Probing of C-terminal lysine variation in a recombinant monoclonal antibody production using Chinese hamster ovary cells with chemically defined media

C-terminal lysine (C-K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C-K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch-to-batch culture performance and product quality reproducibility is a key cell culture development criterion. Understanding the operational parameters affecting C-K levels provides valuable insight into the cell culture process. A CHO cell line X expressing a recombinant antibody was selected as the model cell line due to the exhibited sensitivity of its C-K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C-K level for in-process samples. The effects of operating conditions (i.e., temperature and culture duration) and media trace elements (copper and zinc) on C-K variants were studied. The dominant effect on C-K level was identified as the trace elements concentration. Specifically, increased C-K levels were observed with increase of copper concentration and decrease of zinc concentration in chemically defined medium. Further, a hypothesis for C-K processing with intracellular and extracellular carboxypeptidase activity was proposed, based on preliminary intracellular carboxypeptidase Western blot results and the extracellular HCCF holding study.     

Early prediction of instability of Chinese hamster ovary cell lines expressing recombinant antibodies and antibody-fusion proteins

One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster ovary (CHO) derived production cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a cell line expressing an antibody or antibody-fusion protein declined by 20-30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of cells as detected with flow cytometric analysis of intracellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host cell line from which the candidate production cell lines were derived was apoptotic-resistant. This data suggested that unstable cell lines were more prone to apoptosis, which was confirmed by the fact that unstable cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production cell lines at an early stage of the cell line development process, potentially reducing the cost of biotherapeutic development.     

Identifying bottlenecks in transient and stable production of recombinant monoclonal-antibody sequence variants in chinese hamster ovary cells

The increasing demand for antibody-based therapeutics has emphasized the need for technologies to improve recombinant antibody titers from mammalian cell lines. Moreover, as antibody therapeutics address an increasing spectrum of indications, interest has increased in antibody engineering to improve affinity and biological activity. However, the cellular mechanisms that dictate expression and the relationships between antibody sequence and expression level remain poorly understood. Fundamental understanding of how mammalian cells handle high levels of transgene expression and of the relationship between sequence and expression are vital to the development of new antibodies and for increasing recombinant antibody titers. In this work, we analyzed a pair of mutants that vary by a single amino acid at Kabat position 49 (heavy-chain framework), resulting in differential transient and stable titers with no apparent loss of antigen affinity. Through analysis of mRNA, gene copy number, intracellular antibody content, and secreted antibody, we found that while translational/post-translational mechanisms are limiting in transient systems, it appears that the amount of available transgenic mRNA becomes the limiting event on stable integration of the recombinant genes. We also show that amino acid substitution at residue 49 results in production of a non-secreted HC variant and postulate that stable antibody expression is maintained at a level which prevents toxic accumulation of this HC-related protein. This study highlights the need for proper sequence engineering strategies when developing therapeutic antibodies and alludes to the early analysis of transient expression systems to identify the potential for aberrant stable expression behavior.     

不同方向内含子对重组CHO细胞中神经生长因子表达的影响

为了研究不同方向的嵌合体内含子对重组神经生长因子 (Nerve growth factor,NGF) 基因表达的影响,以人β-珠蛋白第一内含子5?端剪接序列和人免疫球蛋白重链可变区内含子3?端剪接序列组合而成的嵌合体内含子作为研究对象,在NGF基因5?端插入不同方向的嵌合体内含子,构建含不同方向内含子的NGF基因表达载体。转染至CHO细胞后,G418筛选稳定转染的细胞,荧光定量PCR、ELISA和Western blotting检测不同载体NGF基因的表达情况。结果显示内含子可以大幅度提高NGF基因的表达,且正向内含子对NGF基因表达的增强作用无论是在mRNA水平还是在蛋白水平都要高于反向内含子。所以内含子能够提高外源NGF基因的表达,且内含子调控转基因表达具有方向性。     

A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull‐down assay was performed with dual‐tagged (N‐terminal GST‐ and C‐terminal hexahistidine‐tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual‐tagged EPO were then resolved by two‐dimensional gel electrophoresis (2DE) and identified by MALDI‐TOF MS/MS. A total of 27 protein spots including glucose‐regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull‐down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Enhancement of erythropoietin production in recombinant Chinese hamster ovary cells by sodium lactate addition

The stabilization of optimum pH for cells can cause a higher erythropoietin (EPO) production rate and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the optimum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM sodium lactate was added, a specific growth rate was decreased by approximately 22% as compared with the control culture. However the culture longevity was extended to 187 h, and more than a 2.7-fold increase in a final accumulated EPO concentration was obtained at 40 mM of sodium lactate. On the condition that caused the high production of EPO, a specific glucose consumption rate and lactate production rate decreased by 23.3 and 52%, respectively. Activity of lactate dehydrogenase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate, together with the reverse reaction, at the addition of 40 mM sodium lactate. The addition of 40 mM sodium lactate caused the positive effects on a cell growth and an EPO production in the absence of carbon dioxide gas as well as in the presence of carbon dioxide gas by reducing the accumulation of lactate.     

