Statistically-aided development of protein A affinity chromatography for enhancing recovery and controlling quality of a monoclonal antibody.

Protein A chromatography is widely used for isolation of monoclonal antibodies (mAbs) from cell culture components. In this study, the effect of different process parameters of the Protein A purification namely, binding pH, elution pH, flow rate, neutralization pH and tween concentration, on the concentration and quality of the purified mAb were evaluated. Using design of experiments approach, the critical process parameters of protein A chromatography were identified and experimentally optimized. Their impact on quality attributes, such as size variants and charge variants, of the mAb was studied. Multivariate data analysis was subsequently performed using multiple linear regression and partial least squares regression methods. It was observed that the elution pH primarily governed the concentration of the purified mAb and the content of monomers and aggregates, while the tween concentration primarily influenced the main peak of the charge variants. This is the first study that evaluates the impact of tween concentration in buffers on the protein A chromatography purification step. These studies helped in identifying the design space and defining the target robust and optimal setpoints of the responses, which were subsequently verified experimentally. These setpoints not only passed the target criteria but also resulted in the highest recoveries during the investigation. Through this statistically-aided approach, an optimized and robust protein A chromatography process was rationally developed for purification of mAbs, while achieving the desired product quality. This study highlights the influence of multiple parameters of the protein A purification process on critical quality attributes of mAbs, such as the size and charge variants, which has been a very scarcely explored area.

Controlling monoclonal antibody aggregation during cell culture using medium additives facilitated by the monitoring of aggregation in cell culture matrix using size exclusion chromatography.

Controlling monoclonal antibody aggregation at the upstream stage itself can significantly reduce the burden on downstream processing and can improve the process yield. Hence, we have investigated the use of sugar osmolytes (glucose, mannose, sucrose and maltose) and formulation excipients (mannitol, polysorbate 20 and polysorbate 80) as medium additives to reduce protein aggregation during cell culture. Aggregate content in cell culture samples was estimated using a high-resolution size-exclusion chromatography technique, which efficiently resolved the antibody monomer and aggregates in the cell culture matrix i.e., without purification. Glucose, mannose, maltose and the polysorbates effectively reduced the mean aggregate content over the course of the culture. Sugar-based additives exhibited a higher degree of variation during aggregate quantitation as compared to polysorbate additives, rendering the latter a preferred additive. Therefore, this study demonstrated the potential of sugar osmolytes and formulation excipients as media additives during cell culture to reduce aggregate formation, without negatively impacting cell growth and antibody production, facilitated by the monitoring of aggregate content in cell culture samples without purification.

A case study: Correlation of the nutrient composition in Chinese Hamster Ovary cultures with cell growth, antibody titre and quality attributes using multivariate analyses for guiding medium and feed optimization in early upstream process development.

In this case-study, we demonstrate an approach for identifying correlations between nutrients/metabolites in the spent medium of CHO cell cultures and cell growth, mAb titre and critical quality attributes, using multivariate analyses, which can aid in selection of targets for medium and feed optimization. An extensive LC-MS-based method was used to analyse the spent medium composition. Partial least squares (PLS) model was used to identify correlations between nutrient composition and cell growth and mAb titre and orthogonal projections to latent structures (OPLS) model was used to determine the effect of the changing nutrient composition during the culture on critical quality attributes. The PLS model revealed that the initial concentrations of several amino acids as well as pyruvic acid and pyridoxine, governed the early cell growth, while the concentrations of TCA cycle intermediates and several vitamins highly influenced the stationary phase, in which mAb production was maximum. For the first time, with the help of the OPLS model, we were able to draw correlations between nutrients/metabolites during the culture and critical quality attributes, for example, optimizing the supply of certain amino acids and vitamins could reduce impurities while simultaneously increasing desirable glycoforms. The unique correlations obtained from such an exploratory analysis, utilizing conditions that are commonly adopted in early process development, present opportunities for optimizing the compositions of the growth media and the feed media for enhancing cell growth, mAb production and quality, thereby proving to be a useful preliminary step in bioprocess optimization. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00561-z.

Advanced strategies in glycosylation prediction and control during biopharmaceutical development: Avenues toward industry 4.0.

Glycosylation has been shown to define the safety and efficacy of biopharmaceuticals, thus classified as a critical quality attribute. However, controlling glycan heterogeneity has always been a major challenge owing to the multivariate factors that govern the glycosylation process. Conventional approaches for controlling glycosylation such as gene editing and metabolic control have succeeded in obtaining desired glycan profiles in accordance with the Quality by Design paradigm. Nonetheless, the development of smart algorithms and omics-enabled complete cell characterization has made it possible to predict glycan profiles beforehand, and manipulate process variables accordingly. This review thus discusses the various approaches available for control and prediction of glycosylation in biopharmaceuticals. Further, the futuristic goal of integrating such technologies is discussed in order to attain an automated and digitized continuous bioprocess for control of glycosylation. Given, control of a process as complex as glycosylation requires intense monitoring intervention, we examine the current technologies that enable automation. Finally, we discuss the challenges and the technological gap that currently limits incorporation of an automated process in routine bio-manufacturing, with a glimpse into the economic bearing.

A comparison between analytical approaches for molecular weight estimation of proteins with variable levels of glycosylation.

Biotherapeutics, such as mAbs and fusion proteins, are a major and rapidly growing class of pharmaceuticals. Majority of the biopharmaceuticals are glycoproteins, wherein about 1 to 30% of their molecular weight (MW) are contributed by the glycans. Determination of MW of heavily glycosylated proteins, such as Fc-fusion proteins, is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation influences the expected size of the glycoprotein, which leads to disproportionate MW estimation, in size-dependent methods. Hence, in this study, we have demonstrated the advantages and limitations of four widely used MW estimation techniques for three proteins having varying levels of glycosylation. It was proven that glycosylation had least impact on MW determination by SEC-MALS and SV-AUC. However, MW estimation by LC-MS and SDS-PAGE was extensively hampered by the degree of glycosylation. It is, thus, essential to consider the structural characteristics of proteins while selecting a technique for determining their MW.

A Regulatory Perspective on Testing of Biological Activity of Complex Biologics.

Antibody-drug conjugates (ADCs) and bispecific antibodies are becoming increasingly popular. However, their complex structures mandate stringent regulatory guidelines to ensure their safety and efficacy. We have briefly reviewed the existing regulatory guidelines and presented our perspectives on refining them to hasten the transition of these drugs from laboratories to market.