Dopamine Receptors

Peak No

Peak No. charge variants, proline amidation, copper, mass spectrometry Introduction The majority of recombinant monoclonal antibody (mAbs) products contain heterogeneous Nomilin variants. These variants are commonly the result of modifications that occur during cell culture production due to enzymatic processes or spontaneous degradation, and can accumulate during production, purification, formulation and storage.1C8 Heterogeneity in mAbs is represented by charge variants, typically caused by deamidation, isomerization, succinimide formation, oxidation, sialylation, N-terminal pyroglutamic acid or C-terminal lysine (Lys) clipping.9C12 In Nomilin addition to these variants, species of unknown origin may also exist, and these species must be characterized to ensure the safety and efficacy of the products.11,12 Characterization or comparability data must be generated in order to demonstrate the consistency in product quality for regulatory filings.13,14 C-terminal -amidation is a modification recently identified in mAbs. C-terminal proline amidation (pro-amidation) was first identified and characterized in 2007.15 In spite of its relatively widespread occurrence in bioactive proteins and short polypeptides from invertebrates and vertebrates, including human,16C19 the exact biological impact of proline amidation remains to be fully understood. In higher organisms, the amidation reaction is catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PAM). Human PAM expressed in Chinese hamster ovary (CHO) cells has been previously characterized in reference 20, and copper was shown to be critical for the catalytic function of the PAM.21 In addition, the copper also plays an important roles in the structure and molecular trafficking of the PAM.22,23 However, exactly how pro-amidation is mediated by certain ions remains unclear. During the development of a new chemically defined medium (CDM) platform cell culture process, it was found that supplementing copper in the production medium above the original levels in the historical medium formulation helped maintain cell viability and improve mAb titers. Here, we present a case study demonstrating the impact of copper concentration in the production media on the charge profiles of an IgG1. In a copper titration study, the relative abundance of basic variants detected by imaged capillary isoelectric focusing (ICIEF) was found to correlate directly Lepr with the copper concentration in the basal production media. We report that the C-terminal pro-amidation exists as a basic charge variant of the IgG1. In contrast to previous observations that pro-amidation exists as a minor proportion of the basic charge variants,15 pro-amidation constituted the majority of the basic charge variants of this IgG1, in single and double amidation forms at the C-terminus of the heavy chains. To further characterize the pro-amidation and charge variants in the IgG1, a pH gradient cation exchange-high performance liquid chromatography (pH-IEC) was employed to isolate the basic charge variants. Analyses of the basic charge variants from different productions also indicated that the basic peak levels measured by ICIEF and pH-IEC methods correlate well with the pro-amidation level determined by peptide mapping, further supporting the conclusion that the majority of the basic variants were due to Nomilin pro-amidation. Results Observation of basic charged variants. ICIEF profiles of the IgG1 generated with and without carboxyl peptidase B (CpB) treatment are shown in Figure 1. Compared to non-CpB treated sample with a basic peak at 8.1%, the similar basic level at 7.8% with the CpB treatment indicated that very few C-Lys containing variants were present. Consistent with previous observations,21 including the molecule presented here, higher basal media copper concentrations are correlated Nomilin with lower lactate accumulation during the production process. However, since higher copper results in higher levels of basic charge variants, a small scale (2 L bioreactors) study was conducted with variable copper ion concentrations to determine a target concentration for large scale production that would permit a well-controlled upstream process without compromising product quality. The purified antibodies were analyzed by ICIEF, and the resulting chromatograms are shown in Figure 2. While the acidic charge variant profile remained unchanged for all copper concentrations, the relative abundance of certain basic peaks increased with increasing copper concentration over the range tested (inset in Fig. 2B), suggesting an involvement of copper in mediating the basic charge profile of the IgG1. Open in a separate window Figure 1 Imaged capillary isoelectric focusing analysis of an IgG1 before and after CpB treatment. The samples were incubated with CpB at an enzyme to.