Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/244—Interleukins [IL]
  • C07K16/248—IL-6
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]

Definitions

• the present disclosure generally relates to methods for producing desired proteins in yeast and other cells. Included in the disclosure are methods that can be used to express single- and multi-subunit proteins, including antibodies.
• the cells are a yeast, such as Pichia pastoris.
• Embodiments of the subject methods can produce antibodies or other desired proteins with increased yield as compared to conventional methods.
• this invention is related to the area of fermentation. In particular, it relates to fermentation of recombinant yeast cells.
• Fungal hosts such as the methylotrophic yeast Pichia pastoris have distinct advantages for therapeutic protein expression, including that they do not secrete high amounts of endogenous proteins, have strong inducible promoters available for producing heterologous proteins, can be grown in defined chemical media and without the use of animal sera, and can produce high titers of recombinant proteins (Cregg et al., FEMS Microbiol. Rev. 24: 45-66 (2000)).
• Prior work, including work conducted by the present inventors, has helped established P. pastoris as a cost-effective platform for producing functional antibodies that are suitable for research, diagnostic, and therapeutic use. See co-owned U.S.
• Patents 7,927,863 and 7,935,340 each of which is incorporated by reference herein in its entirety.
• Methods are also known in the literature for design of P. pastoris fermentations for expression of recombinant proteins, with optimization having been described with respect to parameters including cell density, broth volume, pH, substrate feed rate, and the length of each phase of the reaction. See Zhang et al., “Rational Design and Optimization of Fed- Batch and Continuous Fermentations” in Cregg, J. M, Ed., 2007, Pichia Protocols (2nd edition), Methods in Molecular Biology, vol. 389, Humana Press, Totowa, N.J., pgs. 43-63, which is hereby incorporated by reference in its entirety.
• the disclosure provides a method of producing a desired protein, comprising: (a) culturing eukaryotic cells comprising one or more genes that provide for the expression of said desired protein at a first temperature; and (b) culturing said eukaryotic cells at a second temperature and allowing said eukaryotic cells to produce said desired protein; wherein said second temperature is different than said first temperature.
• said culturing may comprise the steps of: (a) culturing under fed-batch fermentation conditions a population of yeast cells in a culture medium, wherein each yeast cell comprises a DNA segment encoding a polypeptide, wherein said DNA segment is operably linked to a glyceraldehyde-3 -phosphate (GAP) transcription promoter and a transcription terminator, wherein the protein is not glyceraldehyde-3-phosphate, wherein the fermentation comprises a fermentable sugar feed at a first feed rate and wherein the fermentation is agitated at a first oxygen transfer rate; (b) measuring respiratory quotient (RQ) of the population during the batch fermentation and determining if it is within a desired predetermined range, wherein the desired predetermined range of RQ at about 20 - 40 hours after initiation of the culturing is between about 1.08 and about 1.35; (c) adjusting one or both of the fermentable sugar feed rate to a second feed rate or the oxygen transfer rate to a second oxygen transfer rate to
• Said first temperature may be between about 24 degrees C and about 31.5 grees
• Said first temperature may be between about 27.5 degrees C and about 30 degrees C.
• Said first temperature may be between about 24 degrees C and about 29 grees
• Said first temperature may be between about 27 degrees C and about 28.5 degrees C.
• Said first temperature may be between about 27.5 degrees C and about 28.5 degrees C.
• Said second temperature may be between about 1 degree C and about 6 degrees C higher than said first temperature.
• Said second temperature may be between about 30 degrees C and about 32 degrees C.
• Said second temperature may be between about 30 degrees C and about 31 .5 degrees C.
• Said second temperature may be about 30 degrees C or about 31 degrees C.
• Said desired protein comprises a multi-subunit complex.
• Said method may decrease the relative abundance of product-associated variants having a higher or lower apparent molecular weight than said desired multi-subunit complex as detected by size exclusion chromatography or gel electrophoresis relative to the same method effected without a difference between said first temperature and said second temperature.
• Said method may decrease the relative abundance of complexes having reduced cysteines relative to the same method effected without a difference between said first temperature and said second temperature.
• the end of said batch phase may be determined by exhaustion of the carbon source in the culture medium.
• the batch phase may comprise culturing the eukaryotic cells with a carbon source until said carbon source is depleted.
• Said carbon source may comprise one or more of: glycerol, glucose, ethanol, citrate, sorbitol, xylose, trehalose, arabinose, galactose, fructose, melibiose, lactose, maltose, rhamnose, ribose, mannose, mannitol, and raffinose, and preferably comprises glycerol.
