RESEARCH ARTICLES Biotechnology Aggregation and pH–Temperature Phase Behavior for Aggregates of an IgG2 Antibody ERINC SAHIN, 1 WILLIAM F. WEISS IV, 1 ANDREW M. KROETSCH, 1 KEVIN R. KING, 2 R. KENDALL KESSLER, 2 TAPAN K. DAS, 2 CHRISTOPHER J. ROBERTS 1 1 Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716 2 Pfizer Biotherapeutics Research and Development, Chesterfield, Missouri 63017 Received 28 August 2011; revised 19 December 2011; accepted 27 December 2011 Published online 13 January 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23056 ABSTRACT: Monomer unfolding and thermally accelerated aggregation kinetics to produce soluble oligomers or insoluble macroscopic aggregates were characterized as a function of pH for an IgG2 antibody using differential scanning calorimetry (DSC) and size-exclusion chromatog- raphy (SEC). Aggregate size was quantified via laser light scattering, and aggregate solubility via turbidity and visual inspection. Interestingly, nonnative oligomers were soluble at pH 5.5 above approximately 15 ◦ C, but converted reversibly to visible/insoluble particles at lower tem- peratures. Lower pH values yielded only soluble aggregates, whereas higher pH resulted in insoluble aggregates, regardless of the solution temperature. Unlike the growing body of liter- ature that supports the three-endotherm model of IgG1 unfolding in DSC, the results here also illustrate limitations of that model for other monoclonal antibodies. Comparison of DSC with monomer loss (via SEC) from samples during thermal scanning indicates that the least confor- mationally stable domain is not the most aggregation prone, and that a number of the domains remain intact within the constituent monomers of the resulting aggregates. This highlights continued challenges with predicting a priori which domain(s) or thermal transition(s) is(are) most relevant for product stability with respect to aggregation. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:1678–1687, 2012 Keywords: biotechnology; calorimetry (DSC); physical stability; protein aggregation; solubility INTRODUCTION Monoclonal antibodies (mAbs), like most therapeutic proteins, are subject to both physical and chemical in- stabilities during product manufacture and storage. 1 Physical instabilities often manifest as partial unfold- ing leading to nonnative aggregate formation, with aggregates spanning from soluble dimers and small oligomers to high-molecular-weight (HMW) soluble and/or insoluble aggregates. 1,2 In this context, the Erinc Sahin and Andrew M. Kroetsch’s current address is Drug Product Science and Technology, Research and Development, Bris- tol–Myers Squibb, New Brunswick, New Jersey 08903. William F. Weiss IV’s present address is Biopharmaceutical Research and Development, Eli Lilly and Company, Indianapolis, Indiana 46285. Correspondence to: Christopher J. Roberts (Telephone: +302- 831-0838; Fax: +302-831-1048; E-mail: cjr@udel.edu) Journal of Pharmaceutical Sciences, Vol. 101, 1678–1687 (2012) © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association term soluble indicates that particles (aggregates) are not sufficiently large and/or dense to sediment eas- ily during benchtop centrifugation, 2,3 and does not denote an exact size range. Aggregates of any size must be monitored and characterized for reasons of product quality, pharmaceutical elegance, and regu- latory compliance. 2,3 For a more detailed discussion on the relationship between particle size and solubil- ity, readers are directed to a review article by Das and Nema. 4 Nonnative aggregation is often irreversible under the solution conditions of aggregate formation 2,5 ; as such, the kinetics of aggregation ultimately deter- mine product stability as a function of temperature, protein concentration, and solvent composition. 6–9 Nonnative aggregation (hereafter simply denoted as aggregation) at least putatively involves partial unfolding to create aggregation-prone or “reactive” monomers that then assemble through one or more 1678 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 5, MAY 2012