Specific and high-resolution identification of monoclonal antibody fragments detected by capillary electrophoresis–sodium dodecyl sulfate using reversed-phase HPLC with top-down mass spectrometry analysis

Abstract

In recent years, capillary electrophoresis-sodium dodecyl sulfate (cSDS) has been widely used for high resolution separation and quantification of the fragments and aggregates of monoclonal antibodies (mAbs) to ensure the quality of mAb therapeutics. However, identification of the low-molecular-weight (LMW) and high-molecular-weight (HMW) species detected in cSDS electropherograms has been based primarily on the approximate MWs calculated from standard curves using known MW standards and correlations with fragments and aggregates identified by other methods. It is not easy to collect sufficient amounts of H/LMW species from cSDS for analysis by orthogonal methods and the direct coupling of cSDS with mass spectrometry (MS) is very difficult due to interference from SDS. In this study, we describe the precise identification of H/LMW species detected by cSDS using reversed-phase high performance liquid chromatography (RP-HPLC) coupled with top-down tandem MS analysis. The H/ LMW species were first identified by on-line RP-HPLC MS analysis and the RP-HPLC fractions were then analyzed by cSDS to connect the identified H/LMW species with the peaks in the cSDS electropherogram. With this method, 58 unique H/LMW species were identified from an immunoglobulin G1 (IgG1) mAb. The identified fragments ranged from 10 kDa single chain fragments to 130 kDa triple chain fragments, including some with post-translational modifications. This is the first study to clearly identify the antibody fragments, including the exact clipping sites, observed in cSDS electropherograms. The methodology and results presented here should be applicable to most other IgG1 mAbs.

Identification of a CE-SDS shoulder peak as disulfide-linked fragments from common CH2 cleavages in IgGs and IgG-like bispecific antibodies

“…Clipping within the C H 2 domain, such as the DP (aspartic acid, proline) clipping at D270 (EU numbering), is commonly observed in mAbs, even in unstressed material, as reported in this and many other studies. 2 , 8 , 11 , 15 , 17 , 18 C H 2 cleavages at D270, histidine (H)285, asparagine (N)286, N297, and N315 were detected only by rCE-SDS in this study. Because cleavages at these sites generate fragments that are still linked by the cysteine (C)261-C321 intrachain disulfide bond within the C H 2 domain, the authors hypothesized that these clipped, but not dissociated, species would comigrate with intact IgG in nrCE-SDS.…”

“… 3–11 Owing to difficulties in direct fraction collection or online coupling to a mass spectrometer, studies on CE-SDS peak identification largely rely on: 1) prior knowledge of possible species under certain conditions; 2) sodium dodecyl sulfate–polyacrylamide gel electrophoreses (SDS-PAGE) separation, gel band excision, and in-gel digestion peptide mapping; 3) gel-free fractionation, intact mass, and peptide mapping; 12 4) SEC-based fractionation with offline intact mass, peptide mapping, and CE-SDS; 10 , 13 , 14 and 5) reversed-phase liquid chromatography (RPLC) based fractionation, intact mass, top-down tandem mass spectrometry (MS/MS), or offline peptide mapping and CE-SDS. 15 …”

“…Recently, Wang et al 15 reported a comprehensive CE-SDS peak assignment for an IgG1 mAb. All peaks observed in both nrCE-SDS and rCE-SDS of an IgG1, with and without heat stress (40°C for 3 months), were identified through RPLC fractionation, intact LCMS, and top-down MS/MS.…”

In vitro evaluation of potential interference of lokivetmab with protein electrophoresis and immunofixation

“…Another limitation regarding our methodology is, that after the dilution scheme, the actual amount of mAb in the obtained fraction is unknown. This could have been overcome by performing additional investigations, for example high-performance liquid chromatography [16].…”

Advancements in the co-formulation of biologic therapeutics

“…Capillary electrophoresis (CE) and imaged capillary isoelectric focusing (iCIEF), separate the mixed biologics based on their charge heterogeneity [ 67 , 68 ]. These methods can be used along with chromatography to map out the degradation species in solution.…”