Enhanced cell culture performance using inducible anti-apoptotic genes E1B-19K and Aven in the production of a monoclonal antibody with Chinese hamster ovary cells

Mammalian cells are used for the production of numerous biologics including monoclonal antibodies. Unfortunately, mammalian cells can lose viability at later stages in the cell culture process. In this study, the effects of expressing the anti-apoptosis genes, E1B-19K and Aven, separately and in combination on cell growth, survival, and monoclonal antibody (MAb) production were investigated for a commercial Chinese Hamster Ovary (CHO) mammalian cell line. CHO cells were observed to undergo apoptosis following a model insult, glucose deprivation, and at later stages of batch cell culture. The CHO cell line was then genetically modified to express the anti-apoptotic proteins E1B-19K and/or Aven using an ecdysone-inducible expression system. Stable transfected pools induced to express Aven or E1B-19K alone were found to survive 1-2 days longer than the parent cell line following glucose deprivation while the expression of both genes in concert increased cell survival by 3 days. In spinner flask batch studies, a clonal isolate engineered to express both anti-apoptosis genes exhibited a longer operating lifetime and higher final MAb titer as a result of higher viable cell densities and viabilities. Interestingly, survival was increased in the absence of an inducer, most likely as a result of leaky expression of the anti-apoptosis genes confirmed in subsequent PCR studies. In fed-batch bioreactors, the expression of both anti-apoptosis genes resulted in higher growth rates and cell densities in the exponential phase and significantly higher viable cell densities, viabilities, and extended survival during the post-exponential phase. As a result, the integral of viable cells (IVC) was between 40 and 100% higher for cell lines engineered to express both Aven and E1B-19K in concert, and the operational lifetime of the fed-batch bioreactors was increased from 2 to 5 days. The maximum titers of MAb were also increased by 40-55% for bioreactors containing cells expressing Aven and E1B-19K. These increases in volumetric productivity arose primarily from enhancements in viable cell density over the course of the fed-batch culture period since the specific productivities for the cells expressing anti-apoptosis genes were comparable or slightly lower than the parental hosts. These results demonstrate that expression of anti-apoptosis genes can enhance culture performance and increase MAb titers for mammalian CHO cell cultures especially under conditions such as extended fed-batch bioreactor operation.     

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)‐enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND‐enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies—mAb1, mAb2, and mAb3—at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND‐enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449–1455, 2017     

Bcl-x(L) mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells

Enhanced product yields, reduction in throughput time, improved cost-effectiveness and product quality are examples of benefits gained by delaying apoptotic cell death in bioreactors. To examine the effect on recombinant protein production, bcl-x(L) was overexpressed in a CHO cell line secreting humanized monoclonal antibody directed against the alpha1beta1 integrin. When cell lines overexpressing bcl-x(L) were compared to the parent, cell viability was increased by 20% and titers by 80%. Total viable cell densities were similar and specific productivities were enhanced by almost two-fold on scale-up to bioreactors. Comparison in a chemically defined media demonstrated an even greater sustained viability in bcl-x(L) expressing cells by 50% and up to 90% increase in titer with no impact on product quality. Caspase 3 activities were monitored as a marker for apoptotic cell death. In the presence of Bcl-x(L), caspase activities were reduced to background levels. The role of Bcl-x(L) in protecting cells from premature death was further examined in studies performed in the presence of NaBu, at concentrations known to trigger cell death. Results demonstrated that cells expressing bcl-x(L) retained 88% cell viability with >2 fold increase in titer. Bcl-x(L) was similarly overexpressed in a different CHO cell line producing a humanized mAb against the chemokine MCP1. Once again, production titer was increased by 80% and viability by 75%. Together the studies have shown that overexpression of bcl-x(L) in production cell lines was able to significantly increase the titer by enhancing both the specific activity and total cell viability while maintaining product quality.     

Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3

Sodium butyrate (NaBu) can enhance the expression of genes controlled by some of the mammalian promoters, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, the expression vector of antisense RNA of caspase-3 was constructed and transfected to recombinant Chinese hamster ovary (rCHO) cells producing a humanized antibody. Using this antisense RNA strategy, rCHO cells (B3) producing a low level of caspase-3 proenzyme were established. When batch cultures of both B3 cells and control cells transfected with antisense RNA-deficient plasmid were performed in the absence of NaBu, both cells showed similar profiles of cell growth and antibody production. Compared with control cell culture, under the condition of 5 mM NaBu addition at the exponential growth phase, expression of antisense RNA of caspase-3 significantly suppressed the NaBu-induced apoptosis of B3 cells and extended culture longevity by >2 days if the culture was terminated at cell viability of 50%. However, compared with control cell culture, the final antibody concentration of B3 cell culture was not increased in the presence of NaBu, which may be due to the loss of cellular metabolic capability resulted from the depolarization of mitochondrial membrane. Taken together, this study suggests that, although expression of antisense RNA of caspase-3 does not improve antibody productivity of rCHO cells, it can suppress NaBu-induced apoptotic cell death of rCHO cells and thereby may reduce problems associated with cellular disintegration.     

