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Residual host cell proteins (HCPs) in the drug product can affect product quality, stability, and/or safety. In particular, highly active hydrolytic enzymes at sub-ppm levels can negatively impact the shelf life of drug products but are challenging to identify by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) due to their high dynamic range between HCPs and biotherapeutic proteins. We employed new strategies to address the challenge: (1) native digest at a high protein concentration; (2) sodium deoxycholate added during the reduction step to minimize the inadvertent omission of HCPs observed with native digestion; and (3) solid phase extraction with 50% MeCN elution prior to LC-MS/MS analysis to ensure effective mAb removal. A 50 cm long nanoflow charged surface hybrid column was also packed to allow for higher sample load for increased sensitivity. Our workflow has increased the sensitivity for HCP identification by 10- to 100-fold over previous reports and showed the robustness as low as 0.1 ppm for identifying HCPs (34.5 to 66.2 kDa MW). The method capability was further confirmed by consistently identifying85% of 48 UPS-1 proteins (0.10 to 1.34 ppm, 6.3 to 82.9 kDa MW) in a monoclonal antibody (mAb) and the largest number (746) of mouse proteins from NIST mAb reported to date by a single analysis. Our work has filled a significant gap in HCP analysis for detecting and demonstrating HCP clearance, in particular, extremely low-level hydrolases in drug process development.

Chinese hamster ovary (CHO) cell engineering based on CRISPR/Cas9 knockout (KO) technology requires the delivery of guide RNA (gRNA) and Cas9 enzyme for efficient gene targeting. With an ever-increasing list of promising gene targets, developing and optimizing a multiplex gene KO protocol is crucial for rapid CHO cell engineering. Here, we describe a method that can support efficient targeting and KO of up to ten genes through sequential transfections. This method utilizes Cas9 protein to first screen multiple synthetic gRNAs per gene, followed by Sanger sequencing indel analysis, to identify effective gRNA sequences. Using sequential transfections of these potent gRNAs led to the isolation of single cell clones with the targeted deletion of all ten genes (as confirmed by Sanger sequencing at the DNA level and mass spectrometry at the protein level). Screening 704 single cell clones yielded 6 clones in which all 10 genes were deleted through sequential transfections, demonstrating the success of this decaplex gene editing strategy. This pragmatic approach substantially reduces the time and effort required to generate multiple gene knockouts in CHO cells. This article is protected by copyright. All rights reserved.

Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO-based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell-specific productivity (Qp ) over time for recombinant CHO cells developed using the glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze-thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze-thaw did not result in any significant impact beyond the initial post-thaw passages. Production cultures sourced from cryopreserved cells and their non-cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Qp at increased cell age: 17 of the 24 cell lines displayed ≥20% Qp decline after ∼2-3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Qp decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze-thaw as a cause for Qp decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463-477, 2018.

The extent of afucosylation, which refers to the absence of core fucose on Fc glycans, can correlate positively with the antibody-dependent cellular cytotoxicity (ADCC) activity of a monoclonal antibody (mAb). Therefore, it is important to maintain consistent afucosylation during cell culture process scale-up in bioreactors for a mAb with ADCC activity. However, there is currently a lack of understanding about the impact of partial pressure of carbon dioxide (pCO2 )-a parameter that can vary with bioreactor scale-on afucosylation. Using a small-scale (3 L) bioreactor model that can modulate pCO 2 levels through modified configurations and gassing strategies, we identified three cell culture process parameters that influence afucosylation of a mAb produced by a recombinant Chinese Hamster Ovary (CHO) cell line: pCO 2 , media hold duration (at 37°C), and manganese. These three-independent parameters demonstrated a synergistic effect on mAb afucosylation; increase in pCO 2 , media hold duration, and manganese consistently increased afucosylation. Our investigations into the underlying mechanisms through proteomic analysis indicated that the synergistic interactions downregulated pathways related to guanosine diphosphate-fucose synthesis and fucosylation, and upregulated manganese transport into the CHO cells. These new findings highlight the importance of considering potential differences in culture environment and operations across bioreactor scales, and understanding the impact of their interactions on product quality.

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.

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.

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 O(2) and CO(2) transfer in the system. From these cell-free studies, we identified rock rate and rock angle as key parameters affecting O(2) transfer. We also identified the concentration of CO(2) in the incoming gas and the rate of gas flow into the headspace as key parameters affecting CO(2) 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.

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.

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% to20% 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 (10(7) 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.

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.

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.

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 (q(GSH)) to the cell-specific production rate of THIOMAB (q(p))--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 q(GSH)/q(p) ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high q(GSH)/q(p) 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.