Patent Application
20230340096
The present invention relates to DNA construct suitable for expressing a recombinant protein in a bacterial host cell wherein said DNA construct comprises nucleotide sequences encoding: a rhaBAD promoter, a RhaR transcription activator, a RhaS transcription activator, an antibiotic resistance marker, at least one terminator, and an origin of replication. The DNA construct may also comprise a promoter operably linked to the nucleic acid encoding for the antibiotic resistance marker, and a nucleotide sequence encoding said recombinant protein operably linked to the rhaBAD promoter. The at least one terminator may be an rmB T1 terminator and an rmB T2 terminator. The recombinant protein may be ranibizumab. The present invention further relates to a vector and bacterial host cell comprising said DNA construct as well as a method of producing said recombinant protein by exposing said bacterial host cell to rhamnose and thereby inducing expression of said recombinant protein.
The metabolism of rhamnose involves L-rhamnose being taken up into cells via the permease RhaT and then isomerized into L-rhamnulose by L-rhamnose isomerase (RhaA), and L-rhamnulose is then phosphorylated further by rhamnulokinase (RhaB) and finally hydrolyzed by rhamnulose-1-phosphate aldolase (RhaD) to give dihydroxyacetonephosphate and L-lactaldehyde [1]. The genes rhaA, rhaB and rhaD form an operon referred to as rhaBAD and are transcribed with the aid of the rhaBAD promoter [1]. In comparison with other systems, the rhamnose metabolism pathway is distinguished by the fact that two transcription activators known as RhaS and RhaR are required for regulation as explained below [1].
The rhaBAD operon is a positively regulated catabolic operon which transcribes above mentioned rhaB, rhaA and rhaD genes divergently from the rhaSR operon with approximately 240 bp of DNA separating their respective transcription start sites [1]. The rhaSR operon encodes RhaS and RhaR wherein each monomer of the dimeric RhaS and RhaR proteins contains two helix-turn-helix motifs and contacts two major grooves of DNA. RhaR regulates transcription of rhaSR by binding promoter DNA spanning −32 to −82 bases relative to the rhaSR transcription start site [1]. Subsequent to rhaSR expression, RhaS bind DNA upstream of the rhaBAD operon at −32 to −81 bases relative to the transcription start site to increase rhaBAD expression [1]. Furthermore, the rhaSR-rhaBAD intergenic region contains CRP binding sites at positions −92.5 (CRP 1) relative to the transcription start site of the rhaBAD operon and CRP binding sites at positions −92.5 (CRP 2), −115.5 (CRP 3) and −116.5 (CRP 4) relative to the transcription start site of the rhaSR operon [1]. The cyclic AMP receptor protein (CRP) regulates the expression of more than 100 promoters in Escherichia coli.
DNA constructs comprising DNA sequences encoding RhaS, RhaR and the rhaBAD promoter are known in the art. U.S. Pat. No. 8,138,324 discloses pTACO- and pLEMO-derived plasmids (i.e. DNA constructs) comprising DNA sequences encoding RhaS, RhaR and the rhaBAD promoter. However, U.S. Pat. No. 8,138,324 is silent about using host cells which have a disabled rhamnose metabolism.
DNA constructs based on pRha-derived plasmids comprising DNA sequences encoding RhaS, RhaR and the rhaBAD promoter are also known in the art, for example from Giacalone et al. [5] or Hjelm et al. [2]. Giacalone et al. describe for example the plasmids pRha67A and pRha109A whereas Hjelm et al. disclose the plasmid pRha67K.
Although DNA constructs comprising DNA sequences encoding RhaS, RhaR and the rhaBAD promoter are known in the art there are still many challenges, especially in industrial scale production of recombinant proteins, in particular monoclonal antibodies or fragments thereof. The main challenges are:
Accordingly, there is a need for improved DNA constructs as well as a host cell and method suitable for the efficient production of recombinant proteins, such as monoclonal antibodies or fragments thereof in a high yield, particularly when such recombinant proteins are intended for therapeutic applications.
Thus, the object of the present invention was to provide improved DNA constructs, as well as a bacterial host cell and method suitable for the efficient production of recombinant proteins, such as monoclonal antibodies or fragments thereof in a bacterial host cell in a high yield, particularly when such recombinant proteins are intended for therapeutic applications. The recombinant protein may be ranibizumab.
A first aspect of the invention relates to a DNA construct for expressing ranibizumab in a bacterial host cell, wherein ranibizumab comprises (i) a light chain comprising the amino acid sequence of SEQ ID 3, and (ii) a heavy chain comprising the amino acid sequence of SEQ ID 4, wherein said DNA construct comprises a nucleotide sequence encoding ranibizumab, wherein said DNA construct further comprises at least one nucleotide sequence encoding a signal peptide which is operably linked in the direction of transcription to the nucleotide sequence encoding the light chain of ranibizumab and/or the heavy chain of ranibizumab, wherein said DNA construct comprises nucleotide sequences encoding:
In a preferred embodiment, said DNA construct further comprises a nucleotide sequence encoding a promoter operably linked to the nucleotide sequence encoding the antibiotic resistance marker.
