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

Abstract

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.

Biomanufacturing evolution from conventional to intensified processes for productivity improvement: a case study

“…6 To reduce the manufacturing cost, which would allow wider patient access, process intensification through continuous or semi-continuous biomanufacturing has been extensively studied in both academia and industry. [7][8][9][10][11] Process intensification is defined as the adoption of innovative technologies and methods to achieve major process improvements (e.g., productivity), which significantly reduce manufacturing costs and facility footprints. In the chemical industry, process intensification has successfully been implemented for effective manufacturing of commercial commodities with dramatic reductions in costs and plant footprints over the last several decades.…”

“…6 To reduce the manufacturing cost, which would allow wider patient access, process intensification through continuous or semi-continuous biomanufacturing has been extensively studied in both academia and industry. 7 - 11 …”

Impact of next-generation high productivity perfusion cell culture process on host cell protein profile and a comparison with fed-batch cultures.

“…With perfusion processes, the abundance acidic species is significantly reduced to 13.6% for SS and 15.8% for NSS while more basic forms are produced to 14.6% for SS and 13.4% for NSS compared to FB. Other mAbs also exhibited similar trends in charged variants with perfusion processes due to the increase Cterminal lysine and/or less light chain N-terminal glutamine cyclization (Gomez et al, 2020;Walther et al, 2019). The general consensus according to a number of scientific publications and various conventions is that C-terminal lysine and N-terminal glutamine cyclization, are likely non-CQAs (critical quality attributes), which may not have substantial effects on antigen binding affinity, efficacy and safety of the antibody product (Du et al, 2012;Sing h et al, 2016).…”

Microfluidic chip‐based single‐cell cloning to accelerate biologic production timelines

“…Cation exchange chromatography (CEX) (YMC America Inc, Allentown, PA) was performed to evaluate distribution of charge variants. Methods followed were previously reported 18,21,22 . In short, for SEC, components were eluted isocratically and monitored by UV detection at 220 or 280 nm.…”

“…Methods followed were previously reported. 18,21,22 In short, for SEC, components were eluted isocratically and monitored by UV detection at 220 or 280 nm. Three injections of a universal reference standard were included at the front and end of a sample sequence and one product-specific control injection.…”