Free sulfhydryl in recombinant monoclonal antibodies.

Monoclonal antibody (mAb) therapy applications have been growing rapidly in recent years. Like other recombinant protein drugs, therapeutic mAb's need to be well characterized to ensure their structural and functional integrity. IgG mAb's are composed of two heavy and two light chains covalently linked by interchain disulfide bonds. Each domain of the heavy or light chain contains one additional disulfide bond. Native IgG mAb's, with completely formed disulfide bonds, should not bear any free sulfhydryl. This report describes detection and quantification of free sulfhydryl in recombinant mAb's produced in Chinese hamster ovary (CHO) cells using a fluorescent technique. The method utilizes the fluorescent probe N-(1-pyrenyl)maleimide (NPM). The purified mAb's appear to be homogeneous under native conditions with approximately 0.02 mol of free sulfhydryl per mole of protein. Upon denaturation, minor species related to the mAb's are observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the free sulfhydryl level is determined to be approximately 0.1 mol/mol of protein. These results suggest that a small portion of these recombinant mAb's lack in intermolecular disulfide bonds but remain noncovalently associated under native conditions. The formation of the free sulfhydryl containing mAb species is likely to occur during the culture process and/or protein folding process in the endoplasmic reticulum (ER).

Analysis of isoaspartate in a recombinant monoclonal antibody and its charge isoforms.

Monoclonal antibody (mAb) therapy applications have been growing rapidly in recent years. Like other proteins, therapeutic mAbs can under go various enzymatic and non-enzymatic reactions that can affect their structural integrity and stability. Among the degradation reactions, isoaspartate (isoAsp) formation is one of the major sources of charge heterogeneity of mAbs. This paper reports the detection and quantification of isoAsp in a recombinant mAb and its charge isoforms resolved by cation exchange high performance liquid chromatography. The assay utilizes the enzyme protein isoaspartyl methyltransferase in conjunction with strong cation exchange separation and UV detection (at 260 nm) of S-adenosyl-L-homocysteine, which is produced stoichiometrically in the enzymatic reaction. The mAb is found to contain an average 0.2 mol of isoAsp per mol of protein, however, various charge isoforms were found to contain different levels of isoAsp. The most acidic isoforms contain approximately 0.7 mol of isoAsp per mol of protein, and no isoAsp is detected in the most basic isoform. It appears that the majority of isoAsp in the mAb is formed as a result of asparagine deamidation.

Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody.

Recombinant monoclonal antibodies (mAbs) are an emerging therapeutic area. However, there are few reports on disulfide bond assignment of recombinant mAbs. This work describes the complete disulfide bond assignment of a recombinant immunoglobulin G4 (IgG4) mAb. N-ethylmaleimide (NEM) was used to mask free sulfhydryl groups present in the mAb. Digestion of the mAb with endoproteinase Lys-C without disulfide scrambling was achieved by denaturing the mAb in the presence of NEM in guanidine hydrochloride (GuHCl). The Lys-C digest was subsequently reduced with dithiothreitol (DTT). Native and reduced Lys-C digests were mass analyzed by on-line reversed-phase-high-performance liquid chromatography mass spectrometry (RP-HPLC/MS). Disulfide-containing peptides were sequenced by off-line nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoESI-QTOF MS) and N-terminal Edman sequencing for verifying connectivities. The recombinant IgG4 mAb was found to contain the expected disulfide linkages with the proposed method. The NEM alkylating reagent was critical in minimizing disulfide scrambling during the denaturation and digestion of the mAb. This integrated approach, combining MS and N-terminal Edman sequencing, was capable of assigning the disulfide pattern of the IgG4 mAb rapidly and completely, and should be applicable for disulfide bond assignment and structural analysis of other mAbs and large proteins with multiple disulfide bonds.