Embrace Molecule Complexity with Optimized Expression Technologies

Presented by: Anil Sebastian, group leader, cell culture development; and Joachim Klein, head of microbial strain development and cell banking, Lonza Pharma & Biotech

Anil Sebastian, group leader, cell culture development, Lonza Pharma & Biotech

Although monoclonal antibodies (MAbs) remain the most popular modality in the biopharmaceutical industry, new molecular formats (NMFs) such as multitoxin antibody–drug conjugates and multichain antibodies are gaining ground quickly. Such molecules are imposing new demands on gene-expression technologies.

Joachim Klein, head of microbial strain development and cell banking, Lonza Pharma & Biotech

Sebastian and Klein described how their company’s solutions support upstream development for therapies based on NMFs. Sebastian highlighted the GS Xceed system for mammalian-cell production of complex antibodies. He explained that multichain antibody products require expression vectors that can introduce multiple genes of interest (GoI) to host cells. GS pXC expression technologies are designed to facilitate assembly of multigene vectors (MGVs). To create an MGV, each GoI is introduced into a GS pXC system. The resulting constructs are combined using a proprietary “one-vessel assembly reaction.” Currently, the vectors can accommodate three or four GoIs.

Because GoI arrangement in an MGV can influence product assembly and quality, early product-quality assessment should be performed to optimize vector designs. Sebastian showed the utility of such studies by explaining how Lonza constructed a vector for production of a protein with a common light chain and two different heavy chains. Lonza constructed two MGVs with the same GoIs but in different arrangements. During initial screening, the vectors assembled the target with 89% and 100% success, respectively. The higher-performing vector was introduced to GS Xceed Chinese hamster ovary (CHO) K1SV GS-KO host cells and cultured in stable pools for eight days. Supernatant was collected and analyzed by mass spectrometry, which confirmed the presence of the target’s heterodimers and detected low levels of mispaired variants. Using that MGV ultimately improved protein-product yields, Sebastian noted.

Sebastian emphasized that although early upstream activities follow a systematic pathway, NMF complexity can compel development of bespoke assays that ensure appropriate assembly of target proteins. Thus, understanding of NMF quality attributes is critical to upstream success.

Klein explained that NMF proliferation has prompted Lonza to enhance its Escherichia coli and Pichia pastoris platforms and expand its toolbox of promoters, signal sequences, and helper factors. Lonza also has implemented combinatorial screening for both platforms to increase the predictability of NMF production. Testing begins with gene optimization and determination of a suitable host and inducer. Primary screening involves cloning of cells in 24- or 96-well plates. Selected clones advance to secondary studies performed in fed-batch cultures.

Klein demonstrated that screening process by describing how Lonza selected a host for production of a single-domain antibody-like molecule. Prescreening assessment determined that P. pastoris was well suited to producing the target. Thus, primary screening focused on whether to use a G1-3 glucose-regulated or an AOX1 methanol-inducible strain. In stable pools, the AOX1 platform outperformed the G1-3 system. But after 120 hours of fermentation in fed-batch cultures, G1-3 titers exceeded those from AOX1 cultures. The G1-3 system proved to be simpler and more scalable because it required fewer fermentation steps. It also enabled development of a 60-hour fed-batch culture, with titers >5 g/L after 48 hours. Such expediency is important because minimizing product retention time in the supernatant diminishes risks of undesirable protein-product modifications.

Klein also described how Lonza’s screening process enabled selection of an XS E. coli strain for production of a 200-kDa complex protein. Its developer had struggled to produce it in a soluble, properly folded form using an IPTG-inducible host. During primary screening, Lonza compared production of the target in two E. coli strains and using three different promoters. Lonza’s rhamnose-inducible system advanced to fed-batch cultures, during which it produced much more soluble protein than the customer’s host did, eliminating need for large-scale refolding. In this case, high plasmid stability enabled Lonza’s host to generate high titers of correctly folded proteins.