Improving product quality and productivity of bispecific molecules through the application of continuous perfusion principles.

Bispecific protein scaffolds can be more complex than traditional monoclonal antibodies (MAbs) because two different sites/domains for epitope binding are needed. Because of this increased molecular complexity, bispecific molecules are difficult to express and can be more prone to physical and chemical degradation compared to MAbs, leading to higher levels of protein aggregates, clipped species, or modified residues in cell culture. In this study, we investigated cell culture performance for the production of three types of bispecific molecules developed at Amgen. In particular, we cultured a total of six CHO cell lines in both an approximately 12-day fed-batch process and an approximately 40-day high-density perfusion process. Harvested cell culture fluid from each process was purified and analyzed for product quality attributes including aggregate levels, clipped species, charge variants, individual amino acid modifications and host cell protein (HCP) content. Our studies showed that in average, the intensified perfusion process increased 15-fold the integrated viable cell density and the total harvested product (and fivefold the daily volumetric productivity) compared to fed-batch. Furthermore, bispecific product quality improved in perfusion culture (as analyzed in affinity-capture pools) with reduction in levels of aggregates (up to 72% decrease), clipped species (up to 75% decrease), acidic variants (up to 76% decrease), deamidated/isomerized species in complementarity-determining regions, and HCP (up to 84% decrease). In summary, the intensified perfusion process exhibited better productivity and product quality, highlighting the potential to use it as part of a continuous manufacturing process for bispecific scaffolds.

Perfusion CHO cell culture applied to lower aggregation and increase volumetric productivity for a bispecific recombinant protein.

Secreted recombinant proteins can aggregate during cell culture. We studied a poorly-behaved bispecific scaffold that increasingly aggregated (up to 62% high molecular weight species, HMW) as a function of culture time in a fed-batch and intensified cell culture processes. We identified that protein aggregates increased with accumulated protein concentration inside the bioreactor. Furthermore, results indicated that a maximum product concentration was reached beyond which no additional soluble protein accumulated in culture even when doubling the integrated viable cell density with the intensified process, suggesting additional secreted protein was precipitating. To overcome this limitation and maintain the cell-specific productivity (qp) in culture, we explored a perfusion process where recombinant protein was continuously removed from the bioreactor to maintain low product concentration and consequently, minimize protein aggregation. We studied different viable cell densities (VCDs) inside the bioreactor (one to five-fold) and found a corresponding two-fold modulation of monomer levels. In all VCD conditions, qp was maintained. On the contrary, the previous intensified process showed an "apparent" 2.5-fold decrease in qp at the end of culture because of the presumed limited protein solubility at higher concentrations. The combination of lower aggregate levels and constant qp resulted in up to four to five-fold increase in recoverable product (i.e., monomer) with the improved perfusion process.

A novel mammalian cell line development platform utilizing nanofluidics and optoelectro positioning technology.

Generating a highly productive cell line is resource intensive and typically involves long timelines because of the need to screen large numbers of candidates in protein production studies. This has led to miniaturization and automation strategies to allow for reductions in resources and higher throughput. Current approaches rely on the use of standard cell culture vessels and bulky liquid handling equipment. New nanofludic technologies offer novel solutions to surpass these limits, further miniaturizing cell culture volumes (105 times smaller) by growing cells on custom nanofluidic chips. Berkeley Lights' OptoElectro Positioning technology projects light patterns to activate photoconductors that gently repel cells to manipulate single cells on nanofluidic culturing chips. Using a fully integrated technology platform (Beacon), common cell culture tasks can be programmed through software, allowing maintenance and analysis of thousands of cell lines in parallel on a single chip. Here, we describe the ability to perform key cell line development work on the Beacon platform. We demonstrate that commercial production Chinese hamster ovary cell lines can be isolated, cultured, screened, and exported at high efficiency. We compare this process head to head with a FACS-enabled microtiter plate-based workflow and demonstrate generation of comparable clonal cell lines with reduced resources. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1438-1446, 2018.

Analysis of Tubespins as a suitable scale-down model of bioreactors for high cell density CHO cell culture.

High cell density (HCD) culture increases recombinant protein productivity via higher biomass. Compared to traditional fed-batch cultures, HCD is achieved by increased nutrient availability and removal of undesired metabolic components via regular medium replenishment. HCD process development is usually performed in instrumented lab-scale bioreactors (BR) that require time and labor for setup and operation. To potentially minimize resources and cost during HCD experiments, we evaluated a 2-week 50-mL Tubespin (TS) simulated HCD process where daily medium exchanges mimic the medium replacement rate in BR. To best assess performance differences, we cultured 13 different CHO cell lines in simulated HCD as satellites from simultaneous BR, and compared growth, metabolism, productivity and product quality. Overall, viability, cell-specific productivity and metabolism in TS were comparable to BR, but TS cell growth and final titer were lower by 25 and 15% in average, respectively. Peak viable cell densities were lower in TS than BR as a potential consequence of lower pH, different medium exchange strategy and dissolved oxygen limitations. Product quality attributes highly dependent on intrinsic molecule or cell line characteristics (e.g., galactosylation, afucosylation, aggregation) were comparable in both scales. However, product quality attributes that can change extracellularly as a function of incubation time (e.g., deamidation, C-terminal lysine, fragmentation) were in general lower in TS because of shorter residence time than HCD BR. Our characterization results and two case studies show that TS-simulated HCD cultures can be effectively used as a simple scale-down model for relative comparisons among cell lines for growth or productivity (e.g., clone screening), and for investigating effects on protein galactosylation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:490-499, 2017.