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

Abstract

Background: Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab') 2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab') 2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio ( w / w ) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab') 2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab') 2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab') 2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.

Variability in antivenom neutralization of Mexican viperid snake venoms

“…In an analysis of different antivenom lots produced by the Butantan and Vital Brazil Institutes, bands of similar weights were identified as protein contaminants unrelated to degradation products of IgGs [ 34 ]. However, a different study concluded that bands smaller than 20 kDa in experimental antivenoms represent residues from the digestion of the heavy chain of IgGs and inter-alpha-trypsin inhibitors [ 35 ]. These reports emphasize the need for further assays to fully identify the bands migrating below 20 kDa in antivenoms produced by Birmex.…”

Development of Improved High-Performance Liquid Chromatography Method for the Determination of Residual Caprylic Acid in Formulations of Human Immunoglobulins

“…Pure IgGs are extracted from pooled plasma collected from convalescent donors by sequence of different fractionation steps, among which the most widely used are precipitation and liquid chromatography [ 5 , 6 , 7 , 8 , 9 , 10 ]. Caprylic (octanoic) acid (CA) as a fractionation agent is frequently used to purify IgGs from both animal [ 11 , 12 , 13 ] and human plasma [ 4 , 14 , 15 , 16 , 17 ], particularly in recent developments of SARS-CoV-2 specific immunoglobulin preparations [ 4 , 18 , 19 ]. CA selectively precipitates non-immunoglobulin proteins without affecting IgGs, which remain in solution, preserving their structural and conformational stability [ 6 , 7 , 20 , 21 , 22 ].…”

Roughness of Production Conditions: Does It Really Affect Stability of IgG-Based Antivenoms?

“…As such, it causes high morbidity and mortality, mainly due to limited access to adequate health services and the insufficiency of safe and effective antivenoms [ 1 , 2 , 3 ], which are the only specific treatment tool for this medical issue [ 4 ]. However, the manufacture of antivenoms containing either whole animal-derived pure IgGs or their fragments is of low sustainability due to various reasons [ 5 ]. One of them is the stability of their active components, a feature that significantly affects the quality as well as the safety of these therapeutics [ 6 ].…”