Identification of multiple sources of charge heterogeneity in a recombinant antibody

Abstract

Seven forms of a therapeutic recombinant antibody that binds to the her2/neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.

Introduction

The general structural features of immunoglobulins have been known for decades, including the complete amino acid sequence and disulfide bonding pattern for IgG₁ [1] as well as the typical N-linked oligosaccharides found at a conserved position in the C_H2 domain [2]. For recombinant monoclonal antibodies whose light chain and heavy chain sequences are known in advance of any chemical characterization, there would appear to be little potential for the discovery of novel or interesting features, particularly for a large molecule with four polypeptide chains. However, a number of reports are now available detailing variations of covalent structure that introduce undesirable properties, including methionine oxidation that affects the potency and stability of OKT3 [3], a mutation unintentionally introduced during transfection [4], glycosylation differences that influence F_c effector functions [5], or antibody fragmentation [6]. Assays for the determination of the extent of overall deamidation [7], the types of N-linked oligosaccharides [8], or the extent of heavy chain glycosylation [9] are available. The processing of C-terminal lysine residues from antibody heavy chains has been noted by many [10], but this is not a significant issue except that it introduces charge heterogeneity that can mask other structural variants.

The remaining challenge for the analytical chemist is to resolve minor forms, determine the structural features that distinguish them from the major form, and to assign their potency or pharmacokinetic properties. This task is complicated for monoclonal antibodies by the dimeric nature of the molecule, since often only one light or heavy chain is affected, and by the overall size (approximately 148 kDa). A number of analytical approaches have been described, including peptide mapping [3], [4], [11], mass spectrometry [12], [13], hydrophobic interaction chromatography [14], [15], [16], capillary electrophoresis [9], and cation exchange after papain digestion [17]. Approval by regulatory authorities for therapeutic recombinant antibodies requires the development of methods that can show key structural features, particularly those that reveal instability that could affect potency or pharmacokinetic aspects. In addition, such methods often have value for formulation development and/or in the assignment of product expiration dating [18].

rhuMAb HER2 (Herceptin^®, trastuzumab for injection) has been licensed for the treatment of metastatic breast cancer involving over-expression of the her2/neu gene [19]. In unpublished studies with muMAb 4D5, the murine monoclonal anti-p185^her2/neu antibody [20] that is the parent of the humanized antibody (rhuMAb HER2), we found that light chain Asn30 was prone to deamidation when stored at elevated temperatures in a pH 7.4 phosphate-buffered saline (PBS) excipient; this residue was part of a muMAb 4D5 light chain CDR segment that was transferred to the humanized antibody [21]. Subsequent studies with rhuMAb HER2 showed that a succinimide accumulated at the heavy chain Asp102 position when the humanized antibody was incubated at pH 5 in an acetate-NaCl buffer [14]. We investigated the possibility that cation-exchange chromatography could serve as a single analytical method that would allow monitoring of Asn30 deamidation and Asp102 succinimide formation. In addition to these known charge variants, four other minor peak fractions were isolated by cation-exchange chromatography and characterized.

The cation-exchange method described in this paper is the validated method used for the release of rhuMAb HER2 production lots. Seven forms can be resolved by cation-exchange chromatography. All were assigned using a variety of analytical methods, including trypsin and endoproteinase Asp-N peptide map analyses, hydrophobic interaction chromatography (HIC) after papain digestion, isoelectric focusing in a polyacrylamide gel (IEF), and sequential Edman degradation after alkaline hydroxylamine cleavage. Assignment of peptide fractions required sequential Edman degradation and MALDI–TOF mass spectrometry. The potencies of three forms were determined using an assay that measures the ability of rhuMAb HER2 to inhibit proliferation of a human breast carcinoma (BT-474) cell line that over-expresses the her2/neu gene.