Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed.

Bioreactor scale-up is a critical step in the production of therapeutic proteins such as monoclonal antibodies (MAbs). With the scale-up criterion such as similar power input per volume or O volumetric mass transfer coefficient ( kLa), adequate oxygen supply and cell growth can be largely achieved. However, CO stripping in the growth phase is often inadequate. This could cascade down to increased base addition and osmolality, as well as residual lactate increase and compromised production and product quality. Here we describe a practical approach in bioreactor scale-up and process transfer, where bioreactor information may be limited. We evaluated the sparger kLa and kLaCO2 (CO volumetric mass transfer coefficient) from a range of bioreactor scales (3-2,000 L) with different spargers. Results demonstrated that kLa for oxygen is not an issue when scaling from small-scale to large-scale bioreactors at the same gas flow rate per reactor volume (vvm). Results also showed that sparging CO stripping, kLaCO2, is dominated by the gas throughput. As a result, a combination of a minimum constant vvm air or N flow with a similar specific power was used as the general scale-up criterion. An equation was developed to determine the minimum vvm required for removing CO produced from cell respiration. We demonstrated the effectiveness of using such scale-up criterion with five MAb projects exhibiting different cell growth and metabolic characteristics, scaled from 3 to 2,000 L bioreactors across four sites. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1146-1159, 2017.

As reviewed in this report and exemplified in a case study, recombinant antibodies (Abs) can be expressed to very high levels in lymphoid cells by optimizing gene expression and process conditions. Myeloma and hybridoma cell lines have been constructed that secrete functionally active, recombinant antibodies at up to 15 pg/c/d in non-amplified cell lines and up to 100 pg/c/d in gene-amplified cell lines. Processes have been developed that achieve final titers in fed-batch culture of 1-2 g/L, or reach volumetric productivities in perfusion culture of 0.2-0.6 g/L/d. In the specific example of the production of a humanized anti-CD18 Ab, h1B4, amplified NS0 cell lines were developed that secreted Ab at up to 50 pg/c/d. Periodic addition of concentrated nutrient solutions in fed-batch culture allowed production of the Ab to 1.8 g/L. In perfusion culture, titers of up to 0.5 g/L were obtained with maximum volumetric (i.e. reactor volume) productivities in excess of 0.6 g/L/d. Production of Engineered AntibodiesBecause of their extraordinary molecular recognition capabilities and exquisite specificity, Abs have been considered for many potential therapeutic interventions, including (as discussed below) the short-circuiting of the inflammatory reaction. Abs can be engineered via recombinant DNA technology to optimize their therapeutic efficacy or expression levels (as reviewed in 1-2). For example, the non-binding domains of non-human Abs can be replaced by the corresponding human sequences to minimize potential human antimouse antibody (KAMA) responses (3-4); the isotype of the constant region can be changed to modulate effector function interactions (4-5); the binding domains can be modified to enhance affinity or specificity (6); or Abs of the appropriate specificity can be selected from cloned E. Coli or phage combinatorial libraries (7)(8). Such Abs can be efficiently expressed with proper post-translational modification in a number of mammalian cell systems, the most common being Chinese Hamster Ovary cells (9-11) and, as 1 Current address: DuPont Merck Pharmaceutical Co.,