No abstract

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction.

Cyclooxygenase is involved in the biosynthesis and function of prostaglandins. It is a glycoprotein located in the endoplasmic reticulum and in the nuclear envelope, and it has been found to have two isoforms termed COX-1 and COX-2. This paper reports on the glycosylation site analysis of recombinant COX-2 using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) and nanoelectrospray (nanoESI) quadrupole-TOF (Q-TOF) MS. The nanoESI MS analysis of COX-2 revealed the presence of three glycoforms at average molecular masses of 71.4, 72.7, and 73.9 kDa. Each glycoform contained a number of peaks differing by 162 Da indicating heterogeneity and suggesting the presence of high-mannose sugars. The masses of the glycoforms indicate that oligosaccharides occupy two to four sites and a single N-acetylglucosamine (GlcNAc) residue occupied up to two sites. The MALDI MS analysis of a tryptic digest of the protein showed a number of potential glycopeptides. The peptides differed by 162 Da which further suggested high-mannose sugars. Nanoelectrospray MS/MS experiments confirmed glycosylation at the Asn 53 and Asn 130 sites and confirmed the presence of the peptides Asn 396-Arg 414 + GlcNAc and Thr 576-Arg 587 + GlcNAc containing Asn 580. It was not possible to conclusively determine whether the Asn 396 site was glycosylated via an MS/MS experiment, so the tryptic digest was deglycosylated to confirm the presence of the glycopeptides. Finally, a non-glycosylated tryptic peptide was observed containing the Asn 592.

SummaryThree different analysis platforms using LC-MS were successfully developed for pharmacokinetic (PK) studies of an antibody drug in serum. These analysis platforms can be selectively used for different types of protein drugs, which ranged from a very specific for a particular drug (antibody enrichment), to a less specific for any antibody drugs with Fc domain (protein A enrichment), and to a very generic method that can be used for any protein drugs (albumin depletion method). In this manner the three platforms will be applicable to a wide range of antibody therapeutic studies for different species. The analysis using an albumin depletion method (with SDS-PAGE) achieved the detection of the drug (to 1 ng) in an aliquot of serum (30 µL) with a 5-order magnitude of linearity. The analysis using protein A enrichment (with SDS-PAGE) achieved the detection of the drug at a 50 fold lower level (to 0.02 ng). Without using SDS-PAGE for separation, using protein A enrichment achieved the detection to 10 ng and using the anti-drug antibody enrichment achieved the detection to 0.1 ng, with a similar linear dynamic range. These three analysis platforms produced good agreement with a mimic PK study of the drug in monkey serum, as compared to ELISA approach. In addition, these analysis platforms can be selectively applied for PK studies of drugs with different requirements of development time and resources. Such as, the antibody enrichment method can be used in a high throughput manner but limited to a specific protein drug only. On the other hand, the albumin depletion method can be applied to many types of protein drugs, but with the laborious sample preparation steps (SDS-PAGE and the subsequent in-gel digestion). When anti-drug antibodies are not available for antibody drugs, or the sensitivity requirement is not stringent (e.g. > 10 ng), using protein A enrichment (without using SDS-PAGE) seems to be a good choice for PK studies which require fast throughput.

No abstract