In a preferred embodiment, the DNA construct further comprises nucleotide sequences encoding rmB T1 terminator and rmB T2 terminator.
In a preferred embodiment, the DNA construct is characterized by:
In a preferred embodiment, the DNA construct is characterized by:
In a preferred embodiment, the DNA construct is characterized by:
In a preferred embodiment, the DNA construct is characterized by:
In a preferred embodiment, the DNA construct comprises a multiple cloning site comprising 17 restriction sites cleavable by restriction enzymes EcoRI, NdeI, NotI, XhoI, PspXI, PaeR71, BbsI, StyI, AvrII, BanI, Acc65I, KpnI, Eco53kI, SacI, BamHI, XbaI, SalI, AccI, PstI, SbfI, SphI and HindIII.
In a preferred embodiment, the DNA construct comprises the nucleotide sequence of SEQ ID 1, or a sequence with at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 1.
In a preferred embodiment, the nucleotide sequence encoding ranibizumab is operably linked to the rhaBAD promoter.
In a preferred embodiment, the nucleotide sequence encoding ranibizumab comprises (i) a nucleotide sequence encoding for the light chain of ranibizumab comprising the sequence of SEQ ID 5 or a sequence with at least 90% sequence identity thereto, and/or (ii) a nucleotide sequence encoding for the heavy chain of ranibizumab comprising the sequence of SEQ ID 6 or a sequence with at least 90% sequence identity thereto. In a further preferred embodiment, said nucleotide sequence encoding ranibizumab comprises (i) a nucleotide sequence encoding for the light chain of ranibizumab comprising the sequence of SEQ ID 5, and/or (ii) a nucleotide sequence encoding for the heavy chain of ranibizumab comprising the sequence of SEQ ID 6.
In an embodiment, the nucleotide sequence encoding the signal peptide is operably linked in the direction of transcription to either one or both of the nucleotide sequence of SEQ ID 5 and SEQ ID 6.
In an embodiment, the signal peptide is PelB (pectate lyase B). In a preferred embodiment, the nucleotide sequence encoding the PelB signal peptide comprises a sequence of SEQ ID 7 or a sequence with at least 90% sequence identity thereto; more preferably the nucleotide sequence encoding the PelB signal peptide comprises the sequence of SEQ ID 7.
In one embodiment, the resulting PelB signal peptide comprises an amino acid sequence of SEQ ID 18 [6]: MKYLLPTAAAGLLLLAAQPAMA.
In a preferred embodiment, the DNA construct comprises the sequence of SEQ ID 17 or a sequence with at least 90% sequence identity thereto, preferably comprises the sequence of SEQ ID 17.
A second aspect of the invention relates to an expression vector comprising any of the DNA constructs according to the first aspect of the invention.
A third aspect of the invention relates to a bacterial host cell comprising a DNA construct according to the first aspect of the invention or the expression vector according to the second aspect of the invention, wherein said bacterial host cell is preferably an Escherichia coli cell, more preferably an E. coli K-12 cell.
In a preferred embodiment, said bacterial host cell either comprises (i) a chromosome which comprises a mutation in the nucleic acid sequence of the rhaB gene which renders RhaB inactive, or (ii) a chromosome in which the nucleotide sequence encoding RhaB is deleted.
In a preferred embodiment, said bacterial host cell is an Escherichia coli W3110 cell, preferably comprising a chromosome which comprises a frame shift-mutation in the nucleotide sequence encoding RhaB.
In a preferred embodiment, said bacterial host cell is an Escherichia coli W3110 cell comprising a chromosome which comprises nucleotide sequence of SEQ ID 2 or a sequence with at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 2, wherein optionally the chromosome of the bacterial host cell further comprises a nucleotide sequence encoding RhaT.
A fourth aspect of the invention relates to a method of producing ranibizumab comprising the step of exposing the bacterial host cell according to the third aspect of the invention to rhamnose, thereby inducing expression of ranibizumab. In a preferred embodiment, the method further comprises the step of recovering ranibizumab from the bacterial host cell; and optionally further comprises one or more step(s) of purifying the recovered ranibizumab, preferably by one or more chromatography steps.
A fifth aspect of the invention relates to a method of producing ranibizumab, comprising the steps of:
In a preferred embodiment, the method further comprises the step of exposing the bacterial host cell to rhamnose, thereby inducing expression of the recombinant protein.
In another embodiment, the method further comprises the step of recovering ranibizumab from the bacterial host cell; and optionally further comprises one or more step(s) of purifying the recovered ranibizumab, preferably by one or more chromatography steps.
In a preferred embodiment, said bacterial host cell is an Escherichia coli cell, more preferably an E. coli K-12 cell, most preferably an E. coli W3110 cell.
In a preferred embodiment, said bacterial host cell either comprises (i) a chromosome which comprises a mutation in the nucleotide sequence of the rhaB gene which renders RhaB inactive, or (ii) a chromosome in which the nucleotide sequence encoding RhaB is deleted.
