Monoclonal antibody aggregates are the most significant product related impurity and are often times immunogenic, requiring typical final formulation targets of <1%.  Aggregates, especially dimers, are inherently difficult to separate and pose significant challenges in downstream purification demanding extensive method screening and process development.  Part of the difficulty of developing a dedicated and robust aggregate removal solution is the high variability and often reversible nature of aggregates.  In this work, we develop an approach to generate a reliable, non-reversible aggregate-containing antibody feed stream that can be used for development of advanced purification solutions.Two independent methods were developed: chemical cross-linking and pH shift.  The chemical cross-linking method was developed for generating covalent monoclonal antibody aggregates with a high ratio of  dimer to high molecular weight aggregates. The alternative method (pH shift) developed to form non-reversible aggregates for comparison used a hold at high pH and conductivity in order to simulate �process-induced aggregates.�  The aggregates were characterized using Protein A-HPLC, SDS-PAGE, SEC-HPLC, Weak Cation Exchange Chromatography, Mass Spectrometry, and cIEF.  To determine the relevance of the induced aggregates from a process standpoint, the model feeds and an inherent aggregate-containing feed were purified using an aggregate removal step. The similarities and differences in the performance of the aggregate removal step for the induced and natural aggregate feeds will also be presented.

Comparative characterization of monoclonal antibody aggregates formed by two generation methods for use as model feeds in downstream processing

243rd National Meeting (2012)

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