FUJIFILM Diosynth Biotechnologies UK Ltd.

Chinese hamster ovary (CHO) cells are the industrial workhorse for manufacturing biopharmaceuticals, including monoclonal antibodies. CHO cell line development requires a more data-driven approach for the accelerated identification of hyperproductive cell lines. Traditional methods, which rely on time-consuming hierarchical screening, often fail to elucidate the underlying cellular mechanisms driving optimal bioreactor performance. Big data analytics, coupled with advancements in "omics" technologies, are revolutionizing the study of industrial cell lines. Translating this knowledge into practical methods widely utilized in industrial biomanufacturing remains a significant challenge. This study leverages discovery proteomics to characterize dynamic changes within the CHO cell proteome during a 14-day fed-batch bioreactor cultivation. Utilizing a global untargeted proteomics workflow on both a ZenoTOF 7600 and a Cyclic IMS QToF, we identify 3358 proteins and present a comprehensive data set that describes the molecular changes that occur within a well-characterized host chassis. By mapping relative abundances to key cellular processes, eight protein targets were selected as potential biomarkers. The abundance of these proteins through the production run is quantified using a 15-min targeted triple quadrupole (MRM) assay, which provides a molecular-level QC for cell viability. This discovery to target workflow has the potential to assist engineering of new chassis and provide simple readouts of successful bioreactor batches.

Antibodies are valuable biological reagents used in a wide range of discovery research, biotechnology, diagnostic and therapeutic applications. Currently, both commercial and laboratory-scale antibody production is reliant on expression from mammalian cells, which can be time-consuming and requires the use of specialist facilities and costly growth reagents. Here, we describe a simple, rapid and cheap method for producing and isolating functional monoclonal antibodies and antibody fragments from bacterial cells that can be used in a range of laboratory applications. This simple method only requires access to basic microbial cell culture and molecular biology equipment, making scalable in-house antibody production accessible to the global diagnostics, therapeutics and molecular bioscience research communities.

3D printing offers the unprecedented ability to fabricate chromatography stationary phases with bespoke 3D morphology as opposed to traditional packed beds of spherical beads. The restricted range of printable materials compatible with chromatography is considered a setback for its industrial implementation. Recently, we proposed a novel ink that exhibits favourable printing performance (printing time ∼100 mL/h, resolution ∼200 µm) and broadens the possibilities for a range of chromatography applications thanks to its customisable surface chemistry. In this work, this ink was used to fabricate 3D printed ordered columns with 300 µm channels for the capture and polishing of therapeutic monoclonal antibodies. The columns were initially assessed for leachables and extractables, revealing no material propensity for leaching. Columns were then functionalised with protein A and SO₃ ligands to obtain affinity and strong cation exchangers, respectively. 3D printed protein A columns showed >85 % IgG recovery from harvested cell culture fluid with purities above 98 %. Column reusability was evaluated over 20 cycles showing unaffected performance. Eluate samples were analysed for co-eluted protein A fragments, host cell protein and aggregates. Results demonstrate excellent HCP clearance (logarithmic reduction value of > 2.5) and protein A leakage in the range of commercial affinity resins (<100 ng/mg). SO₃ functionalised columns employed for polishing achieved removal of leaked Protein A (down to 10 ng/mg) to meet regulatory expectations of product purity. This work is the first implementation of 3D printed columns for mAb purification and provides strong evidence for their potential in industrial bioseparations.