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High-throughput analysis of sub-visible mAb aggregate particles using automated fluorescence microscopy imaging

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Abstract

Aggregation of therapeutic proteins is a major concern as aggregates lower the yield and can impact the efficacy of the drug as well as the patient’s safety. It can occur in all production stages; thus, it is essential to perform a detailed analysis for protein aggregates. Several methods such as size exclusion high-performance liquid chromatography (SE-HPLC), light scattering, turbidity, light obscuration, and microscopy-based approaches are used to analyze aggregates. None of these methods allows determination of all types of higher molecular weight (HMW) species due to a limited size range. Furthermore, quantification and specification of different HMW species are often not possible. Moreover, automation is a perspective challenge coming up with automated robotic laboratory systems. Hence, there is a need for a fast, high-throughput-compatible method, which can detect a broad size range and enable quantification and classification. We describe a novel approach for the detection of aggregates in the size range 1 to 1000 μm combining fluorescent dyes for protein aggregate labelling and automated fluorescence microscope imaging (aFMI). After appropriate selection of the dye and method optimization, our method enabled us to detect various types of HMW species of monoclonal antibodies (mAbs). Using 10 μmol L−1 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonate (Bis-ANS) in combination with aFMI allowed the analysis of mAb aggregates induced by different stresses occurring during downstream processing, storage, and administration. Validation of our results was performed by SE-HPLC, UV-Vis spectroscopy, and dynamic light scattering. With this new approach, we could not only reliably detect different HMW species but also quantify and classify them in an automated approach. Our method achieves high-throughput requirements and the selection of various fluorescent dyes enables a broad range of applications.

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Acknowledgments

This research was supported by the German Federal Ministry of Education and Research (Grant No. 0315342A) and by the Cooperative Research Training Group Pharmaceutical Biotechnology stated by the Postgraduate Scholarships Act of the Ministry for Science, Research, and Arts of the federal state government of Baden-Württemberg. Further funding was obtained by the German Research Foundation (DFG) through the International Graduate School in Molecular Medicine at Ulm University. In addition, the authors are grateful to Rentschler Biotechnologie GmbH (Laupheim, Germany) for providing mAb1 and to Boehringer Ingelheim Pharma GmbH & Co. KG (Ingelheim, Germany) for offering mAb2 and mAb3. The mAb4 was kindly donated by Kenneth Siddle (Cambridge University, England).

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Authors and Affiliations

  1. Institute of Applied Biotechnology, Biberach University of Applied Sciences, Hubertus-Liebrecht-Strasse 35, 88400, Biberach, Germany

    Albert Jesuran Paul, Fabian Bickel, Martina Röhm, Lisa Hospach, Bettina Halder, Nina Rettich, René Handrick, Eva Maria Herold, Hans Kiefer & Friedemann Hesse

  2. Medical Faculty, Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany

    Fabian Bickel & Martina Röhm

  3. Faculty of Natural Sciences, Ulm University, Albert-Einstein-Allee 11, 89079, Ulm, Germany

    Lisa Hospach, Bettina Halder & Nina Rettich

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  1. Albert Jesuran Paul
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Correspondence to Albert Jesuran Paul.

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Paul, A.J., Bickel, F., Röhm, M. et al. High-throughput analysis of sub-visible mAb aggregate particles using automated fluorescence microscopy imaging. Anal Bioanal Chem 409, 4149–4156 (2017). https://doi.org/10.1007/s00216-017-0362-2

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