C-terminal lysine (C-K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C-K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch-to-batch culture performance and product quality reproducibility is a key cell culture development criterion. Understanding the operational parameters affecting C-K levels provides valuable insight into the cell culture process. A CHO cell line X expressing a recombinant antibody was selected as the model cell line due to the exhibited sensitivity of its C-K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C-K level for in-process samples. The effects of operating conditions (i.e., temperature and culture duration) and media trace elements (copper and zinc) on C-K variants were studied. The dominant effect on C-K level was identified as the trace elements concentration. Specifically, increased C-K levels were observed with increase of copper concentration and decrease of zinc concentration in chemically defined medium. Further, a hypothesis for C-K processing with intracellular and extracellular carboxypeptidase activity was proposed, based on preliminary intracellular carboxypeptidase Western blot results and the extracellular HCCF holding study.

A new strategy for specifically targeting cysteine-containing peptides in a tryptic digest is described. The method is based on quantitatively derivatizing cysteine residues with a quaternary amine tag (QAT). Tags were introduced into proteins following reduction of disulfide bonds through derivatization of cysteine residues with (3-acrylamidopropyl)trimethylammonium chloride. After trypsin digestion, derivatized cysteine-containing peptides were enriched by strong cation exchange chromatography. The method was validated using model peptides and a protein. The QAT strategy has several advantages over other methods for the selection of cysteine-containing peptides. One is that it increases the ionization efficiency of cysteine-containing peptides. The other is that chromatographic selection is achieved with simple, robust cation exchange chromatography columns. As a result, this new strategy provides a simple way to facilitate enrichment of cysteine-containing peptides, thereby reducing sample complexity in bottom-up proteomics.

Agarose based immobilized metal affinity chromatography (IMAC) columns loaded with copper (II) were evaluated for the selection of histidine-containing peptides in comparative proteomics. Recovery, binding specificity, and reproducibility were investigated with model proteins. Cu(II)-IMAC was found to be highly selective for histidine containing peptides; moreover, a low degree of nonspecific selection was observed. Acylation of the amino-terminus of peptides with either succinic anhydride, N-acetoxysuccinamide, or [3-(2,5)-dioxopyrrolidin-1-yloxycarbonyl)-propyl]-trimethylammonium (quaternary amine) reduced the number of histidine-containing peptides bound by the Cu(II)-IMAC columns. This provides an additional possibility for sample simplification in proteomic applications. The number of acylated peptides selected decreased in the order of quaternary amine > N-acetoxysuccinamide > succinic anhydride derivatization. Although the selection of N-terminally derivatized peptides is biased toward peptides that contain more than one histidine, it is not yet possible to predict selectivity.

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