Anal Bioanal Chem (2014) 406:6257–6264 DOI 10.1007/s00216-014-8065-4
RESEARCH PAPER
Separation and quantification of monoclonal-antibody aggregates by hollow-fiber-flow field-flow fractionation Jun Fukuda & Takafumi Iwura & Shigehiro Yanagihara & Kenji Kano
Received: 20 May 2014 / Revised: 16 July 2014 / Accepted: 24 July 2014 / Published online: 13 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Hollow-fiber-flow field-flow fractionation (HF5) separates protein molecules on the basis of the difference in the diffusion coefficient, and can evaluate the aggregation ratio of proteins. However, HF5 is still a minor technique because information on the separation conditions is limited. We examined in detail the effect of different settings, including the main-flow rate, the cross-flow rate, the focus point, the injection amount, and the ionic strength of the mobile phase, on fractographic characteristics. On the basis of the results, we proposed optimized conditions of the HF5 method for quantification of monoclonal antibody in sample solutions. The HF5 method was qualified regarding the precision, accuracy, linearity of the main peak, and quantitation limit. In addition, the HF5 method was applied to non-heated Mab A and heatinduced-antibody-aggregate-containing samples to evaluate the aggregation ratio and the distribution extent. The separation performance was comparable with or better than that of conventional methods including analytical ultracentrifugation–sedimentation velocity and asymmetric-flow field-flow fractionation.
Keywords Antibodies aggregates . Hollow-fiber-flow field-flow fractionation . Size-exclusion chromatography . Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-8065-4) contains supplementary material, which is available to authorized users. J. Fukuda (*) : K. Kano (*) Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan e-mail: [email protected] e-mail: [email protected] J. Fukuda : T. Iwura : S. Yanagihara Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., Hagiwara-machi, Takasaki, Gunma 370-0013, Japan
Analytical ultracentrifugation . Asymmetric-flow field-flow fractionation
Introduction Study of therapeutic protein products, especially monoclonal antibodies, has recently attracted a great deal of attention from pharmaceutical industries. In the last 20 years over 20 monoclonal antibodies have been approved in a variety of therapeutic categories [1], and six antibody pharmaceuticals have been approved including Humira (adalimumab) and Remicade (infliximab), which were the two best-selling pharmaceuticals in 2012 (http://www.utobrain.co.jp/news/ 20130724.shtml). These biopharmaceutical products have drastically improved the quality of life of patients. Compared with small pharmaceutical molecules, therapeutic protein products are rather large entities with inherent physicochemical complexity. Therefore, many physical and chemical factors may lead to protein degradation and consequently affect the quality of therapeutic protein products during manufacturing, storage, shipping, and handling [2–4]. Chemical degradation of proteins involves fragmentation, oxidation (mostly occurring at methionine and tryptophan residues), deamidation (mostly occurring at asparagine residues), and disulfide scrambling. Physical degradation of proteins involves unfolding, dissociation, denaturation, adsorption, precipitation processes, and aggregates [4]. Aggregates are one of the most hazardous protein impurities of therapeutics because they are associated with high risks of immune response or immunogenicity [5–9]. Therefore, the rapid development of therapeutic protein products leads to a demand for robust analytical methods, for example analytical ultracentrifugation–sedimentation velocity (AUC) and asymmetric-flow field-flow fractionation (AF4), for quantifying protein aggregates to assess the risk they pose [10–14].
6258
The authorities demand that pharmaceutical companies strictly quantify and qualify the aggregates of therapeutic protein products [15]. Size-exclusion high-performance liquid chromatography (SE-HPLC) is a well-known method for quantifying the amount of aggregates, with high-throughput capacity and quantitative performance. Therefore, SE-HPLC is an indispensable method and is frequently used for quantification of protein aggregates. However, SE-HPLC has several challenges, including limited analytical range (
RESEARCH PAPER
Separation and quantification of monoclonal-antibody aggregates by hollow-fiber-flow field-flow fractionation Jun Fukuda & Takafumi Iwura & Shigehiro Yanagihara & Kenji Kano
Received: 20 May 2014 / Revised: 16 July 2014 / Accepted: 24 July 2014 / Published online: 13 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Hollow-fiber-flow field-flow fractionation (HF5) separates protein molecules on the basis of the difference in the diffusion coefficient, and can evaluate the aggregation ratio of proteins. However, HF5 is still a minor technique because information on the separation conditions is limited. We examined in detail the effect of different settings, including the main-flow rate, the cross-flow rate, the focus point, the injection amount, and the ionic strength of the mobile phase, on fractographic characteristics. On the basis of the results, we proposed optimized conditions of the HF5 method for quantification of monoclonal antibody in sample solutions. The HF5 method was qualified regarding the precision, accuracy, linearity of the main peak, and quantitation limit. In addition, the HF5 method was applied to non-heated Mab A and heatinduced-antibody-aggregate-containing samples to evaluate the aggregation ratio and the distribution extent. The separation performance was comparable with or better than that of conventional methods including analytical ultracentrifugation–sedimentation velocity and asymmetric-flow field-flow fractionation.
Keywords Antibodies aggregates . Hollow-fiber-flow field-flow fractionation . Size-exclusion chromatography . Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-8065-4) contains supplementary material, which is available to authorized users. J. Fukuda (*) : K. Kano (*) Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan e-mail: [email protected] e-mail: [email protected] J. Fukuda : T. Iwura : S. Yanagihara Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., Hagiwara-machi, Takasaki, Gunma 370-0013, Japan
Analytical ultracentrifugation . Asymmetric-flow field-flow fractionation
Introduction Study of therapeutic protein products, especially monoclonal antibodies, has recently attracted a great deal of attention from pharmaceutical industries. In the last 20 years over 20 monoclonal antibodies have been approved in a variety of therapeutic categories [1], and six antibody pharmaceuticals have been approved including Humira (adalimumab) and Remicade (infliximab), which were the two best-selling pharmaceuticals in 2012 (http://www.utobrain.co.jp/news/ 20130724.shtml). These biopharmaceutical products have drastically improved the quality of life of patients. Compared with small pharmaceutical molecules, therapeutic protein products are rather large entities with inherent physicochemical complexity. Therefore, many physical and chemical factors may lead to protein degradation and consequently affect the quality of therapeutic protein products during manufacturing, storage, shipping, and handling [2–4]. Chemical degradation of proteins involves fragmentation, oxidation (mostly occurring at methionine and tryptophan residues), deamidation (mostly occurring at asparagine residues), and disulfide scrambling. Physical degradation of proteins involves unfolding, dissociation, denaturation, adsorption, precipitation processes, and aggregates [4]. Aggregates are one of the most hazardous protein impurities of therapeutics because they are associated with high risks of immune response or immunogenicity [5–9]. Therefore, the rapid development of therapeutic protein products leads to a demand for robust analytical methods, for example analytical ultracentrifugation–sedimentation velocity (AUC) and asymmetric-flow field-flow fractionation (AF4), for quantifying protein aggregates to assess the risk they pose [10–14].
6258
The authorities demand that pharmaceutical companies strictly quantify and qualify the aggregates of therapeutic protein products [15]. Size-exclusion high-performance liquid chromatography (SE-HPLC) is a well-known method for quantifying the amount of aggregates, with high-throughput capacity and quantitative performance. Therefore, SE-HPLC is an indispensable method and is frequently used for quantification of protein aggregates. However, SE-HPLC has several challenges, including limited analytical range (
