Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale‐down study of the effect of harvesting by centrifugation on the post‐translational modification profile

Abstract

With the trend towards the generation and production of increasing numbers of complex biopharmaceutical (protein based) products, there is an increased need and requirement to characterize both the product and production process in terms of robustness and reproducibility. This is of particular importance for products from mammalian cell culture which have large molecular structures and more often than not complex post-translational modifications (PTMs) that can impact the efficacy, stability and ultimately the safety of the final product. It is therefore vital to understand how the operating conditions of a bioprocess affect the distribution and make up of these PTMs to ensure a consistent quality and activity in the final product. Here we have characterized a typical bioprocess and determined (a) how the time of harvest from a mammalian cell culture and, (b) through the use of an ultra scale-down mimic how the nature of the primary recovery stages, affect the distribution and make up of the PTMs observed on a recombinant IgG(4) monoclonal antibody. In particular we describe the use of rapid whole antibody analysis by mass spectrometry to analyze simultaneously the changes that occur to the cleavage of heavy chain C-terminal lysine residues and the glycosylation pattern, as well as the presence of HL dimers. The time of harvest was found to have a large impact upon the range of glycosylation patterns observed, but not upon C-terminal lysine cleavage. The culture age had a profound impact on the ratio of different glycan moieties found on antibody molecules. The proportion of short glycans increased (e.g., (G0F)(2) 20-35%), with an associated decrease in the proportion of long glycans with culture age (e.g., (G2F)(2) 7-4%, and G1F/G2F from 15.2% to 7.8%). Ultra scale-down mimics showed that subsequent processing of these cultures did not change the post-translational modifications investigated, but did increase the proportion of half antibodies present in the process stream. The combination of ultra scale-down methodology and whole antibody analysis by mass spectrometry has demonstrated that the effects of processing on the detailed molecular structure of a monoclonal antibody can be rapidly determined early in the development process. In this study we have demonstrated this analysis to be applicable to critical process design decisions (e.g., time of harvest) in terms of achieving a desired molecular structure, but this approach could also be applied as a selection criterion as to the suitability of a platform process for the preparation of a new drug candidate. Also the methodology provides means for bioprocess engineers to predict at the discovery phase how a bioprocess will impact upon the quality of the final product.

End-to-end integrated fully continuous production of recombinant monoclonal antibodies

“…The product quality was steady state over the time of operation. The steady state product quality that results from lower residence time can be a marked advantage over batch processes where product stability over culture days can be a significant issue (Joshi et al, 2014;Mohan et al, 1993;Pacis et al, 2011;Reid et al, 2010;Robinson et al, 1994). The consistent product quality over ∼30 days also indicates the feasibility of using CHO/DHFR cell line for prolonged duration continuous/perfusion cell culture processes.…”

“…Inherently, however, the dynamic nature of the Biologics process results in variable product quality as a function of culture days in both perfusion and batch cell culture processes (Pacis et al, 2011;Reid et al, 2010;Robinson et al, 1994). Mohan et al have specifically shown degradation effects of protease being minimized in continuousflow cultures as opposed to batch processes (Mohan et al, 1993).…”

Application of mass spectrometry to facilitate advanced process controls of biopharmaceutical manufacture

“…Liquid chromatography (LC) at-line with MSD-TOF MS has been used to perform intact antibody analysis for an ultrascale down study, which has been undertaken to investigate a potential impact of downstream processing on the molecular structure of a monoclonal antibody (mAb) and to demonstrate how process operating conditions can impact other unit operations [22]. LC/MSD-TOF analysis allowed for simultaneous detection of the changes that occur to the cleavage of heavy chain C-terminal lysine residues and the glycosylation pattern, as well as the presence of heavy chain/light chain dimers depending on the time of harvest.…”

“…Although LC-MS method with SIM detection certainly permits monitoring and quantitation of major glycoforms in cell culture supernatant; however, it is much more challenging to monitor and quantify lower abundant species, which could contain important information, for example, on fucosylation levels and addition of potentially immunogenic sugars, among others. A more recent publication addresses the deficiencies of both the SIM method [23] and the off-line protein A purification approach [22], by utilizing (LC/electrospray quadrupole time-of-flight-MS (LC/ESI-qTOF-MS) system, coupled with an on-line column-switching system equipped with two columns, a protein A affinity column and a reversed-phase (RP) desalting column [24]. Two column-switching systems have been introduced, one targeting intact mAbs, and the other targeting the light and heavy chains of the mAbs.…”

The future of industrial bioprocessing: Batch or continuous?

“…are minimized. Potential quality issues with stable molecules are addressed as well (Pacis, 2011;Robinson, 1994;Reid, 2010), as continuous processes may be operated with robust control, improving product quality and minimizing heterogeneity.…”