Abstract
ABSTRACTThe development of therapeutic and diagnostic antibodies is a rapidly growing field of research, being the fastest expanding group of products on the pharmaceutical market, and appropriate quality controls are crucial for their application. We have identified and characterized the serine protease termed BspK (Bdellovibrio serine protease K) from Bdellovibrio bacteriovorus and here show its activity on antibodies. Mutation of the serine residue at position 230 rendered the protease inactive. Further investigations of BspK enzymatic characteristics revealed autoproteolytic activity, resulting in numerous cleavage products. Two of the autoproteolytic cleavage sites in the BspK fusion protein were investigated in more detail and corresponded to cleavage after K28 and K210 in the N- and C-terminal parts of BspK, respectively. Further, BspK displayed stable enzymatic activity on IgG within the pH range of 6.0 to 9.5 and was inhibited in the presence of ZnCl2. BspK demonstrated preferential hydrolysis of human IgG1 compared to other immunoglobulins and isotypes, with hydrolysis of the heavy chain at position K226 generating two separate Fab fragments and an intact IgG Fc domain. Finally, we show that BspK preferentially cleaves its substrates C-terminally to lysines similar to the protease LysC. However, BspK displays a unique cleavage profile compared to several currently used proteases on the market.IMPORTANCE The rapid development of novel therapeutic antibodies is partly hindered by difficulties in assessing their quality and safety. The lack of tools and methods facilitating such quality controls obstructs and delays the process of product approval, eventually affecting the patients in need of treatment. These difficulties in product evaluations indicate a need for new and comprehensive tools for such analysis. Additionally, recent concerns raised regarding the limitations of established products on the market (e.g., trypsin) further highlight a general need for a larger array of proteases with novel cleavage profiles to meet current and future needs, within both the life science industry and the academic research community.
Highlights
IMPORTANCE The rapid development of novel therapeutic antibodies is partly hindered by difficulties in assessing their quality and safety
posttranslational modifications (PTMs) can be incorporated in vivo by natural cellular synthesis and processing or in vitro by manufacturing and processing, making quality controls of critical importance to ensure the efficacy and safety of these therapeutics [3, 7, 8]
An initial screening for IgG-modifying activities was performed with crude B. bacteriovorus lysate and revealed enzymatic activity on human IgG, displaying a cleavage pattern suggestive of a single cleavage within the hinge region (Fig. 1A)
Summary
IMPORTANCE The rapid development of novel therapeutic antibodies is partly hindered by difficulties in assessing their quality and safety. The lack of tools and methods facilitating such quality controls obstructs and delays the process of product approval, eventually affecting the patients in need of treatment. These difficulties in product evaluations indicate a need for new and comprehensive tools for such analysis. There are many advantages of protein therapeutics such as MAbs, compared to the classical chemical drugs, including high specificity for their targets, long half-lives, consistent pharmacokinetic properties, and low batch-to-batch variations [2]. Product approvals of biological agents, such as antibodies, that are naturally heterogeneous and undergo a wide range of posttranslational modifications (PTMs) require rigorous controls to guarantee their quality and safety prior to any clinical application. There have been several studies investigating the safety of B. bacteriovorus administration, and accumu-
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