Development of At-Line Assay to Monitor Charge Variants of MAbs During Production M. M. St. Amand and B. A. Ogunnaike Dept. of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716 A.S. Robinson Dept. of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716 Dept. of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA 70118 DOI 10.1002/btpr.1848 Published online December 19, 2013 in Wiley Online Library (wileyonlinelibrary.com) One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post- translational modifications and PK behavior. However, one major restriction to implementa- tion of quality control strategies is the availability of techniques for obtaining on-line or at- line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2D- HPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequen- tial flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and deliv- ered to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions. V C 2013 American Institute of Chemi- cal Engineers Biotechnol. Prog., 30:249–255, 2014 Keywords: monoclonal antibody, charge variants, at-line monitoring, HPLC, bioreactor Introduction Monoclonal antibodies (MAbs) are proteins with a high specificity for a target molecule known as an antigen. This specificity makes MAbs ideal treatments for a variety of dis- eases ranging from rheumatoid arthritis, multiple sclerosis, heart disease and cancer. As such, MAbs comprise the larg- est sector of the biopharmaceutical industry with more than 30 FDA approved therapies currently on the market and global sales of $20.3 billion in 2011. 1 Though MAbs can be used to treat a variety of diseases effectively, their production costs can make them prohibi- tively expensive. In recent years, there has been much debate over the cost effectiveness of such treatments. 2–7 For exam- ple, Bevacizumab, a monoclonal antibody used to treat meta- static colorectal cancer, has an estimated treatment cost of $39,614 per patient. In 2005, the potential lifetime absolute costs to the Canadian health care system for Bevacizumab treatment were estimated at $299 million, an increase in expenditure of 21% over conventional non-MAb treatments. 2 In 2010, the total cost to treat patients with colorectal cancer in the US was $14.4 billion and is estimated to increase to $16.6 billion by 2020. 8 With such high treatment costs for colorectal cancer, some national health care systems have found that Bevacizumab is not a cost effective treatment, and thus Bevacizumab is not available to patients who might have otherwise been helped by the drug. 3 Similar analyses have found that other MAbs are also not cost-effective treatments. 7,9,10 Biosimilars or generics could reduce the treatment costs associated with monoclonal antibody therapeutics and other biopharmaceuticals and hence make these treatments more accessible. However, issues surrounding the quality of biosi- milars and generic biopharmaceuticals remain a topic of debate in the industry. The concerns about the quality of these proteins stem from the presence of protein microheter- ogeneity. Specifically for MAbs, various chemical and Correspondence concerning this article should be addressed to A. S. Robinson at asr@tulane.edu. V C 2013 American Institute of Chemical Engineers 249