Protein modification during anti‐viral heat‐treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose

Abstract

To ensure the optimal safety of plasma derived and new generation recombinant proteins, heat treatment is customarily applied in the manufacturing of such biopharmaceuticals as a means of viral inactivation. In subjecting proteins to anti-viral heat-treatment it is necessary to use high concentrations of thermostabilizing excipients to prevent protein damage, and it is therefore imperative that the correct balance between bioprocessing conditions, maintenance of protein integrity and virus kill is found. In this study we have utilized model proteins (lysozyme, fetuin, and human serum albumin) and plasma-derived therapeutic proteins (factor VIII and factor IX) to investigate the protein modifications that occur during anti-viral heat treatment. Specifically, we investigated the relationship between bioprocessing conditions and the type and extent of protein modification under a variety of industrially relevant wet and lyophilized heat treatments using sucrose as a thermostabilizing agent. Heat treatment led to the formation of disulfide crosslinks and aggregates in proteins containing free cysteine residues. Terminal oligosaccharide sialic acid residues were hydrolyzed from the glycan moieties of glycoproteins during anti-viral heat treatment. Heat treatment promoted sucrose hydrolysis to yield glucose and fructose, leading, in turn, to the glycation of lysine amino groups in those proteins containing di-lysine motifs. During extended hear treatments, 1,2-dicarbonyl type advanced glycation end-products were also formed. Glycation-type modifications were more prevalent in wet heat-treated protein formulations.

Resistance of porcine circovirus and chicken anemia virus to virus inactivation procedures used for blood products

“…3B). These elevated temperatures are well beyond those that are used for viral inactivation procedures for blood products, as they would be highly damaging for the constituent active proteins 31 . Dry heat during manufacturing would therefore be unlikely to be able to inactivate viruses with stabilities similar to PCV2 and CAV from blood products.…”

The effect of peptide glycation on local secondary structure

“…Non-enzymatic glycation of proteins has been shown to be a potential problem during their storage in the food and biotech industries (Davis et al, 2001;Smales et al, 2002), and also in human health as a complication of diseases such as diabetes (Al-Abed et al, 1999;Stitt, 2001). Glycation occurs via the Maillard reaction in which a reducing sugar reacts with an amino group on a protein, either at the NH 2 terminus or at the e-amino group of lysine residues (Tagami et al, 2000;Yeboah et al, 2004).…”

Development of candidate reference reagent for HIV-1 RNA and comparison analysis for different HIV-1 RNA quantitative assay

“…The infectivity of HIV-1 RNA reference reagents must be eliminated without affecting the fidelity of the viral RNA load. Heating of human plasma might cause unacceptable alterations in HIV-1 RNA due to modification of serum proteins (17), but further study of this issue is needed. In our study, we used heat inactivation to eliminate RNA infectivity.…”