EBF Recommendation for stability testing of anti-drug antibodies; lessons learned from anti-vaccine antibody stability studies.

Long- and short-term stability testing of the analyte is one of the key parameters in bioanalytical method validation in support of pharmacokinetics. However, for immunogenicity testing the scientific rationale for long- and short-term stability testing on quality control (QC) samples most often spiked with polyclonal antibody raised in a different species should be questioned. Therefore, the European Bioanalysis Forum (EBF) formed a Topic Team (TT) to discuss the scientific rationale for stability testing of anti-drug antibodies (ADA). A review of EBF member companies’ experience on ADA stability and data from vaccine projects was the basis of this discussion. EBF recommends to perform short-term stability testing but not to perform long-term stability testing of ADAs in non-clinical and clinical studies

Remodeling of the Carbohydrate Chains of hCG by Use of Sialyltransferases: Effects on the Biological Activity

Human chorionic gonadotropin (hCG) is a glycoprotein hormone which contains both N- and O-linked carbohydrate chains. It consists of two subunits, a and 8. The a-subunit contains two N-linked carbohydrate chains: a mono-antenna and a non-fucosylated bi-antenna. The $-subunit contains both (two) N and (four) O-linked carbohydrate structures. Both of the N-linked carbohydrate chainsare biantennary, one of which is fucosylated

SIALYLTRANSFERASES; THEIR SPECIFICITY AND THEIR USE IN CARBOHYDRATE REMODELLING.

In order to apply sialyltransferases in the remodelling of the carbohydrate chains on biologically active glycoproteins, it is a prerequisite to know the fine specificity of these enzymes. In this report the specificity of several sialyltransferses involved in the sialylation of O- and N-linked oligosaccharide chains is reviewed. Also novel results on the branch specificity of a3- and a6-sialyltransferase are reported. The potential application of these enzymes in carbohydrate remodelling was studied using human chorionic gonadotropin (hCG) as a model glycoprotein. Differently sialylated preparations of this hormone were obtained and tested for their stimulatory effect on steroidogenesis in Leydig cells in vitro. Asialo-hCcG appeared to be only 45% as effective as native hCG. a3-Resialylation of the O-linked chains on the ß-subunit of this hormone did not restore the biological activity to a higher level. By contrast, 55% a6- resialylation of the N-linked chains yielded a preparation which was almost as active as native hCG. Interestingly, further sialylation by the a6-sialyltransferase resulted in a decrease of the bio-activity to levels lower than obtained with asialo-hcG. It is concluded that the lectin-carbohydrate binding, which is part of the process that triggers the biological respons of the target cell can be mimicked by N-linked chains carrying a6-linked sialic acid. However, too high a density of such residues interferes with this interaction