Batch-to-batch N-glycosylation study of infliximab, trastuzumab and bevacizumab, and stability study of bevacizumab

Abstract

ObjectivesInfliximab, trastuzumab and bevacizumab are among the most frequently prescribed therapeutic proteins, and like most other therapeutic proteins, are glycosylated. As differences in glycosylation may significantly change the safety and efficacy of therapeutic glycoproteins, it is extremely important to control N-glycosylation consistency. In the first part of this study, the batch-to-batch consistency of the N-glycosylation of infliximab, trastuzumab and bevacizumab was analysed. In the second part, the consistency of the N-glycosylation of bevacizumab stored in polycarbonate syringes (for off-label drug use) for 3 months was examined.MethodsN-glycans were (i) enzymatically released using peptide-N-glycosidase F, (ii) reduced, and (iii) analysed using hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry. Mass spectrometry data were interpreted using principal component analysis combined with two-way analysis of variance and Tukey post hoc tests. The biological activity of infliximab and trastuzumab was examined using enzyme-linked immunosorbent assays.ResultsThe results of both studies make important contributions to the field of hospital pharmacy. All batches of the studied therapeutic glycoproteins (infliximab, trastuzumab and bevacizumab) varied considerably (especially in galactosylation), while the N-glycosylation of bevacizumab remained unchanged during 3-month storage.ConclusionsThreshold values for batch-to-batch N-glycosylation variations should be established and batch-to-batch glycosylation consistency should be regularly tested. In our study, samples with significantly different N-glycosylation profiles showed no significant variations in biological activity, suggesting that the differences are probably not therapeutically significant.

Pathogenic effector functions of ACPA: Where do we stand?

“…It is important to consider that ACPA might be particularly prone to aggregation and interactions with pyrogens since they display some unique features including the presence of negatively charged, N -linked glycans in the variable domain 37 38. Also in this case, the composition of the N -linked glycans present on recombinantly produced antibodies may vary substantially from batch-to-batch and/or between different antibodies when not controlled for and hence could impact on biological outcomes 39–41. The experience of one of us (RT) with isolated ACPA, monoclonal ACPA, as well as control antibody preparations is consistent with this possibility since we also obtained inconsistent and variable effects in similar studies addressing the osteoclastogenic effects of ACPA (unpublished data).…”

The process defines the product: what really matters in biosimilar design and production?

“…Several PTMs, such as glycosylation, can have a direct impact on the clinical properties of therapeutic proteins, potentially influencing their biologic activity (potency), pharmacokinetics (PK), pharmacodynamics (PD) or immunogenicity [ 38 ]. Glycosylation can be considered the most complex PTM, and its potential for clinically relevant impact and its susceptibility to change based on process conditions make it extremely challenging to control [ 39 ]. For example, the degree of fucosylation and mannosylation can have a significant impact on the effector function of a mAb [namely FcRIIIa receptor binding and antibody-dependent cell cytotoxicity (ADCC)], which plays a key role in triggering the killing of disease cells bound by the therapeutic antibody by natural killer cells [ 38 , 40 ].…”

“…As each step of the manufacturing process has multiple process parameters that can alter the quality of the product, the manufacturing process for biologics is highly challenging, with batch-to-batch variability being the norm. For example, a recent study found significant variation in the level of glycosylation in several batches of common originator biologic therapies, such as infliximab, trastuzumab and bevacizumab; a phenomenon that can also be expected in biosimilars [ 39 ]. As such, when developing a biosimilar, the variability associated with the reference product must be well understood, and the manufacturing process for the biosimilar must be carefully controlled, as even minor process alterations may have a potential irreparable impact on the qualities of the biosimilar and its comparability with the reference product [ 28 , 30 , 35 , 42 ].…”

“…The cells are maintained in growth media and are provided with the required nutrients and additives to ensure the viability of the cells. Slightly altering the cell culture conditions can have a significant impact on several CQAs, including glycosylation and impurity profiles, and must be carefully controlled [ 39 ]. To attain the quality target product profile, several parameters are optimized during process development, involving the performance of hundreds of small-scale experiments using sound statistical procedures [ 73 ].…”

Constrained global sensitivity analysis for bioprocess design space identification

“…In a manufacturing context, a key challenge is how to control which sugars are added (glycoform) and ensure that all or most proteins produced have the same sugar structures on them (homogeneous glycoform profile). Current production methods yield a heterogeneous mix of glycoforms [4]. In addition, different glycoforms interact with the immune system in different ways therefore affecting the protein's efficacy as a pharmaceutical product [5][6][7][8][9].…”