Abstract
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.
Highlights
Passive immunotherapy involves the administration of antibodies collected from individuals who have recovered from an infection, or have been vaccinated against it, to a patient susceptible to the disease in question
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 aim of this work was to develop a sensitive RP high-performance liquid chromatography (HPLC) method that would be suitable for determination of residual Caprylic (octanoic) acid (CA) in samples of immunoglobulin G (IgG) purified from convalescent human plasma, complying the principles of good laboratory practices
Summary
Passive immunotherapy involves the administration of antibodies collected from individuals who have recovered from an infection, or have been vaccinated against it, to a patient susceptible to the disease in question. Today, when the whole world is faced with the COVID-19 pandemic induced by the virus named SARS-CoV-2, passive immunotherapy has once again been drawn to attention as the promising treatment option, together with development of refinement strategies for feasible production of effective and safe immunoglobulin G (IgG) preparations [1–4]. 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 [5–10]. Caprylic (octanoic) acid (CA) as a fractionation agent is frequently used to purify IgGs from both animal [11–13] and human plasma [4,14–17], in recent developments of SARS-CoV-2 specific immunoglobulin preparations [4,18,19]. The mechanism of CA-based precipitation is not yet fully understood
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