• the fed batch phase may be initiated after the batch phase.
• the method may decrease the relative abundance of complexes having aberrant stoichiometry relative to the same method effected in the absence of the temperature shift.
• the method may decrease the relative abundance of complexes having aberrant disulfide bonds relative to the same method effected in the absence of the temperature shift.
• Said eukaryotic cell may be a diploid, tetraploid cell, or polyploid.
• the method may further comprise purifying said multi-subunit complex from said eukaryotic cells or from the culture medium.
• Said humanized antibody may be of rabbit origin.
• the relative abundance of undesired side-product(s) may be decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% , at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or down to undetectable levels compared to initial abundance levels, relative to conventional methods.
• Exemplary undesired side-products whose relative abundance may be so decreased may include one or more species having a different apparent molecular weight than the desired multi-subunit complex. For example, apparent molecular weight may be affected by differences in stoichiometry, folding, complex assembly, and/or glycosylation.
• such undesired side products may be detected using size exclusion chromatography and/or gel electrophoresis, and may have a higher or lower apparent molecular weight than the desired multi-subunit complex.
• the undesired side-products may be detected under reducing conditions. In other exemplary embodiments, the undesired side-products may be detected under non-reducing conditions.
• the methylotrophic yeasts of the genus Pichia is Pichia pastoris.
• the eukaryotic cell may be a haploid, diploid or tetraploid cell.
• At least one of the genes encoding said desired protein may be expressed under control of an inducible or constitutive promoter, such as CUPl (induced by the level of copper in the medium; see Roller et al., Yeast 2000; 16: 651-656.), tetracycline inducible promoters (see, e.g., Staib et al., Antimicrobial Agents And Chemotherapy, Jan. 2008, p.
• an inducible or constitutive promoter such as CUPl (induced by the level of copper in the medium; see Roller et al., Yeast 2000; 16: 651-656.), tetracycline inducible promoters (see, e.g., Staib et al., Antimicrobial Agents And Chemotherapy, Jan. 2008, p.
• At least one of said genes that provide for expression of the desired protein, such as the light chain and/or heavy chain of a desired antibody, in said eukaryotic cell may be optimized for expression in said host cell (e.g., by selecting preferred codons and/or altering the percentage AT through codon selection).
• said method may include (a) culturing eukaryotic cells comprising one or more genes that provide for the expression of said desired protein at a first temperature; and (b) culturing said eukaryotic cells at a second temperature and allowing said eukaryotic cells to produce said desired protein; wherein said second temperature may be different than said first temperature; for example, said first temperature may be between about 20 degrees C and about 32 degrees C, between about 24 degrees C and about 31.5 degrees C, between about 27 degrees C and about 31 degrees C, between about 27.5 degrees C and about 30 degrees C, between about 20 degrees C and about 29.5 degrees C, between about 24 degrees C and about 29 degrees C, between about 27 degrees C and about 28.5 degrees C, or between about 27.5 degrees C and about 28.5 degrees C; and/or optionally said second temperature may be between about 1 degree C and about 6 degrees C higher than said first temperature, between about 1 degree C and about 3 degrees C higher than said first temperature, between about 2 degrees C and about 4 degrees C higher than said first temperature, or between about 2
• the method may further comprise delivering ethanol to the yeast cells at about 10 to 14 hours of the culturing to achieve a level in the fermentation of about 8.0 to about 12.0 g/1 ethanol.
• Step (b) of measuring may be performed by sampling the exhaust gas of the fermentation.
• the fed-batch fermentation may be conducted for at least 50 hours, or for at least 70 hours.
• the yeast cells may be Pichia pastoris, such as polyploid, haploid, or diploid Pichia pastoris.
• step (iii) the feed rate may be increased or decreased so as to adjust the RQ value to fall within the specified range.
• FIG. 4 shows the Growth profiles of 3 different Mabl Strains A, B, and C.
• FIG. 6 shows the RQ profiles of Mabl Strain A at 3 different RQ Set points RQ 1.09 - 1.15, 1.19 - 1.25, 1.29 - 1.35.
• the vertical line indicates the time at which RQ control was initiated.
• FIG. 8 shows the Ethanol profiles of Mabl Strain A at 3 different RQ Set points RQ 1.09 - 1.15, 1.19 - 1.25, 1.29 - 1.35.
• FIG. 19 shows the Ethanol profiles of 4 different Mab2 Strains A, B, C and D strains.
• FIG. 23 shows the Agitation profiles of 3 different Mab3 Strains A, B, and C.
• the vertical line indicates the time at which RQ control was initiated.
