Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain.

We investigated the formation and stability of succinimide, an intermediate of deamidation events, in recombinant monoclonal antibodies (mAbs). During the course of an analytical development study of an IgG1 mAbs, we observed that a specific antibody population could be separated from the main product by cation-exchange (CEX) chromatography. The cell-based bioassay measured a approximately 70% drop in potency for this fraction. Liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) and tandem mass spectrometry (LC-MS/MS) analyses showed that the modified CEX fraction resulted from the formation of a succinimide intermediate at Asn 55 in the complementarity determining region (CDR) of the heavy chain. Biacore assay revealed a approximately 50% decrease in ligand binding activity for the succinimide-containing Fab with respect to the native Fab. It was found that the succinimide form existed as a stable intermediate with a half-life of approximately 3 h at 37 degrees C and pH 7.6. Stress studies indicated that mildly acidic pH conditions (pH 5) favored succinimide accumulation, causing a gradual loss in potency. Hydrolysis of the succinimide resulted in a further drop in potency. The implications of the succinimide formation at Asn 55, a highly conserved residue among IgG1 (mAbs), are discussed.

Analysis of human antibody IgG2 domains by reversed-phase liquid chromatography and mass spectrometry.

It has been well documented that papain cleaves an IgG1 molecule to release Fab and Fc domains; however, papain was found unable to release such domains from an IgG2. Here we present a new combinatory strategy to analyze the heterogeneity of the light chain (LC), single chain Fc (sFc), and Fab portion of the heavy chain (Fd) of an IgG2 molecule released by papain cleavage under mild reducing conditions. These domains were well separated on reversed-phase high performance liquid chromatography (RP-HPLC) and analyzed by in-line liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS). In addition, some modifications of these domains were revealed by in-line mass spectrometry, and confirmed by the peptide mapping on LC-MS/MS analysis. This same strategy was proven suitable for IgG1 molecules as well. This procedure provides a simplified approach for the characterization of antibody biomolecules by facilitating the detection of low-level modifications in a domain. In addition, the technique offers a new strategy as an identification assay to distinguish IgG2 molecules on RP-HPLC, by which highly conserved Fc domains remain at a constant retention time (RT) unique to its subisotype, while varying RTs of the light chain and the Fd distinguish the monoclonal antibody from other molecules of the same isotype based on the underlying characteristics of each antibody.

Analysis of post-translational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry.

Characterization and quantitative analysis of modifications in recombinant monoclonal antibodies (mAbs) plays an important role in biopharmaceutical development. This study demonstrates a new approach to assess variants in mAbs, based on individual analysis of subdomains. These subdomains were generated by dithiothreitol reduction and papain cleavage. A reversed-phase LC-MS method was developed that provides efficient separation of subdomains (light and heavy chains, Fab and Fc) containing several specific modifications such as pyroglutamic acid, deamidation, isomerization and oxidation. The best separation was achieved on Zorbax SB C8 columns using linear water-acetonitrile gradients in 0.1% trifluoroacetic acid. Deconvoluted electrospray ionization mass spectra of these domains revealed the modification profiles of these variants with high accuracy and resolution. This study presents a strategy that offers orthogonal approaches to analyze antibody variants, and provide a qualitative and quantitative assessment of mAb heterogeneity.

Identification of naturally processed CD4 T cell epitopes from the prostate-specific antigen kallikrein 4 using peptide-based in vitro stimulation.

Kallikrein (KLK)4 is a recently described member of the tissue kallikrein gene family that is specifically expressed in normal and prostate tumor tissues. The tissue-specific expression profile of this molecule suggests that it might be useful as a vaccine candidate against prostate cancer. To examine the presence of CD4 T cells specific for KLK4 in PBMC of normal individuals, a peptide-based in vitro stimulation protocol was developed that uses overlapping KLK4-derived peptides spanning the majority of the KLK4 protein. Using this methodology, three naturally processed CD4 epitopes derived from the KLK4 sequence are identified. These epitopes are restricted by HLA-DRB1*0404, HLA-DRB1*0701, and HLA-DPB1*0401 class II alleles. CD4 T cell clones specific for these epitopes are shown to efficiently and specifically recognize both recombinant KLK4 protein and lysates from prostate tumor cell lines virally infected to express KLK4. CD4 T cells specific for these KLK4 epitopes are shown to exist in PBMC from multiple male donors that express the relevant class II alleles, indicating that a CD4 T cell repertoire specific for KLK4 is present and potentially expandable in prostate cancer patients. The demonstration that KLK4-specific CD4 T cells exist in the peripheral circulation of normal male donors and the identification of naturally processed KLK4-derived CD4 T cell epitopes support the use of KLK4 in whole gene-, protein-, or peptide-based vaccine strategies against prostate cancer. Furthermore, the identification of naturally processed KLK4-derived epitopes provides valuable tools for monitoring preexisting and vaccine-induced responses to this molecule.