COMMUNICATION TO THE EDITOR
Multiple cell line sources are used as hosts to produce recombinant prophylactic and therapeutic proteins for human use. Recombinant CHO cell lines (Puck et al., 1958) remain a preferred host due to the reliability, robustness and maturity of the technology in generating clonally-derived cell line. Despite the high level of production, batch-to-batch consistency and robustness of a clonally-derived CHO cell line, the approach is challenging due to the burden of time and resources required for stable clone isolation, selection, and provision of tested cell banks for cGMP manufacturing. To overcome this challenge, we wanted to evaluate the possibility of using non-clonal stable CHO cell pools to expediate production of a monoclonal antibody (mAb) against SARS-CoV2, CC6.35, particularly through cGMP manufacturing of a single batch of material for a Phase 1 clinical trial. Since this approach has not been rigorously tested for scaled-up cGMP manufacturing and concerns remain that the cellular and genetic heterogeneity of non-clonal stable CHO cell pools may result in production variability and concomitant heterogeneous product qualities between batches, we present a case study wherein we used non-clonal qualified cell banks and platform processes to accelerate manufacturing of CC6.35 mAb. To do so, we used the novel transposon-based Leap-In Transposase® system (Rajendra et al., 2017; Rajendran et al., 2021) for the development of stable CHO cell lines. The codon-optimized DNA sequence encoding the amino acid sequence for the Heavy chain(HC) and Light chain(LC) of CC6.35 mAb along with corresponding signal peptide and the novel expression constructs based on the Leap-In transposon® system were designed and synthesized. These synthesized DNA constructs along with transposase mRNA (Rajendran et al., 2021; Wilson et al., 2007) were used to co-transfect HD-BIOP3 glutamine synthase (GS) knock-out CHO-K1 host cells. Two promoter components were used to generate two unique sets of CHO cell pools: one using the EF1 promoter and another using the CMV promoter. Post-transfection the recovery of these two CHO cell pools was performed; after the initial recovery phase, the positive CHO cell pools were selected by outgrowth in a glutamine-free formulation at 37°C in 5% CO2 and 70-80% relative humidity and expanded further before cryopreservation and generation of Research Cell Banks (RCBs). In order to estimate productivity of the CC6.35 antibody-expressing stable CHO cell pools, both cell pools were expanded in a small-scale cell culture based on fed-batch process. After 14-days, the harvested supernatant was measured for titer by binding to a Protein-A biosensor on the Octet® System.Table 1 shows the percent (%) viability and expression levels (in g/L) for both EF1-CC6.35 and CMV-CC6.35 stable CHO cell pools.
Based on the higher titer, the CMV-CC6.35 cryopreserved RCB vials were advanced for use in scale-up production. This (CMV-CC6.35) RCB was tested for safety and regulatory acceptance for producing the two pivotal lots of material: one for preclinical safety studies and another for Phase 1 clinical trial.
For producing material for pre-clinical safety study, the RCB vials were thawed and expanded using a seed-train to support a 200 L bioreactor. The key cell culture process indicators included cell growth, cell viability, metabolic profiles (for ammonia and lactate), bioreactor regulation profiles (for pH, glucose, osmolality and pCO2) and antibody titer. After 14 days, the cell culture supernatant was harvested, clarified, and subjected to a 3-column chromatography platform purification process (involving Protein-A column chromatography, Anion-Exchange Column Chromatography and a Cation-Exchange column chromatography) steps to generate purified Drug Substance (DS), formulated at 20 mg/mL in an antibody platform buffer as Drug Product (DP) for intra-venous administration.
For producing material for the Phase 1 clinical study, a similar upstream (cell culture) process strategy was employed except that in this case, the RCB vials were thawed and expanded using a seed-train to support a 2000 L bioreactor. After 14 days, the supernatant from the 2000 L bioreactor was harvested, clarified, and purified via the same 3-column chromatography platform process to generate purified Drug Substance (DS), formulated at 20 mg/mL in a histidine-based buffer as Drug Product (DP) for intra-venous (i.v.) administration.
Both Drug Substances and Drug Products (200L and 2000 L scales) were tested based on a proposed analytical panel and results compared to agreed acceptance criteria. The analytical panel, comprising of quantitative test (protein concentration and biological activity), qualitative tests (physico-chemical and microbiological purity) and compendial methods, was set based on prior knowledge on antibodies of similar structure, and after confirmation of their suitability for the purpose. In addition, a formal analytical comparability was performed to ensure that the two products, from 200L and 2000L scales, are ”essentially similar” despite the changes in their production scale and minor change in processes. The results of the analytical comparability study are reported in Table 2 .
In addition to results in Table 2 , Circular Dichroism (CD) analysis showed that both DS materials are comparable in terms of secondary and tertiary structures (data not shown). Thermal analysis (by Differential Scanning Fluorimetry) showed that both DS samples have similar thermal denaturation profiles with a temperature of onset (Tonset) at 60-61 °C with two inflection points (IP) for both samples, one at 67 °C and another at 78 °C (data not shown). Extensive characterization of glycans (glycan mapping) of the two DS materials showed that the various detected N-linked glycan species are comparable, except for some minor differences in relative distribution of the galactosylated, fucosylated and sialylated species (data not shown). Additionally, LC-MS analysis was performed on the two DS materials for intact mass and deglycosylated/reduced mass. Intact mass LC-MS analysis showed that the main proteoform for both non-cGMP DS batch and the cGMP DS materials was the intact molecule PyroQ-LC + HC[-K] coupled with FA2-FA2 glycans (data not shown). With respect to the expected mass of 150622.60 Dalton, the non-cGMP DS had an experimental mass of 150622.35 Dalton, whereas the cGMP DS had a mass of 150623.80 Dalton. Some other fragments were detected and identified in both samples, amongst which the most abundant was the LC-LC dimer (data not shown). After deglycosylation/reduction, the LC-MS analysis revealed that the two DS materials were overall comparable in terms of intact molecule, except for slightly higher levels of HC C-terminal truncation in the cGMP batch (data not shown). Finally, non-reducing peptide mapping by LC-MS/MS showed that the (nine) detected disulphide bridges were aligned in line with expected (canonical) ones.
In summary, we demonstrate that using non-clonal stable CHO cell pools and use of platform processes can expediate early clinical development of monoclonal antibodies during pandemic outbreaks of emerging infectious such as COVID-19. We show that the antibodies produced from these stable CHO pools at two large scales bioreactors were comparable using platform upstream and downstream processes, and through robust analytical tested they were deemed suitable for clinical use. Since clinical development timeline continue to thwart rapid evaluation of therapeutic and prophylactic interventions during pandemics and improvements in development span essential (Kelley, 2020), we believe that this approach of using non-clonal stable CHO cell pools, which enabled manufacturing of early clinical trial material within 4.5 months, is a feasible alternative for rapid cGMP manufacturing, and a means that can accelerate the pace of therapeutic and prophylactic protein evaluation in clinic.