Predicting the expression of recombinant monoclonal antibodies in Chinese hamster ovary cells based on sequence features of the CDR3 domain

Despite the development of high‐titer bioprocesses capable of producing >10 g L^?1 of recombinant monoclonal antibody (MAb), some so called “difficult‐to‐express” (DTE) MAbs only reach much lower process titers. For widely utilized “platform” processes the only discrete variable is the protein coding sequence of the recombinant product. However, there has been little systematic study to identify the sequence parameters that affect expression. This information is vital, as it would allow us to rationally design genetic sequence and engineering strategies for optimal bioprocessing. We have therefore developed a new computational tool that enables prediction of MAb titer in Chinese hamster ovary (CHO) cells based on the recombinant coding sequence of the expressed MAb. Model construction utilized a panel of MAbs, which following a 10‐day fed‐batch transient production process varied in titer 5.6‐fold, allowing analysis of the sequence features that impact expression over a range of high and low MAb productivity. The model identified 18 light chain (LC)‐specific sequence features within complementarity determining region 3 (CDR3) capable of predicting MAb titer with a root mean square error of 0.585 relative expression units. Furthermore, we identify that CDR3 variation influences the rate of LC‐HC dimerization during MAb synthesis, which could be exploited to improve the production of DTE MAb variants via increasing the transfected LC:HC gene ratio. Taken together these data suggest that engineering intervention strategies to improve the expression of DTE recombinant products can be rationally implemented based on an identification of the sequence motifs that render a recombinant product DTE. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:188–197, 2014     

Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA

We explored the possibility of converting established antibody-producing cells to cells producing high antibody-dependent cellular cytotoxicity (ADCC) antibodies. The conversion was made by constitutive expression of small interfering RNA (siRNA) against alpha1,6 fucosyltransferase (FUT8). We found two effective siRNAs, which reduce FUT8 mRNA expression to 20% when introduced into Chinese hamster ovary (CHO)/DG44 cells. Selection for Lens culinaris agglutinin (LCA)-resistant clones after introduction of the FUT8 siRNA expression plasmids yields clones producing highly defucosylated (approximately 60%) antibody with over 100-fold higher ADCC compared to antibody produced by the parental cells (approximately 10% defucosylated). Moreover, the selected clones remain stable, producing defucosylated antibody even in serum-free fed-batch culture. Our results demonstrate that constitutive FUT8 siRNA expression can control the oligosaccharide structure of recombinant antibody produced by CHO cells to yield antibodies with dramatically enhanced ADCC.     

Effect of chemical chaperone addition on production and aggregation of recombinant flag-tagged COMP-angiopoietin 1 in Chinese hamster ovary cells

To investigate the effect of chemical chaperones on the production and aggregation of flag-tagged cartilage oligomeric matrix protein-Angiopoietin1 (FCA1) in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in serum-free media with various chemical chaperones, 1 mM 4-phenylbutyrate (4-PBA), 200 mM proline, 3% glycerol, 2% dimethyl sulfoxide (DMSO), and without chemical chaperone as control. The addition of chemical chaperones enhanced FCA1 production and specific FCA1 productivity, q(FCA)(1). For example, the q(FCA)(1) at 200 mM proline was fourfold higher than that at control. Unlike q(FCA)(1), the aggregation of FCA1 was strongly affected by which chemical chaperone was added. The addition of 2% DMSO and 200 mM proline significantly reduced the proportion of aggregates, but the addition of 1 mM 4-PBA and 3% glycerol was hardly effective. The proportions of aggregates were 29.5 and 55.6% at 2% DMSO and 200 mM proline, respectively, whereas it was 79.6% at control. The exact mechanism how chemical chaperones affected the aggregation of FCA1 was not investigated in this study, and therefore, more extensive works will be needed to clarify why different chemical chaperones behaved differently in reducing the aggregation of FCA1. Among chemical chaperones tested, DMSO was the most effective one in regard to enhancing the production and reducing the aggregation of FCA1 in CHO cells.     

Effects of phosphatidic acid on recombinant protein production by Chinese hamster ovary cells in serum-free culture

When recombinant Chinese hamster ovary (CHO) cells (ATCC CRL-8200) producing human interferon-γ (hIFN-γ) were incubated with exogenously supplied phosphatidic acid (PA) from egg yolk lecithin in a serum-free medium, PA induced increases in both the density of viable cells and concentration of hIFN-γ secreted into the medium. Dispersion of PA with a non-ionic surfactant, Tween 80, further enhanced both cell growth and hIFN-γ production. Replacement of the culture medium containing PA by fresh medium without PA in the course of a static culture did not influence cell growth indicating that PA is required to be continuously present in a serum-free medium to stimulate cell growth. Using a fresh medium containing PA for replacement resulted in significant enhancement of both cell density and hIFN-γ yield. These results suggest that PA is a promising constituent of low-protein serum-free media for the effective production of recombinant proteins.     

Violacein improves recombinant IgG production by controlling the cell cycle of Chinese hamster ovary cells

Cytotechnology - Chinese hamster ovary (CHO) cells are used as host cells for industrial monoclonal antibody (mAb) production. Cell cycle control is an effective approach to increase mAb production...