In a preferred embodiment, said bacterial host cell is an E. coli W3110 cell comprising a chromosome which comprises a frame shift-mutation in the nucleotide sequence encoding RhaB.
In a preferred embodiment, said bacterial host cell is an E. coli W3110 cell comprising a chromosome which comprises the nucleotide sequence of SEQ ID 2 or a sequence with at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 2, wherein optionally the chromosome of the bacterial host cell further comprises a nucleotide sequence encoding RhaT.
In a preferred embodiment, said nucleotide sequence encoding ranibizumab comprises (i) a nucleotide sequence encoding the light chain of ranibizumab comprising the sequence of SEQ ID 5 or a sequence with at least 90% sequence identity thereto, and/or (ii) a nucleotide sequence encoding for the heavy chain of ranibizumab comprising the sequence of SEQ ID 6 or a sequence with at least 90% sequence identity thereto; preferably said nucleotide sequence encoding the recombinant protein comprises (i) a nucleotide sequence encoding for the light chain of ranibizumab comprising the sequence of SEQ ID 5, and/or (ii) a nucleotide sequence encoding for the heavy chain of ranibizumab comprising the sequence of SEQ ID 6.
In a preferred embodiment, a nucleotide sequence comprising SEQ ID 5 and/or SEQ ID 6 further comprises a nucleotide sequence encoding a signal peptide which is operably linked in the direction of transcription to the nucleotide sequence of SEQ ID 5 and/or SEQ ID 6.
In a preferred embodiment, said nucleotide sequence encoding the signal peptide is a nucleotide sequence encoding a PelB signal peptide, preferably the nucleotide sequence encoding the PelB signal peptide comprises the sequence of SEQ ID 7 or a sequence with at least 90% sequence identity thereto; preferably the nucleotide sequence encoding the PelB signal peptide comprises the sequence of SEQ ID 7 and is fused to the nucleotide sequence encoding the light chain and/or heavy chain of ranibizumab.
In a preferred embodiment, the resulting nucleotide sequence to be introduced into the bacterial host cell comprises the sequence of SEQ ID 17 or a sequence with at least 90% sequence identity thereto, preferably comprises the sequence of SEQ ID 17.
A sixth aspect of the invention relates to a bacterial host cell being an E. coli W3110 cell and comprising a chromosome which comprises a frame shift-mutation in the nucleotide sequence encoding RhaB. In an embodiment, said E. coli W3110 host cell comprises a chromosome which comprises the nucleotide sequence of SEQ ID 2 or a sequence with at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 2. In a further embodiment the chromosome of the bacterial host cell comprises a nucleotide sequence encoding RhaT.
One or more of the above indicated SEQ IDs 1-18 of the various aspects of the invention (and embodiments thereof) may be replaced by a sequence with at least 90% sequence identity thereto. The term “sequence identity” as used herein is used with regard to amino acid or nucleotide sequences and the sequence identity is over the entire length of the specified sequence. A sequence may thus be at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent or at least 99 percent, identical in sequence to the amino acid or nucleotide sequence specified. Such sequences of the invention thus include single or multiple nucleotide or amino acid alterations (additions, substitutions, insertions or deletions) to the sequences of the invention. At the amino acid level preferred sequences with the above defined sequence identity contain up to 5, e.g. only 1, 2, 3, 4 or 5, preferably 1, 2 or 3, more preferably 1 or 2, altered amino acids in the sequences of the invention.
The present invention relates to regulating the L-rhamnose rhaBAD promoter-based production of recombinant proteins.
More specifically, the present invention relates to producing a recombinant protein, preferably ranibizumab, comprising the steps of:
The DNA construct disclosed in the above method may comprise a nucleotide sequence encoding the rhaBAD promoter. In an embodiment of the invention, the nucleotide sequence of the rhaBAD promoter comprises the sequence of SEQ ID 8 (and wherein the sequence is referred to as “rhaBAD” in
The DNA construct may comprise a nucleotide sequence encoding the RhaR transcription activator. In an embodiment of the invention, the nucleotide sequence of the RhaR transcription activator comprises a sequence of SEQ ID 9 (and wherein the sequence is referred to as “rhaR” in
The DNA construct may further comprise a nucleotide sequence encoding an extension of the RhaR transcription activator which is in frame with RhaR because of a missing stop codon. In an embodiment of the invention, the nucleotide sequence of the extension of the RhaR transcription activator comprises the sequence of SEQ ID 10 (and wherein the sequence is referred to as “rhaR extended” in
The DNA construct may comprise a nucleotide sequence encoding the RhaS transcription activator. In an embodiment of the invention, the nucleotide sequence of the RhaS transcription activator comprises the sequence of SEQ ID 11 (and wherein the sequence is referred to as “rhaS” in
The DNA construct may comprise a nucleotide sequence encoding an “antibiotic resistance marker” or “selection marker”. Such a marker is a fragment of DNA that contains a gene whose product confers resistance to an antibiotic (e.g., chloramphenicol, ampicillin, gentamycin, streptomycin, tetracycline, kanamycin, neomycin) or the ability to grow on selective media (e.g., ura (uracil), leu (leucine), trp (tryptophan), his (histidine)). Usually, plasmids contain antibiotic resistance marker to force the bacterial cell to maintain the plasmid. In an embodiment of the invention, the DNA construct may comprise a nucleotide sequence of a kanamycin resistance marker. In a specific embodiment of the invention, the nucleotide sequence for conferring kanamycin resistance comprises the sequence of SEQ ID 12 (and wherein the sequence is referred to as “KanR” in
The DNA construct may comprise a nucleotide sequence encoding a promoter operably linked to the nucleic acid sequence encoding the antibiotic resistance marker. Such a promotor may increase the expression of the antibiotic resistance markers discussed in the previous paragraph. In an embodiment of the invention, the promoter for ampicillin resistance is an AmpR promoter which is not only capable of promoting expression of ampicillin resistance markers but also capable of promoting expression of kanamycin resistance markers. In a specific embodiment of the invention, the nucleic acid sequence of the AmpR promoter comprises the sequence of SEQ ID 13 (and wherein the sequence is referred to as “AmpR promoter” in
The DNA construct may comprise a nucleotide sequence encoding for at least one terminator. The DNA construct may comprise a nucleotide sequence encoding for both of the rmB T1 terminator and the rmB T2 terminator. The rmB T1 and T2 terminators are both efficient transcription terminators in isolated forms, however, when used together, rmB T1 and T2 terminators may more efficiently terminate transcription.
