Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells

Factor IX has been expressed to high levels within a recombinant host cell and the biologically active fraction subsequently purified to homogeneity for characterization. The coding sequence for Factor IX was inserted into a mammalian cell expression vector and transfected into dihydrofolate reductase-deficient Chinese hamster ovary cells. The integrated DNA was amplified to a high copy number by selection for increasingly higher expression levels of the marker gene, dihydrofolate reductase, contained within a co-transfected plasmid. Cloned cell lines secreting over 100 micrograms/ml Factor IX antigen and up to 1.5 microgram/ml native Factor IX antigen have been obtained. Expression of biologically active Factor IX was dependent on the presence of vitamin K in the culture media. The gamma-carboxylated Factor IX was isolated from cell culture fluid by immunoaffinity chromatography using antibodies conformation-specific for the metal-stabilized conformer of Factor IX. This conformation is dependent upon metal ions and gamma-carboxyglutamic acid. Purified recombinant Factor IX migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an electrophoretic mobility equivalent to plasma-derived Factor IX. The purified recombinant Factor IX demonstrated Factor IX coagulant activity, measured in Factor IX-deficient plasma, of 35-75 units/mg. Amino acid analysis of the alkaline hydrolysate of recombinant Factor IX demonstrated an average of 6-7 mol of gamma-carboxyglutamic acid per mol of Factor IX. NH2-terminal sequence analysis of the first 17 residues revealed equivalent amino acid sequences for both purified recombinant and plasma-derived Factor IX. The results represent the first purification and characterization of a biologically active, gamma-carboxylated vitamin K-dependent protein expressed in a recombinant DNA system.     

Expression and characterization of human apolipoprotein A-I in Chinese hamster ovary cells

We produced human apolipoprotein A-I (apoA-I) in Chinese hamster ovary (CHO) cells. The CHO cells were transfected with an expression plasmid which placed the human apoA-I gene under the direction of the human metallothionein II gene promoter. Isolation of a clonal cell line resulted in high level expression of apoA-I. Greater than 30% of total protein secreted by these CHO cells was apoA-I, which enabled us to purify apoA-I with a single step purification scheme. As a result, large quantities of apoA-I can be produced and isolated without having to rely on plasma sources. Structural characterization of the recombinant apoA-I showed it to be identical to authentic apoA-I from human serum high density lipoprotein. Furthermore, we demonstrated approximately equal to 90% of the apoA-I secreted by CHO cells is processed, mature protein. A portion of the secreted recombinant apoA-I was associated with lipid and floated at a density approximately equal to 1.10 g/ml. Additional analysis identified the presence of five isoforms of apoA-I in the CHO cell conditioned medium. Processing and post-translational modification of the recombinant apoA-I occurred in the CHO cell cultures in the absence of serum components. We conclude that the human apoA-I produced by CHO cells is identical to circulating, mature apoA-I in humans and that recombinant mammalian expression offers an opportunity to investigate apoA-I processing.     

Expression of recombinant rat Neurotrophin-3 in Chinese hamster ovary cells

The CHO cell line stably producing recombinant rat NT-3 was established. The insertion of rNT-3 cDNA into transferred cell gonome was analyzed with Southern blot. The expressed protein was identified by Dot ELISA (enzyme-linked immunosorbent assay) and Western blot. Western blot showed a clear specifie band of about 14 ku for NT-3. The mean level of rNT-3 in four NT-3eDNA/CHO cell lines was about 2 100 ng/10~6 cells/48 h determined by EIA. The conditioned-medium (CM) of NT-3cDNA/CHO cells could promote the fiber outgrowth of the dissociated dorsal root ganglion of 8-day-old chick embryos, which shows a dose-response relationship. A half-maximal concentration of the biological activity (EC50) of the recombinant protein was approximately 16.7 ng/mL. The MoAb 3W3 of NT-3 could neutralize the biological activity of the rNT-3.     

Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells

cDNAs containing the entire coding regions of the alpha and beta subunits of calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a rat cerebrum cDNA library, ligated into an expression vector under the control of SV40 early promoter and introduced into Chinese hamster ovary (CHO) cells. To investigate the role of the alpha and beta subunits and their functional domains in CaM kinase II activity, the properties of the kinases expressed in the transfected cells were studied. CaM kinase II activity was detected in the transfected cells when the alpha and beta cDNAs were introduced into CHO cells simultaneously. RNA transfer blot and protein immunoblot analyses demonstrated the expression of the mRNAs and proteins of both alpha and beta subunits in the cloned cells. When alpha or beta cDNA was introduced into CHO cells separately, a significant level of the enzyme activity was also expressed, indicating that the alpha and beta subunits exhibited enzyme activity individually. The apparent Km values for ATP and MAP 2 were almost the same for the alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II. However, there was a slight difference in the affinity for calmodulin between the expressed proteins. The alpha and beta subunits expressed in the same cells polymerized to form alpha beta complex of a size similar to that of brain CaM kinase II. The alpha subunit also polymerized to form an oligomer, which showed almost the same S value as that of alpha beta complex and brain CaM kinase II. In contrast, the beta subunit did not polymerize. The alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II were autophosphorylated with [gamma-32P]ATP in the presence of Ca2+ and calmodulin, which resulted in the appearance of Ca2+-independent activity. The Ca2+-independent activity was 60-75% of the total activity as measured in the presence of Ca2+ plus calmodulin. To examine the functional relationship of peptide domains of the subunits of CaM kinase II, deleted cDNAs were introduced into CHO cells and the properties of the expressed proteins were studied. In cells transfected with alpha or beta cDNA from which the association domain was deleted, a significant level of kinase activity was expressed. However, the expressed proteins showed hardly any autophosphorylation and the appearance of Ca2+-independent enzyme activity was very low, indicating that the association domain was essential for the autophosphorylation and for the appearance of the Ca2+-independent activity.     

Expression of recombinant rat Neurotrophin-3 in Chinese hamster ovary cells

The CHO cell line stably producing recombinant rat NT-3 was established. The insertion of rNT-3 cDNA into transferred cell gonome was analyzed with Southern blot. The expressed protein was identified by Dot ELISA (enzyme-linked immunosorbent assay) and Western blot. Western blot showed a clear specific band of about 14 ku for NT-3. The mean level of rNT-3 in four NT-3cDNA/CHO cell lines was about 2 100 ng/10⁶ cells/48 h determined by EIA. The conditioned-medium (CM) of NT-3cDNA/CHO cells could promote the fiber outgrowth of the dissociated dorsal root ganglion of 8-day-old chick embryos, which shows a dose-response relationship. A half-maximal concentration of the biological activity (EC50) of the recombinant protein was approximately 16.7 ng/mL. The MoAb 3W3 of NT-3 could neutralize the biological activity of the rNT-3.     

Genetic characterization of hydroxyurea-resistance in Chinese hamster ovary cells

Hydroxyurea is an excellent selective agent for obtaining drug-resistant mutants. At a frequency of approximately 1 X 10(-5) it was possible to select, in a single step, colonies that exhibited significant resistance to the cytotoxic effects of the drug. These hydroxyurea-resistant cell lines maintained their resistant phenotype after extensive cultivation in the absence of the drug. Reconstruction experiments indicated that the expression of hydroxyurea-resistance and the frequency of drug-resistant colonies was independent of cell densities up to 5 X 10(5) cells per 100-mm selection plate. Luria-Delbrück fluctuation analyses indicated that the appearance of hydroxyurea-resistant cells in wild type populations occurred spontaneously and at a rate of 4.8 X 10(-6) per cell per generation in the presence of 0.33 mM drug. Studies with the mutagen, ethyl methane sulfonate indicated that it was capable of increasing the frequency of hydroxyurea-resistant cells by a factor of approximately 10. Also, cell-cell hybridization experiments showed that hydroxyurea-resistance behaves as a dominant or codominant trait and that hydroxyurea-resistance was a useful new genetic marker for selection of somatic cell hybrids. Furthermore, similar to many other drug-resistant cell lines hydroxyurea-resistant cells were found to exhibit an altered sensitivity to a number of non-selective agents (guanazole, N-carbamoyloxyurea, formamidoxime, and hydroxyurethane). Except for guanazole these compounds are structurally very similar to hydroxyurea and may be expected to have similar modes of action. The results presented in this paper support the view that hydroxyurea-resistance is expressed as a normal genetic trait and is a useful genetic marker for somatic cell genetic studies.     

