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Comparative Evaluation of Two Methods for Preparative Fractionation of Proteinaceous Subvisible Particles—Differential Centrifugation and FACS

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Abstract

Purpose

The goal of this study was to compare and evaluate two preparative techniques for fractionation of proteinaceous subvisible particles. This work enables future studies to address the potential biological consequences of proteinaceous subvisible particles in protein therapeutic products.

Methods

Particles were generated by heat stress and separated by size using differential centrifugation and FACS (Fluorescence-activated cell sorter). Resulting fractions were characterized by size-exclusion chromatography, light obscuration, flow imaging microscopy and resonant mass measurement.

Results

Here we report the optimization and comprehensive evaluation of two methods for preparative fractionation of subvisible proteinaceous particles into distinct size fractions in the range between 0.25 and 100 μm: differential centrifugation and FACS. Using these methods, well-defined size fractions were prepared and characterized in detail. Critical assessment and comparison of the two techniques demonstrated their complementarity and for the first time—their relative advantages and drawbacks.

Conclusions

FACS and differential centrifugation are valuable tools to prepare well-defined size-fractions of subvisible proteinaceous particles. Both techniques possess unique and advantageous attributes and will likely find complementary application in future research on the biological consequences of proteinaceous subvisible particles.

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Abbreviations

AF4:

Asymmetric flow field-flow fractionation

CCF:

Central composite face-centered

DoE:

Design of experiments

FACS:

Fluorescence-activated cell sorter

FI:

Flow imaging microscopy

FSC:

Forward scattering

IgG:

Immunoglobulin G

LO:

Light obscuration

MAb1:

Monoclonal antibody

PBS:

Phosphate buffered saline

RMM:

Resonant mass measurement

RSM:

Response surface methodology

UV:

Ultraviolet light

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ACKNOWLEDGMENTS AND DISCLOSURES

We thank Tarik Khan for editorial help, Gabriela Quebatte and Adeline Boillon for helpful discussions and Marc Bedoucha for providing access to instrumentation and technical assistance.

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Author notes

    Authors and Affiliations

    1. Pharma Technical Development (Biologics) Europe, Analytical Development and Quality Control, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070, Basel, Switzerland

      Björn Boll, Emilien Folzer, Christof Finkler & Atanas V. Koulov

    2. Pharma Technical Development (Biologics) Europe, Pharmaceutical Development & Supplies, F. Hoffmann-La Roche Ltd., Basel, Switzerland

      Emilien Folzer, Hanns-Christian Mahler & Roland Schmidt

    3. Department of Pharmaceutical Sciences, Division of Pharmaceutical Technology, University of Basel, Basel, Switzerland

      Björn Boll, Emilien Folzer & Jörg Huwyler

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    1. Björn Boll
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    2. Emilien Folzer
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    5. Hanns-Christian Mahler
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    7. Atanas V. Koulov
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    Correspondence to Atanas V. Koulov.

    Additional information

    Björn Boll and Emilien Folzer contributed equally to this work.

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    Boll, B., Folzer, E., Finkler, C. et al. Comparative Evaluation of Two Methods for Preparative Fractionation of Proteinaceous Subvisible Particles—Differential Centrifugation and FACS. Pharm Res 32, 3952–3964 (2015). https://doi.org/10.1007/s11095-015-1755-6

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    • DOI: https://doi.org/10.1007/s11095-015-1755-6

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