In a specific embodiment of the invention, the nucleotide sequence of the rmB T1 terminator comprises the sequence of SEQ ID 14:
In a specific embodiment of the invention, the nucleotide sequence of the rmB T2 terminator comprises the sequence of SEQ ID 15:
The DNA construct may further comprise an origin of replication which is a particular nucleotide sequence at which DNA replication is initiated. DNA replication may proceed from this point bidirectionally or unidirectionally. Some commonly used origins of replication are ColE1, pMB1, pSC101, R6K, pBR322, R6K, p15A, and pUC. In an embodiment of the invention, the origin of replication is pMB1 or derivatives thereof. In a specific embodiment of the invention, the nucleic acid sequence of pMB1 comprises the sequence of SEQ ID 16:
In an alternative embodiment of the invention, the DNA construct is an expression plasmid which comprises a nucleotide sequence encoding one or more of:
In an embodiment of the invention, the above disclosed expression plasmid comprises a nucleotide sequence encoding:
Optionally, the DNA construct further comprises a nucleotide sequence encoding a promoter operably linked to the nucleotide sequence encoding the antibiotic resistance marker and/or nucleotide sequences encoding rmB T1 terminator and rmB T2 terminator.
In an embodiment of the invention, the above disclosed expression plasmid comprises a nucleotide sequence encoding:
Optionally, the nucleotide sequence encoding AmpR promoter is operably linked to the nucleotide sequence encoding the kanamycin resistance marker and/or the DNA construct further comprises nucleotide sequences encoding rmB T1 terminator and rmB T2 terminator.
In an embodiment of the invention, the above disclosed expression plasmid comprises nucleotide sequences encoding:
In an embodiment of the invention, the DNA construct is a plasmid which comprises the nucleotide sequence of SEQ ID 1 and wherein said DNA construct is also referred to as KTXHIS in the present invention.
The nucleotide sequence encoding a recombinant protein which is to be cloned into the DNA constructs described above may comprise a nucleic acid encoding a monoclonal antibody or fragment thereof, preferably ranibizumab or a fragment thereof. In a specific embodiment of the invention, the nucleic acid encoding ranibizumab comprises a nucleic acid encoding the heavy and light chains of ranibizumab. Ranibizumab is a monoclonal antibody fragment (Fab) which is an anti-angiogenic that has been approved to treat the “wet” type of age-related macular degeneration, a common form of age-related vision loss.
In a specific embodiment of the invention, the nucleotide sequence encoding the heavy chain of ranibizumab comprises the sequence of SEQ ID 6:
In a specific embodiment of the invention, the nucleotide sequence encoding the light chain of ranibizumab comprises the sequence of SEQ ID 5:
In a further embodiment of the invention, the nucleic acid encoding the heavy and light chains of ranibizumab further comprises a nucleotide sequence encoding a signal peptide operably linked to either one or both of the nucleotide sequences encoding the heavy and light chains of ranibizumab. The nucleic acid sequence encoding the signal peptide may be a nucleotide sequence encoding the PelB signal peptide. In a specific embodiment of the invention, the nucleotide sequence encoding the PelB signal peptide comprises the sequence of SEQ ID 7:
The resulting nucleotide sequence (i.e. the product of cloning an nucleotide sequence encoding a recombinant protein, preferably ranibizumab, into a DNA construct) is preferably the nucleotide sequence of SEQ ID 17 which is also referred to as KTXHIS-PelbLC-PelbHC in the present invention.
