Characterisation of Stress-Induced Aggregate Size Distributions and Morphological Changes of a Bi-Specific Antibody Using Orthogonal Techniques  Zahra Hamrang, Maryam Hussain, Katie Tingey, Malgorzata Tracka, José R. Casas-Finet, Shahid Uddin, Christopher F. van der Walle, Alain Pluen  Journal of Pharmaceutical Sciences  Volume 104, Issue 8, Pages 2473-2481 (August 2015) DOI: 10.1002/jps.24530 Copyright © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 1 Schematic depicting the relative particle size ranges characterised by several analytical techniques. Asterisk (*) denotes methods that are unable to quantify absolute particle size distributions. SEC refers to size-exclusion chromatography, CE-SDS to capillary electrophoresis-sodium dodecyl sulphate, AUC to analytical ultracentrifugation, FFF to field-flow fractionation and MALLS to multi-angle laser light scattering. Adapted from Hamrang et al.,10 Singh et al.11 and Zölls et al.12 with permission from Elsevier, John Wiley & Sons and John Wiley & Sons, respectively. Journal of Pharmaceutical Sciences 2015 104, 2473-2481DOI: (10.1002/jps.24530) Copyright © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 2 Typical particle images obtained from MFI measurements of 1 and 10mg/mL mAb2 samples subjected to thermal stress (top) and corresponding radar chart plots of particle concentrations for aggregates with an equivalent circle diameter in the 1–2μm and greater than 2, 10, 25 and 50μm size range. Journal of Pharmaceutical Sciences 2015 104, 2473-2481DOI: (10.1002/jps.24530) Copyright © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 3 Aggregates from control and thermally-stressed mAb2 samples labelled with SYPRO red (top) and mAb2 aggregate size distributions obtained from RICS and MFI measurements represented by a box plot for 1 and 10mg/mL samples subjected to thermal stress at 58°C. Sample means are represented by •, outliers by ♦, medians by horizontal lines, minima/maxima by asterisks, upper and lower range by vertical lines. Journal of Pharmaceutical Sciences 2015 104, 2473-2481DOI: (10.1002/jps.24530) Copyright © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 4 Typical particle images obtained from MFI measurements of 10mg/mL mAb2 samples subjected to the controlled freeze–thaw (F–T) stress protocol (top) and corresponding cumulative particle counts (mean and mean−SD) for aggregates of 1 and 10mg/mL mAb2 samples subjected to controlled F–T stress with an equivalent circle diameter in the 1–2μm, and greater than 2, 10, 25 and 50μm size range (bottom). Journal of Pharmaceutical Sciences 2015 104, 2473-2481DOI: (10.1002/jps.24530) Copyright © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 5 Typical particle images obtained from MFI measurements of 10mg/mL mAb2 samples subjected to the uncontrolled freeze–thaw (F–T) stress protocol (top) and corresponding particle counts (mean, and mean−SD) for aggregates of 1 and 10mg/mL mAb2 samples subjected to uncontrolled F–T stress with an equivalent circle diameter in the 1–2μm, and greater than 2, 10, 25 and 50μm size range. Journal of Pharmaceutical Sciences 2015 104, 2473-2481DOI: (10.1002/jps.24530) Copyright © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 6 Aggregates from control and freeze-thaw stressed 10mg/mL mAb2 samples labelled with SYPRO red (left) and mAb2 aggregate size distributions obtained from RICS and MFI measurements represented by a box plot for 1mg/mL (a) and 10mg/mL (b) samples subjected to controlled freeze–thaw and 1mg/mL (c) and 10mg/mL (d) samples subjected to uncontrolled freeze–thaw stress (right). Sample means are represented by •, outliers by ♦, medians by horizontal lines, minima/maxima by asterisks, upper and lower range by vertical lines. Journal of Pharmaceutical Sciences 2015 104, 2473-2481DOI: (10.1002/jps.24530) Copyright © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 7 Radar chart plots of morphological parameters obtained from MFI data for mAb2 samples subjected to thermal stress at 58°C overnight across the five particle size classes. Journal of Pharmaceutical Sciences 2015 104, 2473-2481DOI: (10.1002/jps.24530) Copyright © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 8 Radar chart arrays of morphological parameters versus particle size for mAb2 sub-visible aggregates generated from controlled and uncontrolled freeze–thaw stress treatment as measured by MFI. Journal of Pharmaceutical Sciences 2015 104, 2473-2481DOI: (10.1002/jps.24530) Copyright © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions