Inn H. Yuk

Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.

Unveiling a Glycation Hot Spot in a Recombinant Humanized Monoclonal Antibody

Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.

Controlling glycation of recombinant antibody in fed‐batch cell cultures

Protein glycation is a non‐enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in‐house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post‐translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose—the only source of reducing sugar in our fed‐batch cultures—would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2‐L and 400‐L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second‐order chemical reaction that is first order with respect to both glucose and non‐glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture. Biotechnol. Bioeng. 2011;108: 2600–2610.

Culture temperature modulates aggregation of recombinant antibody in cho cells.

During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing. Strikingly, data analysis showed an inverse correlation of lower cell culture temperature producing higher aggregate levels. All cultures at 37°C exhibited ≤5% aggregates at harvest. Aggregate levels increased 4–12‐fold in 33°C cultures when compared to 37°C, with a corresponding 2–4‐fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37°C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 25–75% at 33°C versus 37°C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33°C for this cell line. Finally, we identified a 2–5‐fold increase in mAb aggregate formation at 33°C compared to 37°C cultures for three additional CHO cell lines. Taken together, our observations indicate that low culture temperature can increase antibody aggregate formation in CHO cells by increasing LC and HC transcripts coupled with limited ER machinery. Biotechnol. Bioeng. 2012;109: 125–136.

Effects of copper on CHO cells: Insights from gene expression analyses

Copper concentration can impact lactate metabolism in Chinese Hamster ovary (CHO) cells. In our previous study, a 20‐fold increase in initial copper concentration enabled CHO cultures to shift from net lactate production to net lactate consumption, and achieve higher cell growth and productivity. In this follow‐up study, we used transcriptomics to investigate the mechanism of action (MOA) of copper that mediates this beneficial metabolism shift. From microarray profiling (days 0–7), the number of differentially expressed genes increased considerably after the lactate shift (>day 3). To uncouple the effects of copper at early time points (days 0–3) from that of lactate per se (>day 3), and to validate microarray hits, we analyzed samples before the lactate shift by RNA‐Seq. Out of 6,398 overlapping genes analyzed by both transcriptomic methods, only the early growth response 1 gene—coding for a transcription factor that activates signaling pathways in response to environmental stimuli—satisfied the differential expression criteria (fold change ≥1.5; P < 0.05). Gene expression correlation and biological pathway analyses further confirmed that copper differences exerted minimal transcriptional impact on the CHO cultures before the lactate shift. By contrast, genes associated with hypoxia network and oxidative stress response were upregulated after the lactate shift. These upregulations should boost cell proliferation and survival, but do not account for the preceding shift in lactate metabolism. The findings here indicate that the primary MOA of copper that enabled the shift in lactate metabolism is not at the transcriptional level.

Effect of temperature, pH, dissolved oxygen, and hydrolysate on the formation of triple light chain antibodies in cell culture.

THIOMABs are recombinant antibodies with reactive cysteine residues used for forming THIOMAB–drug conjugates (TDCs). We recently reported a new impurity associated with THIOMABs: one of the engineered cysteines forms a disulfide bond with an extra light chain (LC) to generate a triple light chain antibody (3LC). In our previous investigations, increased LC expression increased 3LC levels, whereas increased glutathione (GSH) production decreased 3LC levels. In this work, on three stably transfected CHO cell lines, we investigated the effects of temperature, pH, dissolved oxygen (DO), and hydrolysate on 3LC formation during THIOMAB fed‐batch cell culture production. Although pH between 6.8 and 7.0 had no significant impact on 3LC formation, temperature at 35°C instead of 33 or 31°C generated the lowest 3LC values for two cell lines. The decreased 3LC level correlated with increased GSH production. We implemented a 35°C temperature process for large‐scale (2,000 L) production of a THIOMAB. This process reduced 3LC levels by ∼50% compared with a 33°C temperature process. By contrast, DO and hydrolysate had modest effect on 3LC levels for the model cell line studied. Overall, we did not find significant changes in LC expression under the conditions tested, whereas changes in GSH production were more evident. By investigating the impact of bioreactor process and medium conditions on 3LC levels, we identified strategies that reduced 3LC levels.

More similar than different: Host cell protein production using three null CHO cell lines

To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell‐specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time‐course profiles of HCP (as measured by multi‐analyte ELISA) and those of two intracellular HCP species, phospholipase B‐like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC‐MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to <20% and extracellular LDH levels increased several‐fold. Our findings indicate that the CHO‐derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>107 cells/mL)—operated in fed‐batch mode and exhibiting high viabilities (>70%) throughout the culture duration—can accumulate a considerable amount of immunogenic HCP (∼1–2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC‐MS/MS to overcome the limitations associated with ELISA and 2D‐PAGE for HCP analysis. Biotechnol. Bioeng. 2015;112: 2068–2083.

Effects of copper on CHO cells: cellular requirements and product quality considerations.

Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes.

Triple light chain antibodies: factors that influence its formation in cell culture.

THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species—Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression—evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC)—correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production—evaluated as the ratio of the cell‐specific production rate of GSH (qGSH) to the cell‐specific production rate of THIOMAB (qp)—corresponded to decreased 3LC levels. In time‐lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and qGSH/qp ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high qGSH/qp ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity. Biotechnol. Bioeng. 2010. 105: 748–760.

Overcoming challenges in WAVE bioreactors without feedback controls for pH and dissolved oxygen

The biopharmaceutical industry is increasing its use of the WAVE Bioreactor for culturing cells. Although this disposable bioreactor can be equipped to provide real‐time pH and dissolved oxygen (DO) monitoring and control, our goal was to develop a process for culturing CHO cells in this system without relying on pH and DO feedback controls. After identifying challenges in culturing cells without controlling for pH and DO in the WAVE Bioreactor, we characterized O2 and CO2 transfer in the system. From these cell‐free studies, we identified rock rate and rock angle as key parameters affecting O2 transfer. We also identified the concentration of CO2 in the incoming gas and the rate of gas flow into the headspace as key parameters affecting CO2 transfer—and therefore pH—in the disposable culture chamber. Using a full‐factorial design to evaluate the rock rate, rock angle, and gas flow rate defined for this WAVE Bioreactor process, we found comparable cell growth and pH profiles in the ranges tested for these three parameters in two CHO cell lines. This process supported cell growth, and maintained pH and DO within our desired range—pH 6.8–7.2 and DO exceeding 20% of air saturation—for six CHO cell lines, and it also demonstrated comparable cell growth and viability with the stirred‐tank bioreactor process with online pH and DO control. By eliminating the use of pH and DO probes, this process provides a simple and more cost‐effective method for culturing cells in the WAVE Bioreactor.