Expression, purification, and characterization of human recombinant thrombopoietin in Chinese hamster ovary cells

Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO, including the native signal peptide sequence, was amplified by PCR from a human fetal liver cDNA library. The product was cloned into a mammalian expression vector under the control of the SV40 early promoter and enhancer. Secreted rhTPO was purified in three conventional chromatography steps. It migrates on SDS-PAGE as a broad band, characteristic of a heavily glycosylated protein, with an average molecular mass of 85 kDa. rhTPO expressed in CHO cells is biologically active in vitro as demonstrated by its ability to stimulate the proliferation of a megakaryocytic cell line and to trigger the JAK/STAT signal transduction pathway. rhTPO also shows activity in vivo as judged by the elevation of platelet count in treated mice.     

Characterization of recombinant human antithrombin III synthesized in Chinese hamster ovary cells

Biochemical and physiochemical properties of recombinant human antithrombin III were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human plasma when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Comparison of the NH2-terminal sequences of recombinant and human plasma-derived antithrombin III showed that on synthesis and secretion of the recombinant protein from Chinese hamster ovary cells the signal peptide is correctly cleaved by the corresponding endoplasmic signal peptidase. The recombinant antithrombin III has identical properties in heparin binding and biological activities as determined in vitro by two-dimensional immunoelectrophoresis, progressive inhibitor, and heparin cofactor assays. Analysis of the carbohydrate portion of recombinant antithrombin III synthesized in Chinese hamster ovary cells revealed glycosylation of the complex type. Characterization of the oligosaccharide chains present in the recombinant protein reveals three major fractions, A (20%), B (60%), and C (20%). Fraction A contains tri- and tetraantennary complex-type oligosaccharides, fraction B contains biantennary oligosaccharides, and fraction C partially truncated biantennary structures. Pharmacokinetic studies with recombinant and plasma-derived antithrombin III in rabbits showed that the clearance behavior of both proteins is very similar and can be described by a double exponential decrease with almost identical kinetic parameters.     

Purification and characterization of lysosomes from Chinese hamster ovary cells

Lysosomes were isolated from Chinese hamster ovary cells by fractionation of a postnuclear supernatant in consecutive density gradients. By marker enzyme analysis, the preparation was 63-fold enriched for lysosomes compared to the homogenate and contained at most trace amounts of marker activities for plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, cytosol, and mitochondria. The lysosomes were intact as indicated by greater than 95% latency of beta-hexosaminidase activity, and the yield was about 12% relative to the homogenate. By electron microscopy, the lysosomal preparation contained very few mitochondrial profiles. By cytochemistry, greater than 80% of the organelle profiles were positive for the native lysosomal marker, acid phosphatase, and profiles were positive for long-term internalized horseradish peroxidase, an endocytic marker for lysosomes. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lysosomal preparation displayed a unique pattern of polypeptides and was devoid of mitochondrial contamination. Lysosomes were fractionated into membrane and lumenal compartments by Na2CO3 treatment. Each compartment contained 20-30 distinct electrophoretic species ranging from 18 to 200 kDa. Each polypeptide could be assigned to either the membrane or lumenal compartment. A comparison of silver-stained polypeptides with those metabolically labeled with [35S]methionine indicated that, with the possible exception of an 18-kDa species, all of the major lysosomal polypeptides in both compartments were derived by endogenous synthesis in these exponentially growing fibroblasts.     

Expression and function of beta-tubulin isotypes in Chinese hamster ovary cells

The availability of isotype-specific antisera for beta-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of beta-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor beta-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype-specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V. All three isotypes assemble into both cytoplasmic and spindle microtubules and are similar in their responses to cold, colcemid, and calcium-induced depolymerization. They have comparable turnover rates and are equally sensitive to depression of synthesis upon colchicine treatment. These results suggest that beta-tubulin isotypes are used interchangeably to assemble microtubule structures in CHO cells. However, of 18 colcemid-resistant mutants with a demonstrable alteration in beta-tubulin, all were found to have the alteration in isotype I, thus leaving open the possibility that subtle differences in isotype properties may exist.     

Expression of enzymatically active rat dipeptidyl peptidase IV in Chinese hamster ovary cells after transfection

