Characterization of IgG1 Immunoglobulins and Peptide−Fc Fusion Proteins by Limited Proteolysis in Conjunction with LC−MS

Abstract

A combinatory approach for the characterization of post-translational and chemical modifications in high molecular weight therapeutic proteins like antibodies and peptide-Fc fusion proteins (MW > or = 50 000 Da) is presented. In this approach, well-established techniques such as limited proteolysis, reversed-phase (RP) high-performance liquid chromatography (HPLC), and in-line mass spectrometry (MS) were combined for the characterization of a monoclonal IgG1 antibody and three different peptide-Fc fusion proteins. The one commonality of these molecules is the presence of a similarly accessible lysine residue either located in the flexible hinge region of the antibody or in the flexible linker of the peptide-Fc fusion proteins. Applying limited proteolysis using endoproteinase Lys-C resulted in the predominant cleavage C-terminal of this lysine residue. The created fragments, two identical Fab domain fragments and one Fc domain fragment derived from the IgG1 antibody and one Fc domain fragment and each of the three individual peptide moieties generated from the peptide-Fc fusion proteins, were readily accessible for complete separation by RP-HPLC and detailed characterization by in-line MS analysis. This approach facilitated rapid detection of a variety of chemical modifications such as methionine oxidation, disulfide bond scrambling, and reduction as well as the characterization of various carbohydrate chains. We found limited proteolysis followed by RP-HPLC-MS to be less time-consuming for sample preparation, analysis, and data interpretation than traditional peptide mapping procedures. At the same time, the reduced sample complexity provided superior chromatographic and mass spectral resolution than the analysis of the corresponding intact molecules or a large number of enzymatically generated fragments.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

“…A method using Endoproteinase Lys-C has been described for generating Fab fragments. 21 Therefore, we investigated the use of Endoproteinase Lys-C to generate an h2E2 Fab fragment, followed by batch ion exchange removal of the Fc fragment, and size exclusion chromatography, to remove any undigested antibody and aggregates. Conditions for the Endo Lys-C digestion were optimized in terms of time, temperature, buffer, and ratio of protease to antibody, to achieve nearly complete generation of Fab fragments, with only very small amounts of nonselective over-digestion.…”

The use of native cation‐exchange chromatography to study aggregation and phase separation of monoclonal antibodies

“…Fab and Fc fragments are easily produced from IgG1 molecules via Lys-C or papain digestion. 15,29 However, limited proteolysis of IgG2 molecules is much more difficult and requires optimization. The protocol shown in the Materials and Methods section was derived from systematic experimentation on IgG2-A aimed at maximizing the yield of native Fab and Fc fragments.…”

“…Limited proteolysis of an IgG1 mAb using endoproteinase Lys-C Fab and Fc fragments from an IgG1 antibody (IgG1-B) were obtained by limited proteolysis using endoproteinase Lys-C. 15,29 The antibody sample was diluted to a concentration of 1 mg/mL in 100 mM Tris-HCl buffer (pH 7.5) and incubated with Lys-C at an enzyme-to-substrate ratio of 1:200 (w/w) at 37 C for 10 min. The digestion reaction was quenched by adding 1/4 reaction volume of 150 mM ammonium acetate buffer (pH 4.7).…”

Peptide mapping of therapeutic monoclonal antibodies: Improvements for increased speed and fewer artifacts

“…Analytical characterization of these molecules is extremely difficult and often very time consuming. One common method to evaluate the primary structure (identity) of a monoclonal antibody and other protein-based pharmaceuticals such as FC fusion proteins and immunoconjugates is peptide mapping [2][3][4][5][6][7]. This method was often reserved for comparability but recently is moving into many modern stability programs and lot release tests making it necessary for a quick yet effective method [8].…”