The bacterial host cell to be used for producing the recombinant protein comprises a chromosome having a mutation or modification which disables rhamnose metabolism. The bacterial host cell may be an E. coli cell. In a preferred embodiment, the bacterial host cell is an E. coli K-12 cell, more preferably the bacterial host cell is an E. coli W3110 cell. The disabled rhamnose metabolism is achieved by a mutation in the nucleotide sequence encoding RhaB which renders RhaB inactive. Alternatively, the disabled rhamnose metabolism is achieved by using a bacterial host cell having a chromosome in which the nucleotide sequence encoding RhaB is deleted; this can e.g. be achieved by deleting the nucleotide sequence encoding RhaB. Preferably, the chromosome of the bacterial host cell comprises the nucleic acid sequence encoding RhaT, i.e. the RhaT gene is intact.
In an embodiment of the invention, the disabled rhamnose metabolism is achieved by a frame shift-mutation in the nucleotide sequence encoding RhaB. Such a mutation results in a bacterial host cell having a chromosome which comprises the nucleotide sequence of SEQ ID 2:
The bacterial host cell having a chromosome which comprises the nucleotide sequence of SEQ ID 2 is in the present invention referred to as XdevK.
In one embodiment of the invention the method for developing the XdevK host cell comprises the steps of:
For constructing the pRhaBFS plasmid the two primers to PCR amplify parts of the rhaBAD operon from the chromosome of the E. coli BL21 strain (names of primers in parenthesis) are preferably:
The following embodiments are also covered:
The present invention has multiple aspects, illustrated in examples 1-8 in the below non-limiting EXAMPLES section. It should be understood that these examples, relating to the use of the XdevK host cell and/or the KTXHIS plasmid, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above disclosed embodiments of the invention and the following examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various types of therapeutic antibodies and immunoglobulins. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. An example of such a modification is that one or more of the above indicated SEQ IDs 1-18 may be replaced by a sequence with at least 90% sequence identity thereto.
The XdevK host cell is an E. coli W3310 derivative with a frameshift in the chromosomal copy of RhaB making it unable to utilize rhamnose as a carbon source. The XdevK host cell was developed as a strain for a rhamnose inducible system where the nucleotide sequence encoding the recombinant protein of interest is cloned into the KTXHIS plasmid and expressed under the control of a rhamnose inducible promoter.
The starting strain for the XdevK host cell was E. coli W3110 and a similar method of producing the XdevK host cell is described in Wilms et al. in the section titled “Construction of the Production Strain E. coli BW3110” [3].
The method for developing the XdevK host cell comprises the steps of:
Step (a)—Construction of the pRhaBFS Plasmid:
The following two primers were used to PCR amplify parts of the rhaBAD operon from the chromosome of the E. coli BL21 strain (names of primers in parenthesis):
The PCR product was purified and digested with BamHI and SalI and ligated into a pLemo plasmid (such as pLemo described in U.S. Pat. No. 8,138,324) which had been digested with BamHI and SalI. The pLemo plasmid with the insert was then digested with ClaI. The digest was incubated with T4 polymerase to make blunt ends and then ligated. The resulting pLemo plasmid with the inserted rhaBAD operon genomic region with two additional bases was incubated with BamHI and SalI. The fragment containing the rhaBAD operon genomic region with 2 additional bases was purified and ligated with the pmak705 plasmid digested with BamHI and SalI. The plasmid resulting from the ligation was named pRhaBFS and the sequence was verified by sequencing.
Step (b)—Construction of the RhaB Frame Shift Mutant.
E. coli W3110 cells were transformed with the gene replacement plasmid pRhaBFS and then plated on LB agar vegitone containing chloramphenicol (20 μg/ml) and incubated at 30° C. for 20 hours. A single colony was picked and placed in LB vegitone and grown in 30° C. until the OD600 reached 0.5. 5 μl were transferred from the culture to a LB agar vegitone plate containing chloramphenicol (20 μg/ml) and incubated at 43° C. for 16 hours. Colonies were tested for correct insert into the chromosome using PCR. Colonies with correct insert were grown in LB vegitone containing chloramphenicol (20 μg/ml) to saturation three times and then grow 20 hours in LB vegitone. One μl was then plated onto a LB vegitone plate. The LB vegitone plate was used as a template for replica plating onto a McConkey agar plate supplemented with 1% Rhamnose to verify that the cells cannot utilize Rhamnose as a carbon source. One colony was picked and grown in LB vegitone (Sigma) and then aliquoted and named XdevK expression strain. The chromosomal copy of the rhaB gene from XdevK was amplified by means of PCR. The PCR product was sequenced and the correct insertion of two bases (CG) was confirmed (see underlined CG bases in SEQ ID 2):