• FIG. 24 shows the Ethanol profiles of 3 different Mab3 Strains A, B, and C.
• FIG. 29 shows the Ethanol profiles of Mab3 Strain B at different feed rates.
• FIG. 36 shows the Whole Broth profiles of Mab4 Strain A strain at different feed rates.
• FIG. 39 shows the SDS-PAGE Non-Reduced and Reduced gels of Mab 1 Strain A at fermentation times 62, 69, and 85 hours.
• FIG. 45A-C details purity of the recombinant antibodies produced as shown in FIG. 40 for the culture shifted upward by 3° C to 31° C.
• Panel A size exclusion chromatography traces and tabulated results for non-reduced samples.
• Panel B Coomassie stained gel electrophoresis results for non-reduced (lane 1) and reduced (lane 3) samples. Lanes 2 and 4 show a size marker.
• Panel 3 size exclusion chromatography traces and tabulated results for reduced samples. Chromatography results are tabulated and summarized in FIG. 41.
• FIG. 50 shows improvement in titer of Ab-A resulting from a temperature shift during culture.
• Whole broth antibody titer (arbitrary units) is plotted versus time for cultures which were subjected to a temperature shift during culture or control cultures for which a temperature shift was not performed.
• Four cultures were shifted from 28 °C to 30 °C after initiating feed addition (label "30C") and five control cultures were unshifted and maintained at 28 °C throughout culturing.
• Each of the shifted cultures exhibited higher titers than the non-shifted cultures.
• the average increase in titer resulting from the temperature shift was about 47%.
• FIG. 51 summarizes the purity of the recombinant antibodies produced with a culture temperature shift as shown in FIG. 50. Purity was assessed by size exclusion chromatography of protein A-purified antibody harvested at the end of antibody production. Labels are as described in FIG. 41.
• This disclosure describes methods of improving the yield and/or purity of recombinantly expressed proteins, including antibodies and other multi-subunit proteins. Methods are provided wherein a temperature shift is effected during cell culture. The inclusion of a temperature shift is demonstrated below to improve the yield and purity of recombinantly expressed antibodies, compared to expression in the absence of the shift. [222] Though not intending to be limited by theory, it is hypothesized that a temperature shift can cause sustained changes in gene expression which confer a lasting improvement in recombinant protein production.
• the undesired side products may include an HlLl or "half antibody” species (i.e., containing a heavy chain and a light chain, wherein the heavy chain is not linked by a disulfide bond to another heavy chain), and/or a H2L1 species (i.e., containing two heavy chains and one light chain, but lacking a second light chain).
• HlLl or "half antibody” species i.e., containing a heavy chain and a light chain, wherein the heavy chain is not linked by a disulfide bond to another heavy chain
• H2L1 species i.e., containing two heavy chains and one light chain, but lacking a second light chain
• Transformation of haploid and diploid P. pastoris strains and genetic manipulation of the P. pastoris sexual cycle may be performed as described in Pichia Protocols (1998, 2007), supra.
• Presence of the desired protein gene or each expected subunit gene may be confirmed by Southern blotting, PCR, and other detection means known in the art. Additionally, expression of an antibody or other desired protein may also be confirmed by a colony lift/immunoblot method (Wung et al. Biotechniques 21 808-812 (1996) and / or by FACS.
• the mating plate can then be incubated (e.g., at 30° C.) to stimulate the initiation of mating between strains. After about two days, the cells on the mating plates can be streaked, patched, or replica plated onto media selective for the desired diploid strains (e.g., where the mated strains have complementary autotrophies, drop-out or minimal medium plates may be used). These plates can be incubated (e.g., at 30° C.) for a suitable duration (e.g., about three days) to allow for the selective growth of the desired diploid strains. Colonies that arise can be picked and streaked for single colonies to isolate and purify each diploid strain.
• a suitable duration e.g., about three days
• the process using the feedback control mechanism is applicable to the production of full-length, correctly assembled recombinant monoclonal antibodies, as well as to antibody fragments and other recombinant proteins, i.e., not glyceraldehyde-3-phosphate.
• the control mechanism that we employ is easy to mechanize and render automatic, thus eliminating much labor in monitoring and adjusting fermentation conditions.
• the process is applicable to production of a variety of antibodies and other desired proteins and is readily scalable to accommodate commercial, e.g., large scale, production needs.
• RQ ranges that may be desirable include about 1.08 - 2.0; about 1.08 - 1.85; about 1.08 - 1.65; about 1.08 - 1.45; about 1.08 - 1.35; about 1.08 - 1.25; about 1.08 - 1.2; and about 1.08 - 1.15.
