Industry Review of Cell Separation and Product Harvesting Methods

Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

“…A secretion of the mAb product for purification is suboptimal for several lower organism expression platforms such as E. coli due to poor productivity and harsh culture conditions that promote product degradation. Protein is therefore produced intracellularly, as inclusion bodies and harvest involves further processing steps such as cell lysis, inclusion body recovery, protein solubilization, and renaturation prior to further downstream purification steps [19,20]. Despite these pitfalls, the development of lower organism expression systems is of high commercial interest due to the simplified culture conditions, cheaper media requirements, rapid organism growth, and higher product yield as compared to mammalian expression systems [16,21].…”

“…Established lower organism expression systems for fragment or recombinant fusion mAb therapeutics include bacteria such as E. coli , yeasts such as S. cerevisiae and P. pastoris , plants such as tobacco, algae, and insects such as silkworm [5,16,52,53,54]. Expression platforms that use E. coli in particular are considered high risk due to the potential endotoxin contamination in the mAb product, of which complete endotoxin removal requires further purification steps [19]. However, several approved mAb fragment- and recombinant-based therapies are produced in an E. coli -based expression system, including pegol conjugated Fab’ certolizumab pegol (Cimzia ® , Celltech UCB, Brussels, Belgium), Fab ranibizumab (Lucentis ® , Genentech, San Francisco, CA, USA), and recombinant Fc fusion romiplostim (Nplate ® , Amgen, Thousand Oaks, CA, USA).…”

“…In perfusion and continuous culture systems, a feed is removed from the culture simultaneously to the media addition, the difference being that the cell dilution rate in continuous culture is optimized to remain equal or higher than the cell growth rate, which allows the perpetuation of mAb expression and requires the continuous harvest of the supernatant [92,93,94]. The harvest of the supernatant requires clarification technologies such as the use of precipitants/flocculants (e.g., polyethylene glycol, diethylaminoethyl dextran, caprylic acid, and polyethylenimine), high throughput centrifugation, and filtration methods to remove cells and biological debris, for the lowering the loading burden in the lead up to mAb capture through affinity chromatography [19,95,96,97,98,99,100]. The mechanical stresses from centrifugation and filtration are unavoidable, although they can induce mild mAb product loss through fragmentation and aggregation.…”

Process analytical technology implementation for protein refolding: GCSF as a case study

“…Conventionally, the outcome of refolding is only assessed after the completion of the refolding process by testing the refolded samples using reversed‐phase high‐performance liquid chromatography (RP‐HPLC; Pathak, Dixit, Muthukumar, & Rathore, ; Pieracci, Armando, Westoby, & Thommes, ). In industry, refolding is typically performed using batch dilution at protein concentrations as low as <0.1 mg/ml (Eiberle & Jungbauer, ).…”

“…samples using reversed-phase high-performance liquid chromatography (RP-HPLC; Pathak, Dixit, Muthukumar, & Rathore, 2016;Pieracci, Armando, Westoby, & Thommes, 2018). In industry, refolding is typically performed using batch dilution at protein concentrations as low as <0.1 mg/ml (Eiberle & Jungbauer, 2010).…”