The PelB signal peptide comprising the nucleotide sequence of SEQ ID 7 was fused in front of nucleic acids encoding both the light and the heavy chains of ranibizumab and then cloned into the KTXHIS expression plasmid. The resulting nucleotide sequence comprises a nucleotide sequence of SEQ ID 17 and is referred to as KTXHIS-PelbLC-PelbHC in the present invention. Moreover, the plasmid map of KTXHIS-PelbLC-PelbHC is illustrated in
The nucleotide sequence of SEQ ID 17 is disclosed below and the sequence of the nucleic acid encoding the light chain fused to PelB signal sequence is underlined and the sequence of the nucleic acid encoding the heavy chain fused to PelB signal sequence is in bold (see also
AGCACAGCCTGCAATGGCAGATATTCAGCTGACCCAGAGCCCGAGCAGCCTGAG
CGCAAGCGTTGGTGATCGTGTTACCATTACCTGTAGCGCAAGCCAGGATATTAGC
AATTATCTGAATTGGTATCAGCAGAAACCGGGTAAAGCACCGAAAGTGCTGATCT
ATTTTACCAGCAGCCTGCATAGCGGTGTTCCGAGCCGTTTTAGCGGTAGCGGTAG
TGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCAACC
TATTATTGTCAGCAGTATAGCACCGTTCCGTGGACCTTTGGTCAGGGCACCAAAG
TTGAAATTAAACGTACCGTTGCAGCACCGAGCGTTTTTATCTTTCCGCCTAGTGAT
GAACAGCTGAAAAGCGGCACCGCAAGCGTTGTTTGTCTGCTGAATAACTTTTATC
CGCGTGAAGCAAAAGTTCAGTGGAAAGTTGATAATGCACTGCAGAGCGGTAATA
GCCAAGAAAGCGTTACCGAACAGGATAGCAAAGATAGCACCTATAGCCTGAGCA
GCACCCTGACCCTGAGCAAAGCAGATTATGAAAAACACAAAGTGTATGCCTGCG
AAGTTACCCATCAGGGTCTGAGCAGTCCGGTTACCAAAAGTTTTAATCGTGGTGA
ATGCTAATAATCTAGAACTGTTATCGATATCAGGAGGATTAGCATATGAAATATC
TGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTGCAA
TGGCAGAAGTTCAGCTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTG
GTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTATGATTTTACCCATTATGG
TATGAATTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGAATGGGTTGGTTG
GATTAATACCTATACCGGTGAACCGACCTATGCAGCAGATTTTAAACGTCGT
TTTACCTTTAGCCTGGATACCAGCAAAAGCACCGCATATCTGCAGATGAATA
GCCTGCGTGCAGAGGATACCGCAGTGTATTATTGTGCAAAATATCCGTATTA
TTACGGCACCAGCCATTGGTATTTCGATGTTTGGGGTCAGGGCACCCTGGTT
ACCGTTAGCAGCGCAAGCACCAAAGGTCCGAGCGTTTTTCCGCTGGCACCG
AGCAGCAAAAGTACCAGCGGTGGCACCGCAGCACTGGGTTGTCTGGTTAAA
GATTATTTTCCGGAACCGGTTACCGTGAGCTGGAATAGCGGTGCACTGACCA
GCGGTGTTCATACCTTTCCGGCAGTTCTGCAGAGCAGCGGTCTGTATAGCCT
GAGCAGCGTTGTTACCGTTCCGAGCAGCAGCCTGGGCACCCAGACCTATATT
TGTAATGTTAATCATAAACCGAGCAATACCAAAGTGGATAAAAAAGTGGAAC
CGAAAAGCTGCGATAAAACCCATCTGTAATAAAAGCTTGGCTGTTTTGGCGGA
The amino acid sequence of the light chain of ranibizumab expressed by the expression vector KTXHIS-PelbLC-PelbHC comprises the sequence of SEQ ID 3:
The amino acid sequence of the heavy chain of ranibizumab expressed by the expression vector KTXHIS-PelbLC-PelbHC comprises the sequence of SEQ ID 4:
Moreover, the resulting PelB signal peptide comprises an amino acid sequence of SEQ ID 18 [6]: MKYLLPTAAAGLLLLAAQPAMA
3 different batches comprising ranibizumab were produced according to the below described method.
Minimal media was prepared and inoculated with overnight culture of the XdevK host cell comprising the expression vector KTXHIS-PelbLC-PelbHC from a shake flask. Fermentation temperature was set to 30° C. and the cultures were grown for 22 h in batch phase. This phase was followed by a growth phase for 10 h. The temperature was adjusted to 28.5° C. and the growth rate was then decreased by controlling the glucose feed. The culture was induced with 500 μM of rhamnose injected into the fermenter. The glucose feed was also supplemented with rhamnose for continuous induction throughout the induction phase. Material was harvested after 36 h of induction. The total fermentation time was 68 h and harvested volume was 8530 ml.
After harvest, all material, media and cells, were homogenized. 5500 ml were stored in −80° C. and 3000 ml were clarified and washed using TFF. The cells were washed with a volume three times the initial volume for increased release of the product. The resulting product comprising ranibizumab was purified by using capto L affinity chromatography which is also the chromatography method used in Kumar et al. [4] (see sections 2.4 and 3.2 in Kumar et al.) and then the concentration of purified ranibizumab was determined.
The first, second and third batches comprised a ranibizumab titer of 531 mg/L, 487 mg/L and 518 mg/L, respectively.
