No abstract

We present here an improved analytical method for the analysis of glycation events in proteins. Nonenzymatic glycation of an IgG2 monoclonal antibody was studied using affinity chromatography, mass spectrometry, and chemical derivatization. Analysis of both forced-degraded and bulk-drug substance (BDS) samples showed the presence of glycated protein. A new peptide mapping procedure, incorporating derivatization using sodium borohydride, allowed the development of a sensitive method for detecting and identifying the sites of modification. When combined with tandem mass spectrometry, peptides glycated by glucose showed dramatically improved MS/MS spectra as compared to underivatized controls. Using these methods we were able to map a number of glycation sites in both forced-degraded and BDS samples that were distributed across both light and heavy chain subdomains. The combination of affinity chromatography, high-resolution mass spectrometry, and a simple derivatization procedure should allow the facile analysis of glycation for other antibody and protein samples.

A fast and improved sample preparation scheme was developed for protein analysis using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) with laser-induced fluorescence detection. This CE-SDS method was developed as a purity assay for recombinant monoclonal antibodies (rMAbs). In this assay, rMAbs are derivatized with the fluorogenic reagent 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ) in the presence of a nucleophile (CN-), which fluoresces only upon covalent binding to the protein. Purification after labeling is therefore not necessary to remove unreacted reagents. Proteins are incubated at 75 degrees C for 5 min to facilitate denaturation and labeling. For nonreduced preparation, rMAbs are labeled at pH 6.5 with a dye-to-protein (D/P) molar ratio of 50:1, which forms conjugates having 6 +/- 4 FQ labels. For reduced preparation, rMAbs are labeled at pH 9.3 with a D/P molar ratio of 10:1, which generates light chain conjugates incorporated with 3 +/- 2 FQ labels. Labeling artifacts such as fragmentation or aggregation are absent with use of alkylation reagents. This efficient labeling scheme generates detection limits for FQ-labeled rMAbs as low as 10 ng/mL. In comparison to other labeling strategies, labeling proteins with FQ has the advantage of speed, ease of use, and robust quantification.

Mass analysis of recombinant protein therapeutics is an important assay for product characterization. Intact mass analysis is used to provide confirmation of proper translation of the DNA sequence and to detect the presence of post-translational modifications such as amino acid processing and glycosylation. We present here a method for the rapid mass analysis of antibodies using a polyhydroxyethyl aspartamide column operated in size-exclusion mode and coupled with ESI-MS. This method allows extremely efficient desalting of proteins under acidic conditions that are optimal for subsequent mass analysis using standard ESI conditions. Furthermore, this technique is significantly faster and more sensitive than rpHPLC methods, typically considered the standard chromatography approach for mass analysis of proteins. This method is flexible and robust, and should prove useful for applications where a combination of speed and sensitivity are required. M ass analysis of recombinant proteins is a key characterization assay used to evaluate the entire amino acid sequence of the molecule and the presence of post-translational modifications. As part of protein drug development, intact mass analysis supports the characterization package for regulatory filings and may be used to evaluate lot-to-lot consistency on a whole molecule level. Mass analysis of intact or reduced antibodies has been used to evaluate the degree of processing of C-terminal lysine on the heavy chain subunit [1]; evaluate N-terminal heterogeneity such as pyroglutamic acid formation [2,3]; profile N-linked carbohydrate heterogeneity [3,4]; and to detect instabilities in the molecule such as oxidation [5], succinimide formation from aspartic acid [6], glycation [7], internal cleavage [8], and thioether formation [9]. Reversed-phase HPLC (rpHPLC) separation followed by electrospray ionization-mass spectrometry (ESI-MS) analysis is the typical method for analyzing antibodies under both non-reduced and reducing conditions. This method is highly resolving and advantageous for the detection of minor product impurities and the resolution of different amino acid sequences or heterogeneous post-translational modifications. However, rpHPLC of antibodies has several distinct disadvantages. Due to the large size and relatively hydrophobic nature of antibodies, high temperatures are typically employed to improve elution and peak shape profiles [1,4]. However, high temperatures may lead to artifactual degradation of the sample during analysis. Inclusion of TFA as a mobile phase additive in rpHPLC is often necessary to obtain good chromatography [1], but may result in decreased ionization [10,11]. Additionally, reduction of antibodies and analysis of the constituent light and heavy chains often results in tailing of the heavy-chain component and the potential for carry-over problems. These issues can lead to lengthened run times or the inclusion of blank runs between samples to increase confidence in the results.We optimized the use of a commercially available polyhydroxyet...

A capillary isoelectric focusing (cIEF) method has been developed as an alternative to cation exchange chromatography to determine charge heterogeneity for a therapeutic antibody. Characterization of the cIEF profile is important to understand the charged isoform distribution. A variety of preparative IEF methods have been developed over the years but have had various limitations including high levels of contaminating ampholytes and complex fractionation and isolation procedures. More recently, an off-line method that uses pI-based separation on immobilized pH gradients was developed to preparatively isolate material with convenient liquid phase recovery. This method uses the Agilent OFFGEL 3100 Fractionator and was optimized to produce fractions of antibody charge isoforms differing by as little as 0.1 pI units. The isolation of highly resolved fractions then allowed for the identification of N- and C-terminal basic charge modifications including noncyclized glutamine, signal peptide extensions, and various levels of C-terminal lysine processing and high mannose structures. These species could then be correlated to specific peaks in the cIEF profile. This work shows that a preparative IEF method using immobilized pH gradients can be optimized to generate highly resolved, pI-based fractions in solution which can be used for successful cIEF profile characterization. Access to preparative amounts of discrete charged species allows for a better understanding of the underlying covalent modifications responsible for the charge differences and facilitates evaluation of the impact of these modifications on stability and potency of therapeutic antibodies.