 to D3, from the ion exchange separation, containing the PEGylated protein were pooled concentrated and dialyzed in 20 mM sodium acetate, 5% mannitol, pH 4.5 buffer, under nitrogen at 20 psi, in a 10 ml Amicon ultrafiltration stirred cell (Millipore Corp., Billerica, Mass.), using an OMEGA ultrafiltration membrane disc filter (PALL Life Sciences, Ann Arbor, Mich.), having a molecular weight cut-off of 3000. The sample volume was brought down to approximately 3.5 ml final volume.
F. Sterile Filtration
The concentrated sample was sterile filtered through 0.22 μm Acrodisc HT Tuffryn membrane syringe filter, and sterilely filled into autoclaved glass vials. All vials were stored at 4° C. Approximately a total of 1.2 mg of PEG30k-BMP7 were obtained from the purification, as determined by the protein assay described below.
G. Protein Determination Assay:
The protein determination assay was based on the fluorescent characteristic of the protein intrinsic tryptophan. BMP7 was used as a standard, and serial dilutions were made at 6.25, 12.5, 25, 50, 100, and 200 μg/ml in 20 mM sodium acetate, 5% mannitol, pH 4.5 buffer. The mPEG30k-BMP7 sample was diluted 1:10 and 1:20 in the same buffer. The standards and test samples were transferred to a black microtiter plate, at 200 μl/well, in triplicates with the results shown in Table 3 and
H. HPLC-SEC Analysis
The purified mPEG30k-BMP7 sample was analyzed by size exclusion chromatography using a Superose-6 10/300 GL column described above. The sample was diluted to 50 μg/ml in the mobile phase, and 50 μl were injected to the column. The flow rate was set to 0.5 ml/min, and elution off the column was monitored by a fluorescence detector set at an excitation wavelength of 295 nm, and emission wavelength of 360 nm (bandwidth 15 nm) with the results shown in
I. SDS-PAGE Analysis
The mPEG30k-BMP7 sample was analyzed by gel electrophoresis under denaturing conditions, using NuPAGE® Bis-Tris 4-12% gradient gel and MOPS-SDS running buffer (Invitrogen Life Technology, Carlsbad, Calif.). Samples and controls were loaded on 2 gels at 10 μl/well containing 1.5 to 5 μg of protein. The gels were run at a constant voltage of 200 volts for 55 minutes. One gel was stained in iodine for PEG detection (
A 10 mM stock solution of mPEG30k-NPC was prepared in acetonitrile. The lysozyme was prepared at a stock solution of 4.16 mg/ml in 20 mM sodium citrate buffer at pH 6.9, containing 100 mM NaCl. Three MOPS buffer solutions were prepared at 100 mM, and the pH was adjusted to 6.5, 7.0, and 8.0 respectively, with 6 N HCl. Three MOPS/HOSu buffer solutions were prepared at 100 mM MOPS/100 mM HOSu, and the pH was adjusted to 6.5, 7.0, and 8.0 respectively, with 6 N NaOH. A stock solution of glycine was prepared with 500 mM glycine in 10 mM TRIS pH 7.5 buffer.
A total of 6 conjugation reactions were assembled. In the first set, lysozyme was reacted to mPEG30k-NPC in MOPS buffer at pH 6.5, 7.0, or 8.0. In the second set, lysozyme was reacted to mPEG30k-NPC in MOPS/HOSu buffer at pH 6.5, 7.0, or 8.0. All of the reactions were carried out at room temperature (21-22° C.) for 4 hours, while mixing, then transferred to 4° C. for an additional 18 hours.
The final concentrations in the reaction vials were 2 mg/ml (0.14 mM) for lysozyme and 0.4 mM for mPEG30k, resulting in a molar ratio of 3/1 PEG/lysozyme. The final acetonitrile amount was 4%. In the reactions including MOPS/HOSu buffer, the final HOSu concentration was 25 mM, which is equivalent to 62 molar excess over mPEG-NPC.
Samples were analyzed with a Superose-6 10/300 GL column (Amersham Biosciences, Piscataway, N.J.) and fluorescence detector essentially as described in Example 2 to determine the amount of conjugation of the PEG and lysozyme. The results are described in Table 4 below.
CNTO528, an Epo receptor agonist as described in PCT Publication No. WO 04/002417, was selected as a model biomolecule (mimetibody). In CNTO528, the sequence of an Epo mimetic peptide (EMP-1) known to require dimerization for bioactivity is fused to the hinge and Fc portion of IgG1, resulting in an active Epo receptor agonist. There are 21 lysine residues in the Fc and hinge portion of CNTO528 and the Epo mimetic peptide has one lysine residue. In addition, there are two amino terminal groups on a single mimetibody molecule (46 total potential sites). Although the Fc portion contributes to a longer circulation time compared to the free peptide, even longer circulation may be desired for improved dosing regimens.
Amine-directed PEGylation of CNTO528 was performed as follows, and according to the reaction scheme described above. A 10 mM solution of PEG30k-NPC in acetonitrile was prepared just prior to use. The mimetibody CNTO528 (Lot# FV2413A) was prepared as described in PCT Publication Nos. WO 04/002417; WO 04/002424; WO 05/081687; and WO 05/032460. A buffer of 100 mM HEPES and 100 mM N-hydroxysuccinimide (HOSu), pH 7.5 was prepared.
PEG30k-NPC was used in 10-fold molar excess to CNTO528. PEG solution was added to CNTO528 in buffer and water to a final protein concentration of 4 mg/mL, a final buffer concentration of 25 mM HEPES/HOSu, and a final PEG concentration of 0.645 mM. The reaction was allowed to proceed at room temperature (21-22° C.) in the dark on a rocking mixer for 4 hours and then placed in 4° C. overnight. The reaction was stopped with 30 mM final concentration of glycine.
The crude reaction material was dialyzed in 10 mM citrate, pH 5.0, and then purified by cation exchange chromatography (SP HP 1 mL or 5 mL column, Amersham Biosciences) using a NaCl elution gradient. The reaction material was characterized by size-exclusion chromatography (SEC) using Superose-6 (Amersham Biosciences) in a 50 mM sodium phosphate and 100 mM NaCl, pH 7.4 mobile phase. Following analysis by SEC, fractions were pooled to obtain the desired species ratio. The SEC chromatogram details are summarized in Table 5 below.
Next, the pooled conjugate mixture was dialyzed into phosphate buffered saline (PBS), pH 7.2, and filtered using a 0.2 μm pore-size membrane to sterilize. PEG30K-CNTO 528 was placed in a sterile glass vial at 2 mL±0.1 mL, as determined by A280 (1.0 mg/mL). This conjugate mixture was characterized by SEC as above and the results are shown in Table 6 below.
Conjugate was prepared as described in Example 7, with the following changes to the amounts of the reaction components. Final concentrations for CNTO528, HEPES/HOSu buffer, and PEG30k-NPC were 5.6 mg/mL, 35 mM, and 0.9 mM respectively. The reaction was stopped with 40 mM final concentration of glycine.
The conjugate was characterized by size-exclusion chromatography (SEC) using Superose-6 (Amersham Biosciences) in a 50 mM sodium phosphate and 150 mM NaCl, pH 7.0 mobile phase and the results from the chromatograms for crude and purified materials are shown in the Tables 7 and 8 below.
While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/686,738, filed Jun. 1, 2005, which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60686738 | Jun 2005 | US |
