A generic method for intact and subunit level characterization of mAb charge variants by native mass spectrometry.

Monoclonal antibodies (mAbs) are heterogeneous macromolecules that display a complex isoform profile as a result of the large series of modifications they can undergo. Product-related charge variants that are associated with a loss of biological activity or affected half-life and immunogenicity are especially important. Consequently, they are often considered critical quality attributes such that acceptance criteria and controls should be established. The characterization of mAbs charge variants has long been a time and resource consuming task. Recent successes in the use of salt mediated pH gradient ion exchange chromatography with volatile mobile phases have shown there to be significant promise in using online mass spectrometric (MS) detection to facilitate peak detection. In this study, a newly developed 3 µm non-porous cation exchange column technology was investigated for its capability to be hyphenated to MS for the purpose of characterizing mAb charge variants. A 2 mm ID format was selected for the ease of configuring it to classical MS ESI ion sources. A monoclonal antibody reference material from NIST (RM 8671; NISTmAb) was used in its intact and IdeS/IgdE-digested forms to test for column performance and MS sensitivity. Furthermore, three different mAbs with highly basic isoelectric points (pI) were analyzed in their native and proteolyzed forms to demonstrate the straightforward application of the developed technique even with mAbs having strong retention on cation exchange media. The MS detection of low-abundance charge variant species (<0.1%) demonstrated there to be acceptable sensitivity and dynamic range even from routine analyses. The capability of the column to separate different mAbs having high basic pI was demonstrated, and it was found that slight adjustment of ammonium acetate concentration in the eluent can be a convenient way to rapidly optimize a separation if necessary. Linearity was shown to exist between protein mass loads of 2.5 and 50 µg while an optimal balance between chromatographic resolution and MS sensitivity was observed between 5 and 10 µg. Excellent run-to-run and column-to-column repeatability was achieved in terms of retention times, resolution and recovery.

Biochemical characterization of LR769, a new recombinant factor VIIa bypassing agent produced in the milk of transgenic rabbits.

BACKGROUND: The bypassing agent factor VII (FVIIa) is a first-line therapy for the treatment of acute bleeding episodes in patients with haemophilia and high-titre inhibitors. FVIIa is a highly post-translationally modified protein that requires eukaryotic expression systems to produce a fully active molecule. A recombinant FVIIa was produced in the milk of transgenic rabbits to increase expression and provide an efficient, safe and affordable product after purification to homogeneity (LR769). AIM: To present the biochemical and functional in vitro characteristics of LR769. RESULTS: Mass spectrometric analyses of the intact protein and of heavy and light chains revealed a fully activated, mature and properly post-translationally modified protein notably regarding N/O-glycosylations and γ-carboxylation. Primary structure analysis, performed by peptide mapping, confirmed 100% of the sequence and the low level or absence of product-derived impurities such as oxidized, deamidated and glycated forms. Low levels of aggregates and fragments were observed by different chromatographic methods. Higher order structure investigated by circular dichroism showed appropriate secondary/tertiary structures and conformational change in the presence of Ca2+ ions. Finally, activated partial thromboplastin time and thrombin generation assays showed the ability of LR769 to decrease coagulation time and to generate thrombin in haemophiliac-A-plasmas, even in the presence of inhibitors. CONCLUSION: The innovative expression system used to produce LR769 yields a new safe and effective rhFVIIa for the treatment of haemophilia A or B patients with inhibitors.

Differential investigations from plasma-derived and recombinant Factor IX revealed major differences in post-translational modifications of activation peptides.

Post-translational modifications (PTMs) located on the activation peptide (AP) of recombinant FIX (rFIX, BeneFIX(®) ) and plasma-derived FIX (pdFIX, Betafact(®) ) have been investigated by mass spectrometry to review the structural differences between these two products. Three major structural differences were pointed out. rFIX contains a low amount of phosphorylated and sulphated AP (4% for rFIX vs. 70% for pdFIX); rFIX N-glycans are only sialylated in the α2-3 linkage, whereas pdFIX N-glycans contain both type of α2-3 and α2-6 linkages, and rFIX does not contain any sialyl Lewis(X) glycoantigens contrary to pdFIX. These variations might participate in the in vivo potential different behaviours of the two molecules.