• Alternative carbon sources other than glucose can achieve an RQ less than 1. Such carbon sources include acetate and glycerol.
• Other suitable RQ ranges include 1.08 to 1.35, and 1.15 to 1.25.
• RQ can be monitored and controlled during any desired portion of the fermentation, for example from 0 to 1 10 hours, from 20-40 hours, from 20-70 hours, from 20-90 hours, from 20- 1 10 hours, or any other desired time period.
• Large scale fermentation processes are those typically used in commercial processes to produce a useful product. Typically these are greater than 100 liters in volume.
• Fed-batch fermentation is a process by which nutrients are added during the fermentation to affect cell density and product accumulation.
• the methods described herein are readily adapted to other desired proteins including single subunit and multi-subunit proteins. Additionally, the present methods are not limited to production of multi-protein complexes but may also be readily adapted for use with ribonucleoprotein (RNP) complexes including telomerase, hnRNPs, Ribosomes, snRNPs, signal recognition particles, prokaryotic and eukaryotic RNase P complexes, and any other complexes that contain multiple distinct protein and / or RNA subunits.
• RNP ribonucleoprotein
• the host cell that expresses a multi-subunit complex may be produced by methods known in the art. For example, a panel of diploid or tetraploid yeast cells containing differing combinations of gene copy numbers may be generated by mating cells containing varying numbers of copies of the individual subunit genes (which numbers of copies preferably are known in advance of mating).
• Mating competent yeast species In the present invention this is intended to broadly encompass any diploid or tetraploid yeast which can be grown in culture. Such species of yeast may exist in a haploid, diploid, or other polyploid form. The cells of a given ploidy may, under appropriate conditions, proliferate for an indefinite number of generations in that form. Diploid cells can also sporulate to form haploid cells. Sequential mating can result in tetraploid strains through further mating or fusion of diploid strains. The present invention contemplates the use of haploid yeast, as well as diploid or other polyploid yeast cells produced, for example, by mating or fusion (e.g., spheroplast fusion).
• mating or fusion e.g., spheroplast fusion
• homozygotic heterothallic a/a and alpha/alpha diploids and in Pichia by sequential mating of haploids to obtain auxotrophic diploids For example, a [met his] haploid can be mated with [ade his] haploid to obtain diploid [his]; and a [met arg] haploid can be mated with [ade arg] haploid to obtain diploid [arg]; then the diploid [his] can be mated with the diploid [arg] to obtain a tetraploid prototroph. It will be understood by those of skill in the art that reference to the benefits and uses of diploid cells may also apply to tetraploid cells.
• phytohaemagglutinin (PHA) (MKKNRMMMMIWSVGVVWMLLLVGGSYG (SEQ ID NO:22)), Silkworm lysozyme (MQKLIIFALVVLCVGSEA (SEQ ID NO:23)), Human lysozyme (LYZ1) (MKALIVLGLVLLSVTVQG (SEQ ID NO:24)), activin receptor type-1 (MVDGVMILPVLIMIALPSPS (SEQ ID NO:25)), activin type II receptor
• Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion and/or sequenced.
• Such methods utilize intermolecular DNA recombination that is mediated by a mixture of lambda and E. coli- encoded recombination proteins. Recombination occurs between specific attachment (att) sites on the interacting DNA molecules.
• att sites See Weisberg and Landy ( 1983) Site-Specific Recombination in Phage Lambda, in Lambda II, Weisberg, ed. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press), pp. 21 1 -250.
• a monocistronic gene encodes an RNA that contains the genetic information to translate only a single protein.
• a polycistronic gene encodes an mRNA that contains the genetic information to translate more than one protein.
• the proteins encoded in a polycistronic gene may have the same or different sequences or a combination thereof.
• Dicistronic or bicistronic refers to a polycistronic gene that encodes two proteins.
• Folding refers to the three-dimensional structure of polypeptides and proteins, where interactions between amino acid residues act to stabilize the structure. While non-covalent interactions are important in determining structure, usually the proteins of interest will have intra- and/or intermolecular covalent disulfide bonds formed by two cysteine residues. For naturally occurring proteins and polypeptides or derivatives and variants thereof, the proper folding is typically the arrangement that results in optimal biological activity, and can conveniently be monitored by assays for activity, e.g. ligand binding, enzymatic activity, etc.
• sequences may be constitutively or inducibly expressed in the yeast host cell, using vectors, markers, etc. as known in the art.
• sequences, including transcriptional regulatory elements sufficient for the desired pattern of expression are stably integrated in the yeast genome through a targeted methodology.
