Issues and Challenges of Sub-visible and Submicron Particulate Analysis in Protein Solutions: Supplementary Information

Thomas M. Scherer,1,2 Stephenie Leung,1 Laura Owyang,1 and Steven J. Shire1

1Late Stage Pharmaceutical Development, Process Research and Development, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA

2To whom correspondence should be addressed. Thomas Scherer, Late Stage Pharmaceutical Development, Genentech Inc., 1 DNA Way, MS 56-1A, South San Francisco, CA 94080, USA, Telephone (650) 467-3338, e-mail: scherer.thomas@gene,com

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Supplemental Table I. Methods Evaluated for the Characterization of Particulates in High-Concentration Antibody Solutions

Method / Model / Manufacturer
Light obscuration / HIAC Royco 9703 / Hach Company, Loveland, CO, USA
Flow microscopy / Micro-Flow ImagingTM (MFI) / Brightwell Technologies, Inc., Ottowa, Canada
UV-Vis spectrophotometry / 8453/ G1103A / Agilent, Palo Alto, CA, USA,
Dynamic light scattering / Dynapro Titan, 30mW Dawn EOS / Wyatt Technology Corporation, Santa Barbara, CA, USA
BI-DS, 2.4W Argon-ion Laser / Brookhaven Instruments, Holtsville, NY, USA
Nanotrac Ultra / Microtrac Inc. Montgomeryville, PA, USA
Laser diffraction / LiQuilaz 02 / Particle Measurement Systems, Inc., Boulder, CO, USA
Bluewave Particle Size Analyzer / Microtrac Inc., Montgomeryville, PA, USA
LS320 / Beckman Coulter, Inc. Brea, CA, USA

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Supplemental Fig. 1. (a) Vials of MAb-A photographed at time points of 0, 8, 24, and 72 hours (from left to right) during agitation stress. (b) Image of 72-hour agitation stressed MAb-A (10-fold diluted) from microscope flow imaging analysis.

Supplemental Figure 2: Total sub-visible particle counts > 1 um by microscope flow imaging (high magnification) of MAb-A agitation-stressed (48 hours) sample diluted 20-fold in buffer as a function of the duration of the vacuum degassing procedure.

Supplemental Fig. 3: Absorbance vs. wave number of monodisperse polystyrene microsphere suspensions (0.002 % wt fractions) for particle size diameters of 20, 100, 560 and 980 nm. The vertical black line indicates the absorbance wavelength of 350 nm for reference.


Supplemental Fig. 4: Absorbance at 350 and 690 nm wavelengths measured as a function of 980-nm and 560 nm diameter PS bead standards measured as a function weight fraction in suspension. Good linearity of AU vs. weight fraction was observed, however with different slopes in for each sample and wavelength. PS polystyrene

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