Dipeptidyl peptidase IV (DPPIV) is a cell surface membrane glycoprotein expressed in many tissues. We have subcloned the coding region of a full-length cDNA for DPPIV into the inducible eukaryotic expression vector pMSG. The resulting construct was used to transfect Chinese hamster ovary (CHO) cells. Stable transformants were found to express DPPIV, and the expression is enhanced by dexamethasone. Metabolic labeling of the transfected cells with [35S]Met followed by immunoprecipitation revealed the presence of two specific products of apparent Mr 100,000 (100-kDa form) and 110,000 (110-kDa form), respectively. Pulse-chase experiments demonstrated that the 100-kDa form can be chased into the 110-kDa form, suggesting the 100-kDa form is the precursor of the 110-kDa form. Further studies with endo H treatment demonstrated that the carbohydrate structures are of the high-mannose type, and of the complex type for the 100- and 110-kDa forms, respectively. The 110-kDa form is present at the cell surface as shown by its accessibility to cell surface iodination. The DPPIV expressed on the cell surface is resistant to digestion by relatively high concentrations of trypsin. Studies also demonstrated that the surface DPPIV is fairly stable with a half-life for turnover of about 40 h. Furthermore, the DPPIV produced in the transfected cells displays specific dipeptidyl peptidase activity. The stably transfected cells that express enzymatically active DPPIV in an inducible manner will provide an excellent system for further biochemical, functional, and cell biological characterizations of DPPIV.     

Isolation and characterization of recombinant human prorenin in Chinese hamster ovary cells

Recombinant human prorenin (rh-prorenin) was purified from supernatants of Chinese hamster ovary (CHO) cell line transfected with the cDNA for rh-prorenin by employing a simple two-step procedure which consisted of ammonium sulfate precipitation and immunoaffinity chromatography using a monoclonal antibody specific for the profragment of human prorenin. About 100-fold purification with 35% recovery was achieved after the two steps. Purified rh-prorenin migrated as a single protein band with apparent molecular weights of 46,000-47,000 and about 50,000 on SDS-PAGE and gel filtration (HPLC), respectively, although it consisted of multiple components (pI values, 5.6-6.4) that could be resolved by isoelectric focusing (IEF). The treatment of rh-prorenin with endo-beta-N-acetylglucosaminidase converted the rather broad protein band to a sharp band on SDS-PAGE and reduced the number of multiple pI peaks on IEF. Amino-terminal sequence analysis of both the purified rh-prorenin and rh-renin revealed Leu-Pro-Thr-Asp- and Leu-Thr-Leu-Gly-, respectively, which agreed with those predicted from the base sequences of their cDNA. These data suggested that microheterogeneity of rh-prorenin is due to the carbohydrate moiety, but not to the protein moiety. Purified rh-prorenin was almost inactive, but was cleaved at the carboxyl end of a dibasic pair Lys-2-Arg-1 by trypsin and converted to active renin. However, at the early stage during trypsin activation, new intermediate forms between rh-prorenin and rh-renin were formed, suggesting multiple activation steps of rh-prorenin in addition to the one step activation.     

Expression of biologically active human follitropin in Chinese hamster ovary cells

To study the structure-function relationships of follitropin (FSH), we expressed the hormone in a heterologous cell system. A genomic clone bearing a 3.7-kilobase FSH beta insert containing the entire coding sequence was transfected alone or together with the alpha subunit gene into Chinese hamster ovary cells and stable lines expressing either FSH beta or FSH dimer were selected. Pulse-chase experiments revealed that, when transfected alone FSH beta was very slowly secreted similar to lutropin beta and thyrotropin beta but unlike choriogonadotropin beta which is efficiently secreted. However, cotransfection of the FSH beta and alpha subunit genes resulted in "rescue" of the beta subunit and rapid secretion of dimer. These data support the hypothesis that the glycoprotein hormones of pituitary origin have determinants for secretion that differ from those on the placental hormone, choriogonadotropin. Recombinant FSH stimulated steroidogenesis comparable to purified human FSH isolated from pituitaries in an in vitro rat granulosa cell assay and appears more homogeneous by chromatofocusing. Human FSH produced by this cell line provides a source of bioactive FSH for experimental and clinical use.     

Biochemical characterization of theO-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells

Alterations inN- andO-linked glycosylation affect cell surface expression and antigenicity of recombinant glycophorin A expressed in transfected Chinese hamster ovary (CHO) cells. To understand these effects further, glycophorin A was purified by immunoaffinity chromatography from transfected wild type and glycosylation deficient CHO cells. TheO-glycans were characterized both biochemically, using gel filtration and high performance anion exchange chromatography, and immunologically, using carbohydrate specific monoclonal antibodies to probe Western blots. TheO-glycans of human erythrocyte glycophorin A consist mainly of short oligosaccharides with one, two, or three sialic acid residues linked to a common disaccharide core, Gal1-3GalNAc1-Ser/Thr, with the disialylated structure being the most abundant. With the exception of the trisialylated derivative, the same structures were found on recombinant glycophorin A expressed by wild type CHO cells. However, in contrast to human crythrocyte glycophorin A, the monosialylated oligosaccharide was the most abundant structure on the recombinant protein. Furthermore, recombinant glycophorin A was shown to express a small amount of the Tn antigen (GalNAc1-Ser/Thr). Recombinant glycophorin A had the sameO-glycan composition, whether purified from clones expressing high or moderate levels of the recombinant glycoprotein. This indicates that the level of expression of the transfected glycoprotein did not affect itsO-glycan composition. Deletion of theN-linked glycosylation site at Asn₂₆, by introducing the Mi.I mutation (Thr₂₈ Met) by site-directed mutagenesis, did not markedly affect theO-glycan composition of the resulting recombinant glycoprotein expressed in wild type CHO cells. This demonstrates that the presence or absence of theN-glycan did not influenceO-linked glycosylation of the recombinant glycoprotein. Finally, theO-glycans on recombinant glycophorin A expressed in the Lec 2 and Lec 8 glycosylation deficient CHO cells were characterized. TheO-glycans on Lec 2 cell glycophorin A were predominantly Gal1-3GalNAc1-Ser/Thr (T antigen), while those on Lec 8 glycophorin A were exclusively GalNAc1-Ser/Thr (Tn antigen). These results will lead to a better understanding of the cell biology and immunology of this important human erythrocyte glycoprotein.     

Expression and purification of recombinant cynomolgus monkey cholesteryl ester transfer protein from Chinese hamster ovary cells

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2g/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band ofM _r , ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.     

Isolation and characterization of nitrogen mustard-sensitive mutants of Chinese hamster ovary cells

Three nitrogen mustard-sensitive lines of Chinese hamster ovary cells were isolated from mutagenized cultures using the procedure of Thompson et al. (1980). The lines, designated NM1, NM2 and NM3, were 2.1-, 17- and 6.8-fold more sensitive to nitrogen mustard, respectively, than their parent, wild-type, line as determined by the dose required to kill 90% of the cells, IC90. Patterns of cross-sensitivity to other DNA-damaging agents including ultraviolet light, cis-diamminedichloroplatinum, and other alkylating agents were determined for each line. Analysis of these results suggests that the phenotypes of the mutant lines are different from those lines reported previously.     

Isolation and preliminary characterization of bleomycin-resistant mutants from Chinese hamster ovary cells

Stable mutants resistant to an anticancer antibiotic, bleomycin-A2, were selected in Chinese hamster ovary (CHO) cell either spontaneously or after ethylmethane sulfonate mutagenesis. Fluctuation analysis showed that bleomycin resistance occurs in CHO at a rate of 6.50--6.58 x 10(-7) mutations per cell per generation. Bleomycin-A2-resistant cell lines exhibited increased resistance to bleomycin analogs--bleomycin-A5, -B2, -B4, and pepleomycin. Colchicine, mitomycin C, and ultraviolet light irradiation inhibited colony formation equally in CHO cells and in bleomycin-resistant mutants. Cell-cell hybridization tests showed that bleomycin-resistance behaves as a dominant trait. Bleomycin-inactivating activity in the mutant cell extracts was three to fourfold higher than that in extracts of the parental CHO cell.     

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4‐O‐Bu₃ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4‐O‐Bu₃ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ～20% decrease in maximal viable cell density and ～30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4‐O‐Bu₃ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4‐O‐Bu₃ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up‐regulated more in sodium butyrate treated wild‐type, erythropoietin, or IgG expressing CHO‐K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4‐O‐Bu₃ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4‐O‐Bu₃ManNAc‐treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4‐O‐Bu₃ManNAc‐treatment. These findings demonstrate that 1,3,4‐O‐Bu₃ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4‐O‐Bu₃ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.

     

Hyperosmolarity enhances transient recombinant protein yield in Chinese hamster ovary cells

The effect of hyperosmolarity on transient recombinant protein production in Chinese hamster ovary (CHO) cells was investigated. Addition of 90 mM NaCl to the production medium ProCHO5 increased the volumetric yield of recombinant antibody up to 4-fold relative to transfection in ProCHO5 alone. Volumetric yields up to 50 mg l⁻¹ were achieved in a 6 day batch culture of 3 l. In addition, hyperosmolarity reduced cell growth and increased cell size. The addition of salt to cultures of transiently transfected CHO cells is a simple and cost-effective method to increase TGE yields in this host.