• aberrant inter-chain disulfide bonds may result in abnormal complex stoichiometry, due to the absence of a stabilizing covalent linkage, and/or disulfide linkages to additional subunits. Additionally, aberrant disulfide bonds (whether inter-chain or intra-chain) may decrease structural stability of the antibody, which may result in decreased activity, decreased stability, increased propensity to form aggregates, and/or increased immunogenicity.
• Product-associated variants containing aberrant disulfide bonds may be detected in a variety of ways, including non-reduced denaturing SDS-PAGE, capillary electrophoresis, cIEX, mass spectrometry (optionally with chemical modification to produce a mass shift in free cysteines), size exclusion
• a "heterologous" region or domain of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
• the heterologous region encodes a mammalian gene
• the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
• Another example of a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
• immunoglobulins are generated either by hybridomas or by B cells.
• the CDRs in each chain are held in close proximity by framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site.
• the CDRs there are select amino acids that have been described as the selectivity determining regions (SDRs) which represent the critical contact residues used by the CDR in the antibody-antigen interaction (Kashmiri, S., Methods, 36:25-34 (2005)).
• Examples 1 through 10 show the applicability of the method to the production of four different humanized monoclonal antibodies.
• Each antibody is produced in Pichia pastoris using the glyceraldehyde-3 -phosphate (GAP) promoter system.
• GAP glyceraldehyde-3 -phosphate
• any chemical (X nH 2 0; n>0) can be replaced by another chemical containing the same activated ingredient but various amount of water (X kH 2 0; k ⁇ n).
• Ab-A sequences are as follows: [412] Ab-A heavy chain polynucleotide sequence:
• control culture was maintained at 28° C, i.e., without temperature shift.
• the total fermentation time was approximately 86-87 hours.
• P. pastoris engineered to express Ab-A was grown in cultures maintained at 28° C during an initial growth phase with glycerol as a carbon source. After exhaustion of the glycerol, a continuous glucose feed was initiated and the culture temperature was rapidly shifted upward or downward to a new set-point temperature between 25° C and 34° C which was maintained for the duration of the culture. One culture was maintained at 28° C as a control.
• antibody purity at the end of culture i.e., at 86-87 hours was determined.
• protein-A purified antibody from each culture was assessed by exclusion chromatography (SEC) and by gel electrophoresis with Coomassie staining (FIGS. 42-47), which were conducted for both reduced and non-reduced samples.
• SEC analysis of non-reduced samples detected relative abundance of complexes having aberrant
• Ab-B was expressed from engineered P. pastoris strains containing 3 copies of the light chain-encoding gene and 3 copies of the heavy chain-encoding gene (H3/L3) or 3 copies of the light chain-encoding gene and 4 copies of the heavy chain-encoding gene (H4/L3). Cultures were initially grown at 28° C with glycerol as a carbon source. After exhaustion of the glycerol, a continuous glucose feed was initiated and the culture
• the average fraction of protein contained in the full antibody peak was 74.39%, in the Prepeak HHL was 4.26%, and the 75 kD HL was 12.65%, compared to 83.44%, 4.75%, and 6.15% for the shifted H4/L3 sample, respectively.
• the average fraction of protein contained in the full antibody peak was 82.80%, in the Prepeak HHL was 5.31%, and the 75 kD HL was 4.76%, compared to 91.63%, 2.94%, and 1.44%, respectively.
• the non-reducing SEC analysis demonstrated that the temperature shift (1) increased the average amount of antibody contained in the full antibody peak, and (2) decreased the average amount of the aberrant complexes in three out of four instances.
• the reduced samples permitted detection of the relative abundance of protein contained in the full-length heavy and light chains as well as product-associated variants which were observed in three discrete elution peaks. Unlike the observed decrease in aberrant complexes observed with the non-reduced analysis, the reduced samples did not show a consistent improvement in the amount of antibody contained in the full-length heavy and light chains. Overall, for the two strains the average relative abundance of the heavy chain was increased by about 1-3% in the shifted cultures compared to the average of the unshifted cultures, and the average relative abundance of the light chain was unchanged or decreased by about 0.9% for the two strains.
• Ab-A was expressed from a P. pastoris strain containing 4 integrated copies of the heavy chain gene and 3 integrated copies of the light chain gene as described in Example 1 1, except that five cultures were maintained at 28° C (i.e., unshifted) and four cultures were shifted to 30° C. As in Example 11, the temperate shift was effected, if at all, at a time five minutes after feed initiation. The total fermentation time was approximately 87 hours.

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• 2018
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