J Chromatogr B Analyt Technol Biomed Life Sci. 2026 May 12;1280:125111. doi: 10.1016/j.jchromb.2026.125111. Online ahead of print.
ABSTRACT
Therapeutic F(ab’)2 fragments derived from polyclonal equine antisera remain essential emergency biologics for addressing snake envenomation, tetanus, and viral infections. Traditional production methods rely on pepsin digestion, which is limited by non-specific proteolysis, harsh acidic conditions (pH 2.0-4.0), risk of contamination from animal sources, and significant batch variability, often complicating downstream preparative separations. In this study, we introduce a synergistic dual-endopeptidase platform that integrates hinge-specific biocatalysis with affinity chromatography to facilitate high-yield, homogeneous F(ab’)2 production under mild, near-physiological conditions (pH 7.8-8.2). We discovered that IdeZ2, derived from Streptococcus equi ssp. zooepidemicus, effectively cleaved the majority of equine IgG subclasses but was unable to process the structurally resistant IgG(T) fraction (IgG3 and IgG5). The incorporation of IdeS from Streptococcus pyogenes provided orthogonal specificity, selectively targeting the IdeZ2-resistant IgG(T) pool. In a unified reaction, this dual-enzyme approach achieved near-complete conversion of the substrate. Following Protein A affinity column removal of Fc-bearing species, the product attained >97% F(ab’)2 purity by SEC-HPLC with exceptional recovery (92.2% protein yield and 77.1% total potency yield from plasma). For industrial applications, IdeZ2 and IdeS were covalently immobilized on CNBr-activated agarose, creating a reusable heterogeneous biocatalyst that maintained 90% recovery through at least six reuse cycles. Collectively, these findings demonstrate a robust, mechanism-driven alternative to traditional pepsin processes, supporting the production of consistently high-quality equine F(ab’)2 therapeutics for global health applications.
PMID:42142439 | DOI:10.1016/j.jchromb.2026.125111