Multiple functions of caprylic acid-induced impurity precipitation for process intensification in monoclonal antibody purification

Trapp, Anja; Faude, Alexander; Hörold, Natalie; Schubert, Sven; Faust, Sabine; Grob, Thilo; Schmidt, Stefan

doi:10.1016/j.jbiotec.2018.05.001

Cited by 15 publications

(11 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although the share of aggregates in CAP-purified samples was low, 2.3% (Table 2), their presence indicated that CAP does not completely prevent aggregation. Similarly, increased levels of aggregation were noticed in some preparations of affinity-purified monoclonal antibodies following CA polishing treatment [34]. It should be emphasized that the CAP purification work-flow did not induce IgG subclass redistribution (Figure 5), as already reported by Halassy et al [35], which is especially important with regard to the IgG(T) isotype as the most important one for the toxin neutralization activity that will be discussed in more detail later.…”

Section: Precipitation-based Purification Proceduressupporting

confidence: 76%

“…We believe that the former could have been completely eliminated if the precipitation was performed with higher CA concentration, which would probably adversely affect other sample properties such as turbidity, as mentioned above. The main putative mechanism of CA action is based on its hydrophobicity that enables precipitation of acidic proteins, while basic ones, like IgGs, are protected by charge mediated hydration, therefore remaining in the solution [33,34]. According to MS results, alpha-1B-glycoprotein, although slightly acidic, exhibited resistance to the impact of CA (Figure 3D).…”

Section: Precipitation-based Purification Proceduresmentioning

confidence: 99%

See 1 more Smart Citation

Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches

Lukačević

Kurtović

Balija

et al. 2020

Toxins

View full text Add to dashboard Cite

Whole IgG antivenoms are prepared from hyperimmune animal plasma by various refinement strategies. The ones most commonly used at industrial scale are precipitation by sodium or ammonium sulphate (ASP), and caprylic acid precipitation (CAP) of non-immunoglobulin proteins. The additional procedures, which have so far been used for experimental purposes only, are anion-exchange (AEX) and cation-exchange chromatography (CEX), as well as affinity chromatography (AC) using IgG’s Fc-binding ligands. These protocols extract the whole IgG fraction from plasma, which contains both venom-specific and therapeutically irrelevant antibodies. Such preparations represent a complex mixture of various IgG subclasses whose functional and/or structural properties, as well as relative distribution, might be affected differently, depending on employed purification procedure. The aim of this work was to compare the influence of aforementioned refinement strategies on the IgG subclass distribution, venom-specific protective efficacy, thermal stability, aggregate formation and retained impurity profile of the final products. A unique sample of Vipera ammodytes ammodytes specific hyperimmune horse plasma was used as a starting material, enabling direct comparison of five purification approaches. The highest purity was achieved by CAP and AC (above 90% in a single step), while the lowest aggregate content was present in samples from AEX processing. Albumin was the main contaminant in IgG preparations obtained by ASP and CEX, while transferrin dominantly contaminated IgG sample from AEX processing. Alpha-1B-glycoprotein was present in CAP IgG fraction, as well as in those from ASP- and AEX-based procedures. AC approach induced the highest loss of IgG(T) subclass. CEX and AEX showed the same tendency, while CAP and ASP had almost no impact on subclass distribution. The shift in IgG subclass composition influenced the specific protective efficacy of the respective final preparation as measured in vivo. AC and CEX remarkably affected drug’s venom-neutralization activity, in contrary to the CAP procedure, that preserved protective efficacy of the IgG fraction. Presented data might improve the process of designing and establishing novel downstream processing strategies and give guidance for optimization of the current ones by providing information on potency-protecting and purity-increasing properties of each purification principle.

show abstract

Section: Precipitation-based Purification Proceduressupporting

confidence: 76%

Section: Precipitation-based Purification Proceduresmentioning

confidence: 99%

Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches

Lukačević

Kurtović

Balija

et al. 2020

Toxins

View full text Add to dashboard Cite

show abstract

“…Refinement principles employing caprylic acid have been successfully implemented into preparations of a whole series of highly efficacious equine or ovine IgG-based antivenoms [13–17]. They have also been proven beneficial for purification of F(ab') 2 derivatives [18, 19] and monoclonal antibodies [20].…”

Section: Introductionmentioning

confidence: 99%

Refinement strategy for antivenom preparation of high yield and quality

et al. 2019

View full text Add to dashboard Cite

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.

show abstract

“…Residual CA has been considered a process-related impurity; thus, it has to be removed from the final product, and its absence has to be reliably documented. Determination of CA traces has usually been performed by high-performance liquid chromatography (HPLC), gas chromatography, and colorimetric methods [ 11 , 24 , 25 , 26 , 27 ]. Herrera et al [ 11 ] described a reverse-phase (RP) HPLC method that was applicable for the antivenom production process.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

View full text Add to dashboard Cite

Quality control of human immunoglobulin formulations produced by caprylic acid precipitation necessitates a simple, rapid, and accurate method for determination of residual caprylic acid. A high-performance liquid chromatography method for that purpose was developed and validated. The method involves depletion of immunoglobulins, the major interfering components that produce high background noise, by precipitation with acetonitrile (1:1, v/v). Chromatographic analysis of caprylic acid, preserved in supernatant with no loss, was performed using a reverse-phase C18 column (2.1 × 150 mm, 3 μm) as a stationary phase and water with 0.05% TFA–acetonitrile (50:50, v/v) as a mobile phase at a flow rate of 0.2 mL/min and run time of 10 min. The developed method was successfully validated according to the ICH guidelines. The validation parameters confirmed that method was linear, accurate, precise, specific, and able to provide excellent separation of peaks corresponding to caprylic acid and the fraction of remaining immunoglobulins. Furthermore, a 24−1 fractional factorial design was applied in order to test the robustness of developed method. As such, the method is highly suitable for the quantification of residual caprylic acid in formulations of human immunoglobulins for therapeutic use, as demonstrated on samples produced by fractionation of convalescent anti-SARS-CoV-2 human plasma at a laboratory scale. The obtained results confirmed that the method is convenient for routine quality control.

show abstract

Multiple functions of caprylic acid-induced impurity precipitation for process intensification in monoclonal antibody purification

Cited by 15 publications

References 24 publications

Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches

Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches

Refinement strategy for antivenom preparation of high yield and quality

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

Contact Info

Product

Resources

About