Concept of sample-specific correction of immunoassay results for precise and accurate IgG quantification in horse plasma

Halassy, Beata; Kurtović, Tihana; Balija, Maja Lang; Brgles, Marija; Tunjić, Monika; Sviben, Dora

doi:10.1016/j.jpba.2018.10.020

Cited by 7 publications

(11 citation statements)

References 24 publications

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“…Since caprylic acid precipitation or pepsin digestion do not change IgG subclass distribution, ELISA with a sample-specific correction of results [33] was found suitable for precise quantification of active principle. It has been chosen over other methods, such as SEC where overlapping of peaks or influence of protein type on surface area due to varying absorption and/or column adsorption characteristics is highly probable, leading to inaccurate result interpretation [40].…”

Section: Discussionmentioning

confidence: 99%

“…Throughout the isolation procedure total protein concentration was estimated spectrophotometrically by use of the Eq (2) [32], where Ehresmann's factor " f " for equine IgG of 0.2553 was used [33]. Appropriate dilution of each sample was independently prepared three times to obtain the mean value of the measured concentrations for further calculation of yield and purity.…”

Section: Methodsmentioning

confidence: 99%

“…Considering composition differences in subclass and/or venom-specific antibody distribution between each investigated sample and the standard, a recently developed principle for IgG or F(ab') 2 estimation that uses sample-specific correction was applied [33]. Namely, for IgG determination a highly pure IgG-based product (pure IgG sample in Fig 9), which was processed from the respective HHP and precisely quantified, served as internal, sample-specific reference.…”

Section: Methodsmentioning

confidence: 99%

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Refinement strategy for antivenom preparation of high yield and quality

et al. 2019

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Refinement strategy for antivenom preparation of high yield and quality

et al. 2019

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“…Its implementation not only contributes to virus safety [ 20 ], but also, as we demonstrated, enables effective F(ab') 2 preparation with rational pepsin amount even at a lower temperature, which opens up space for simplification of the production equipment and reduction of cost. For monitoring of the process efficiency and purity profiling, ELISA with sample-specific correction of results [ 32 ] for IgG quantification in plasma was preferentially chosen over other quantification methods such as SEC and densitometry, where overlapping of peaks/bands due to poor resolution or influence of protein type on surface area and intensity of developed color, respectively, is highly probable, leading to inaccurate result interpretation [ 35 , 36 , 37 ].…”

Section: Discussionmentioning

confidence: 99%

“…European viper venom antiserum (Zagreb antivenom; Institute of Immunology Inc., Croatia) was used as the standard. Considering composition differences in subclass and/or venom-specific antibody distribution between each investigated sample and the standard, a recently developed principle for F(ab') 2 estimation that uses sample-specific correction was applied [ 32 ]. Namely, F(ab') 2 preparation of the highest purity (referred to as ultrapure F(ab') 2 ), which was processed from the respective HHP and precisely quantified, served as the internal reference.…”

Section: Methodsmentioning

confidence: 99%

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

Kurtović

Brgles

Balija

et al. 2020

J. Venom. Anim. Toxins incl. Trop. Dis

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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.

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