RESUMO
Bispecific antibodies have received wide attention as promising immunotherapeutic agents because of their high specificity and the ability to target immune cells to tumors. However, analysis of bispecific antibodies is challenging because multiple forms of antibodies are potentially generated during production in cell culture. Most analyses of bispecific antibodies rely on liquid chromatography with mass spectrometry (LC-MS), which could miss detection or becomes less quantitative if those forms are not physically separated. Here, we report a novel and sensitive mixed mode size exclusion chromatography (MM SEC) coupled with multiangle light scattering (MALS) to analyze different forms of bispecific IgG molecules under native conditions. The method displayed great ability to separate various antibody forms with peak resolutions unmatched by other methods we tested, isolating desired bispecific molecules, parental homodimers, half molecules, and antibodies with mispaired light and heavy chains. Each peak was analyzed by online MALS and then identified and confirmed by intact and reduced LC-MS of isolated forms. MM SEC in this study performs by a novel mechanism through the interactions of resin with protein surface hydrophobic clusters distributed across CDRs of light chains. This novel MM SEC allows quantitative detection of even low abundance forms and provides a new tool for screening expression profiles of cell culture clones, monitoring purification, and evaluating drug substance purity.
Assuntos
Anticorpos Biespecíficos/análise , Espectrometria de Massas/métodos , Animais , Anticorpos Biespecíficos/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Difusão Dinâmica da Luz , Imunoglobulina G/análise , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismoRESUMO
Maximizing production potential of recombinant proteins such as monoclonal antibodies (mAbs) in Chinese Hamster Ovary (CHO) cells is a key enabler of reducing cost of goods of biologics. In this study, we explored various strategies to utilize adenosine mediated effects in biologics manufacturing processes. Results show that supplementation of adenosine increases specific productivity by up to two-fold while also arresting cell growth. Introducing adenosine in intensified perfusion processes in a biphasic manner significantly enhanced overall productivity. Interestingly, adenosine effect was observed to be dependent on the cell growth state. Using specific receptor antagonists and inhibitors, we identified that ENTs (primarily Slc29a1) mediate the uptake of adenosine in CHO cell cultures. Transcriptomics data showed an inverse correlation between Slc29a1 expression levels and peak viable cell densities. Data suggests that in fed-batch cultures, adenosine can be produced extracellularly. Blocking Slc29a1 using ENT inhibitors such as DZD and DP alone or in combination with CD73 inhibitor, PSB12379, resulted in a twofold increase in peak viable cell densities as well as productivities in fed batch - a novel strategy that can be applied to biologics manufacturing processes. This is the first study that suggests that adenosine production/accumulation in CHO cell cultures can potentially regulate the transition of CHO cells from exponential to stationary phase. We also demonstrate strategies to leverage this regulatory mechanism to maximize the productivity potential of biologics manufacturing processes.
RESUMO
During a recent manufacturing campaign for a monoclonal antibody using a fed-batch process, poor cell culture performance was observed across two manufacturing sites with similar scales and equipment. Root cause analysis indicated that the poor cell culture performance was linked to the production basal media. Genealogy of the precursor raw materials used in the media revealed that a particular lot of Poloxamer 188 (P188) was the common link to the poor-performing media lots. P188 serves a critical role in protecting cells against shear in cell culture bioprocesses. However, the small-scale studies suggested that the poor cell culture performance was cytostatic in nature rather than being caused due to lack of shear protection. Several P188 lots were tested analytically using SEC-MS and RP-LC-MS methods and a unique low molecular weight species was identified in the suspect lot of poloxamer. The impurity was identified to be polypropylene oxide (PPO), a reaction intermediate in P188 synthesis. Spiking studies with PPO further confirmed its cytostatic nature. This case study highlights yet another scenario where lot-to-lot variability continues to impact bioprocesses and re-emphasizes the need for robust analytical and cell-culture raw material screening methods.
Assuntos
Citostáticos , Poloxâmero , Anticorpos Monoclonais , Técnicas de Cultura de Células , Meios de Cultura , Peso MolecularRESUMO
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 O2 volumetric mass transfer coefficient ( kLa), adequate oxygen supply and cell growth can be largely achieved. However, CO2 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 (CO2 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 CO2 stripping, kLaCO2, is dominated by the gas throughput. As a result, a combination of a minimum constant vvm air or N2 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 CO2 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.