Refinement strategy for antivenom preparation of high yield and quality

Abstract

Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational use of plasma of animal origin. We report on development of an efficient refinement strategy for F(ab') 2 -based antivenom preparation. Process design was driven by the imperative to keep the active principle constantly in solution as a precautionary measure to preserve stability of its conformation (precipitation of active principle or its adsorption to chromatographic stationary phase has been completely avoided). IgG was extracted from hyperimmune horse plasma by 2% ( V / V ) caprylic acid, depleted from traces of precipitating agent and digested by pepsin. Balance between incomplete IgG fraction breakdown, F(ab') 2 over-digestion and loss of the active principle's protective efficacy was achieved by adjusting pepsin to substrate ratio at the value of 4:300 ( w / w ), setting pH to 3.2 and incubation period to 1.5 h. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. Developed manufacturing strategy gave 100% pure and aggregate-free F(ab') 2 preparation, as shown by size-exclusion HPLC and confirmed by MS/MS. The overall yield of 75% or higher compares favorably to others so far reported. This optimised procedure looks also promising for large-scale production of therapeutic antivenoms, since high yield of the active drug and fulfillment of the regulatory demand considering purity was achieved. The recovery of the active substance was precisely determined in each purification step enabling accurate estimation of the process cost-effectiveness.

Streamlined downstream process for efficient and sustainable (Fab')2 antivenom preparation

“…Initially, all experimental conditions were adopted from our previously developed downstream processing strategy in which extraction of IgGs and their subsequent enzymatic cleavage were performed as two separate operating units with an interposed diafiltration-mediated removal of precipitating agent [ 28 ]. Namely, heat-treated HHP, acidified to pH 3.2, was fractionated and digested at once by simultaneous incubation with 2% caprylic acid ( V / V ) and pepsin, the latter in a quantity 75-times lower than the measured IgG content (1:75, w / w ).…”

“…Optimal conditions were adopted from our previous research, i.e. , exclusive adsorption of the enzyme and other acidic impurities to the column while the target compound passes unhindered [ 28 ]. Final polishing produced an active drug (ultrapure F(ab') 2 ) of approximately 97% purity and without any loss ( Fig.…”

“…Excised protein spots obtained by 2D gel electrophoresis of F(ab') 2 sample were prepared and analyzed by MALDI MS/MS on an ultrafleXtreme (Bruker, Germany), as described by Kurtović et al [ 28 ]. Proteins were considered to be confidently identified after submitting peptide sequencing data to Mascot (taxonomy "other mammalians") and receiving statistically significant scores for at least two peptides per protein.…”

“…F(ab') 2 content in samples from HHP processing was determined by ELISA according to the protocol from Kurtović et al [ 28 ]. European viper venom antiserum (Zagreb antivenom; Institute of Immunology Inc., Croatia) was used as the standard.…”

“…Recently, we have proposed a refinement strategy [ 28 ] as an attempt to offer a new perspective for overcoming cost-efficiency difficulties associated with antivenom production. It has been demonstrated on equine plasma and consisted of caprylic acid-mediated fractionation, depletion of precipitating agent from the IgG-enriched fraction, pepsin digestion, diafiltration of the F(ab') 2 -based preparation and its final refinement by the flow-through chromatography.…”