As a comparison, in prior art document Kumar et al (which discloses similar purification methods), the ranibizumab titer ranges from 5 mg/L to 25 mg/L as indicated in
Consequently, the method according to the present invention (as well as the use of XdevK host cell and expression vector KTXHIS-PelbLC-PelbHC therein) provides unexpectedly higher levels of ranibizumab titer when compared to prior art methods.
The XdevK was transformed with KTXHIS-PelbLC-PelbHC. A single colony was picked and grown in Defined Bioreactor Medium (DBM) overnight, the following day 20% glycerol was added, the culture was aliquoted and stored in −80° C. One aliquot (50 μl) was used to inoculate 100 ml of Defined Bioreactor Medium (DBM) and cultured for 24h in 30° C., temperature was shifted to 35° C. and the cells were cultured for an additional 6h to increase OD. OD A600 nm=4.44. 20% of glycerol was added, culture was aliquoted (100 μl aliquots) and frozen in −80° C. as RCB100.
Aliquots of the strain were sent for phage testing and microbial purity testing at Charles River Laboratories. The report stated: “It was concluded that RCB100 is free of bacteriophage when tested for both lysogenic prophage and free bacteriophage. The limit of the number of free bacteriophage is less than 1 plaque forming unit per mL of an overnight culture.”
The following three strains were used in the comparative experiments:
The following four expression plasmids were used to express the nucleic acid sequence for ranibizumab, i.e. sequences encoding the heavy and light chains of ranibizumab were cloned into the following expression plasmids:
The resulting expression vectors were confirmed by full sequencing.
Moreover, the resulting expression vectors were introduced into the XdevK, XB201 and MG1655 strains (i.e. host cells) to test the different combinations of strains and plasmids to understand the influence on titratability, titer, quality and control of expression.
Experimental Setup for Titratability Comparisons
The expression vectors comprising the heavy and light chains of ranibizumab were transformed into XdevK, XB201 and MG1655 using standard protocols. Overnight cultures were prepared by inoculating a single colony in 3 ml of LB-veg liquid media containing 100 μg/ml ampicillin or 50 μg/ml kanamycin depending on the construct. Cultures were incubated in a 15 ml falcon tube at 37° C. with shaking for 16 hours. Cultures were then back-diluted (1:50) into 5 ml of LB-veg plus antibiotics in a 24-well growth plate, and incubated as before until an OD600 of approximately 0.3-0.5 was reached. Expression was induced by addition of 5, 50, 100, 250, 500, 5000 μM rhamnose and incubation for 5 hours at 30° C. with shaking followed. A volume of cells corresponding to an OD600 of 0.2 units was collected, resuspended in 20 μl 2× Laemlli loading buffer, boiled for 5 mins after which 7.5 μl was analyzed by 12% mini SDS-PAGE. The gel was then immediately transferred to a nitrocellulose/PVDF membrane and probed using anti-Fab antibody for Western blot analysis.
It should be noted that the combinations of:
Hence, in conclusion, the XdevK in combination with KTXHIS-PelbLC-PelbHC provides a superior titratability compared to the setups known from prior art. Thus, this allows for a precisely tuning of recombinant protein production rates and therefore the recombinant protein production rates can be set precisely and stably.
The expression vectors comprising the heavy and light chains of ranibizumab were transformed into XdevK, XB201 and MG1655 using standard protocols to produce Research Cell Bank (RCB) of respective combination of strain and plasmid. Pre-culture (in shake flask) was inoculated from a single vial of RCB and grown overnight (O/N) in defined reactor media; Potassium dihydrogen phosphate (KH2PO4) 4.5 g/L, di-Potassium hydrogen phosphate (K2HPO4) 3.0 g/L, Ammonium sulfate ((NH4)2SO4) 3.75 g/L, tri-Sodium citrate dihydrate (HOC(COONa)(CH2COONa)2·2H2O) 1.88 g/L, Iron(III) chloride hexahydrate (FeCl3·6H2O) 0.0525 g/L, Zinc sulfate heptahydrate (ZnSO4·7H2O) 0.0158 g/L, Copper(II) sulfate pentahydrate (CuSO4·5H2O) 0.00397 g/L, Manganese sulfate monohydrate (MnSO4·1H2O) 0.0198 g/L, Calcium chloride dihydrate (CaCl2·2H2O) 0.0207 g/L add 17.1 mL sterile 60% (v/w) glucose solution, 2.432 mL 2.5 M MgSO4·7H2O, 1 mL 50 mg/mL Kanamycin solution. The pH was adjusted by adding 1.5 mL 25% ammonium hydroxide solution (to ca. 6.95). A DAS box fermenter was prepared with defined reactor media and inoculated with O/N pre-culture. The cells growth was controlled by feeding the culture with 60% (v/w) sterile glucose. Production phase was initiated by the addition of rhamnose as the inducer. If necessary, the feed profile was adjusted and salts were added to the culture. The cells were harvested after 20-42 h of induction followed by batch purification. The harvest samples were added to a 96-well deep-well microtiter plate that contained wells with pre-frozen water to guarantee cooling of the sample. Empty wells were filled with MilliQ water (MQ). Sonication was performed using a 24-element probe on a sonics vibra-cell device for 3 cycles of 20 sec each at an amplitude of 40% with 30 sec pause in-between cycles. The sonicated samples were transferred to 2 mL spin tubes and centrifuged for 20 min at 20 000×g at 4° C. in a centrifuge 5430 R. The supernatant was removed carefully by pipetting and added to a 50% slurry of the pre-equilibrated Capto L affinity resin. Equilibration had been performed three times with 20 mM sodium phosphate, 150 mM NaCl, pH 7.2 (EQ). 1.9 mL of supernatant was used to load 100 μL of Capto L slurry, which corresponds to a load rate of 9.5 mL/mL resin.
