Within the European Immunogenicity Platform (EIP) (http://www.e-i-p.eu), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.

Monoclonal antibody and Fc fusion protein drugs are complex heterogeneous mixtures of numerous different protein variants and modifications. N-glycosylation as one of the most complex post-translational modification influences the structural characteristics of the antibodies Fc part thereby potentially modulating effector function and pharmacokinetics. Several investigations on the relationship between N-glycosylation and pharmacokinetics have been published. However, this structure-function relationship is not fully understood. In this review potential alterations with focus on N-glycosylation of mAbs and Fc fusion proteins and the possible effects on the pharmacokinetics are reviewed and the current understandings of the underlying mechanisms are described.

Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, b-1,3 N-acetylglucosaminyltransferase 1 and b-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes.

Abstract-Published software quality models either provide abstract quality attributes or concrete quality assessments. There are no models that seamlessly integrate both aspects. In the project Quamoco, we built a comprehensive approach with the aim to close this gap.For this, we developed in several iterations a meta quality model specifying general concepts, a quality base model covering the most important quality factors and a quality assessment approach. The meta model introduces the new concept of a product factor, which bridges the gap between concrete measurements and abstract quality aspects. Product factors have measures and instruments to operationalise quality by measurements from manual inspection and tool analysis. The base model uses the ISO 25010 quality attributes, which we refine by 200 factors and 600 measures for Java and C# systems.We found in several empirical validations that the assessment results fit to the expectations of experts for the corresponding systems. The empirical analyses also showed that several of the correlations are statistically significant and that the maintainability part of the base model has the highest correlation, which fits to the fact that this part is the most comprehensive. Although we still see room for extending and improving the base model, it shows a high correspondence with expert opinions and hence is able to form the basis for repeatable and understandable quality assessments in practice.

PurposeFollowing two cases of neutralizing antibodies to epoetin alfa in an investigational clinical study, a small number of individual syringes of two drug product batches were found to contain unusually high levels of aggregation at the end of the clinical trial.MethodsWe undertook an extensive analytical approach to determine the root-cause of the increased aggregation in the affected batches.ResultsSoluble tungsten was found in the syringes, most likely derived from the pins used to manufacture the syringes. Spiking of epoetin alfa with sodium polytungstate or an extract of tungsten pins used to manufacture the syringes induced the formation of aggregates, both dimers that appeared to be covalently linked by disulphide bonds as well as higher-order aggregates. Sodium polytungstate had also a strong denaturing effect on the protein.ConclusionsWe propose tungsten-mediated unfolding and aggregation of epoetin alfa in pre-filled syringes as a potential root cause for increased immunogenicity. This finding may be more broadly applicable to this and other classes of therapeutic proteins.