The supernatant and the resin were incubated for 15 min at room temperature (RT) while shaking at 1500 rpm on a ThermoMixer C followed by centrifugation for 1 min at 2000×g at RT. The supernatants were discarded. The resin was washed twice by adding 500 μL of EQ and separating the resin by centrifugation at 2000×g, as above. During the third wash step using 300 μL of EQ, the resin was transferred to cellulose acetate filter spin cups suitable for 400 μL and centrifuged as above. The fourth wash step was performed with 150 μL EQ. The spin cups were transferred to new collection tubes and elution with 50 mM sodium acetate pH 3.0 was performed in three cycles using 0.4 μL of per μL resin in total (e.g. 3×40 μL for 50 μL slurry used). The protein concentration in the eluates was analyzed using an affinity HPLC setup. The quality of the samples was analyzed by SDS-PAGE and SCX-HPLC.
Samples were analyzed using SDS-PAGE as illustrated in
The amount of produced material was assessed using affinity HPLC and is summarized in
Quality of the batch purified material was assessed with SCX-HPLC and is summarized in table 1. The material analyzed from the setup with the XdevK strain in combination with the expression vector based on the pRha67A plasmid is not reliable since this setups produces so little material that it is difficult to analyze. The material from the setup with XdevK strain in combination with the expression vector based on the pRha109A plasmid was harvested after a shorter induction time since the cells grew very slow as compared to the other cells. The SCX HPLC data summarized in table 1 clearly shows that the quality of the material produced in and purified from the XdevK strain in combination with KTXHIS-PelbLC-PelbHC is of superior purity (i.e. quality) as compared to the other setups.
Hence, in conclusion, the XdevK strain in combination with KTXHIS-PelbLC-PelbHC is superior compared to the other setups both in the quality of the produced ranibizumab and the yield of the produced ranibizumab.
The expression vectors comprising the heavy and light chains of ranibizumab were transformed into XdevK and MG1655 using standard protocols. Overnight cultures were prepared by inoculating a single colony in 3 ml of LB-veg liquid media containing 100 μg/ml ampicillin or 50 μg/ml kanamycin depending on the plasmid used. Cultures were incubated in a 15 ml falcon tube at 37° C. with shaking for 16 hours. Cultures were then back-diluted (1:50) into 5 ml of LB-veg plus antibiotics in a 24-well growth plate, and incubated as before until an OD600 of approximately 0.3-0.5 was reached and different expression conditions were analyzed. One set of the cultures was grown in the presence of 0.2% glucose. Expression was induced by addition of 250 μM rhamnose in a subset of the cultures and incubated at 30° C. with shaking. A volume of cells corresponding to an OD600 of 0,2 units was collected (spun down) after 24 hours, resuspended in 20 μl 2× Laemlli loading buffer, boiled for 5 mins and analyzed (7.5 μl) by 12% mini SDS-PAGE. The gel was then immediately transferred to a nitrocellulose/PVDF membrane and probed using anti-Fab antibody.
When the accumulation of ranibizumab is compared between the different setup in
Interestingly, as indicated in lanes 3 in
Moreover, of the setups that resulted in the expression of ranibizumab in lanes 3 (i.e. the setups in which the XdevK strain was used), the XdevK strain in combination with KTXHIS-PelbLC-PelbHC resulted in the lowest expression of ranibizumab when grown in the presence of 0.2% glucose as indicated in lanes 2. This suggest that the XdevK strain in combination with KTXHIS-PelbLC-PelbHC is less prone to leaky expression in the absence of inducer. Leaky expression can have a negative effect on growth of the cells leading to lower overall yield. Leaky expression can also affect the quality of the produced material leading to a mixture of different species of the accumulated expressed protein that can be problematic to handle in a downstream purification step and therefore lead to a lower yield and a complicate purification process
Hence, the control of ranibizumab expression in the XdevK strain in combination with KTXHIS-PelbLC-PelbHC is superior when compared to the other setups.
In conclusion, the experimental results shown in Examples 4, 6, 7 and 8 demonstrate that the present invention provides the following advantageous and unexpected effects when compared with prior art expression vectors and/or host cells and therefore in particular is suitable for the industrial production of recombinant proteins
Bioeng. 2001; 73: 95-103
| Number | Date | Country | Kind |
|---|---|---|---|
| 20201096.3 | Oct 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/077820 | 10/8/2021 | WO |
