Utility of High Resolution NMR Methods to Probe the Impact of Chemical Modifications on Higher Order Structure of Monoclonal Antibodies in Relation to Antigen Binding.

PURPOSE: An understanding of higher order structure (HOS) of monoclonal antibodies (mAbs) could be critical to predicting its function. Amongst the various factors that can potentially affect HOS of mAbs, chemical modifications that are routinely encountered during production and long-term storage are of significant interest. METHODS: To this end, two Pfizer mAbs were subjected to forced deamidation stress for a period of eight weeks. Samples were aliquoted at various time points and high resolution accurate mass liquid chromatography-mass spectrometry (LC-MS/MS) was performed using low-artifact trypsin digestion (LATD) peptide mapping to identify and quantify chemical modifications. 2D backbone amide and sidechain methyl NMR spectra were acquired to gauge the effect of HOS changes upon chemical modification. Differential scanning calorimetry was also performed to assess the effect of thermal stability of mAbs upon modification. Finally, functional studies via target-binding based ELISA were performed to connect HOS changes to any loss of potency. RESULTS: The extent of deamidation in the mAb domains were quantified by LC-MS/MS. The HOS changes as obtained from 2D NMR were mostly localized around the affected sites leaving the overall structure relatively unchanged. The antigen-antibody binding of the mAbs, in spite of deamidation in the Fab region, remains unchanged. CONCLUSION: This case study provides an integrated approach of relating chemical modifications in mAb domains with possible changes in HOS. This can be potentially used to assess a possible loss of potency within the structure-function paradigm of proteins in an orthogonal manner.

Reactivity-driven cleanup of 2-Aminobenzamide derivatized oligosaccharides.

N-glycan profiling is commonly accomplished by the derivatization of the enzymatically released oligosaccharides with a fluorophore, thereby facilitating their analysis by hydrophilic-interaction liquid chromatography (HILIC). These fluorescent dyes are often present in large excess during derivatization reactions, and their removal is typically required to minimize chromatographic interference. Herein, we report a reactivity-driven 2-phase extraction protocol with the aldehyde reagent octanal, which demonstrated efficient 2-aminobenzamide cleanup as well as high derivatized N-glycan recovery. This cleanup method can be performed within minutes, and therefore provides an alternative sample preparation route for N-glycan profiling with improved time efficiency and operational simplicity.

Nonclinical Evaluation of PF-06438179: A Potential Biosimilar to Remicade® (Infliximab).

INTRODUCTION: PF-06438179, a potential biosimilar to Remicade® (infliximab, Janssen Biotech, Inc.), is a chimeric mouse-human monoclonal antibody targeting human tumor necrosis factor alpha (TNF). METHODS: Analytical (small subset reported here) and nonclinical studies compared the structural, functional, and in vivo nonclinical similarity of PF-06438179 with Remicade sourced from the United States (infliximab-US) and/or European Union (infliximab-EU). RESULTS: The peptide map profiles were superimposable, and peptide masses were the same, indicating identical amino acid sequences. Data on post-translational modifications, biochemical properties, and biological function provided strong support for analytical similarity. Administration of a single intravenous (IV) dose (10 or 50 mg/kg) of PF-06438179 or infliximab-EU to male rats was well tolerated. There were no test article-related clinical signs or effects on body weight or food consumption. Systemic exposures [maximum drug concentration (C max) and area under the concentration-time curve (AUC)] in rats administered PF-06438179 or infliximab-EU were similar, with mean exposure ratio of PF-06438179 relative to infliximab-EU ranging from 0.88 to 1.16. No rats developed anti-drug antibodies. A 2-week IV toxicity study was conducted with once-weekly administration of 10 or 50 mg/kg of PF-06438179 to male and female rats. PF-06438179-related hyperplasia of sinusoidal cells occurred in the liver in rats administered 50 mg/kg, but was not adverse based on its minimal to mild severity. The no-observed adverse-effect level for PF-06438179 was 50 mg/kg. At this dose, C max was 1360 µg/mL and AUC at 168 h was 115,000 µg h/mL on day 8. CONCLUSIONS: The analytical and nonclinical studies have supported advancement of PF-06438179 into global comparative clinical trials. FUNDING: Pfizer Inc.

Development and application of a robust N-glycan profiling method for heightened characterization of monoclonal antibodies and related glycoproteins.

A highly robust hydrophilic interaction liquid chromatography (HILIC) method that involves both fluorescence and mass spectrometric detection was developed for profiling and characterizing enzymatically released and 2-aminobenzamide (2-AB)-derivatized mAb N-glycans. Online HILIC/mass spectrometry (MS) with a quadrupole time-of-flight mass spectrometer provides accurate mass identifications of the separated, 2-AB-labeled N-glycans. The method features a high-resolution, low-shedding HILIC column with acetonitrile and water-based mobile phases containing trifluoroacetic acid (TFA) as a modifier. This column and solvent system ensures the combination of robust chromatographic performance and full compatibility and sensitivity with online MS in addition to the baseline separation of all typical mAb N-glycans. The use of TFA provided distinct advantages over conventional ammonium formate as a mobile phase additive, such as, optimal elution order for sialylated N-glycans, reproducible chromatographic profiles, and matching total ion current chromatograms, as well as minimal signal splitting, analyte adduction, and fragmentation during HILIC/MS, maximizing sensitivity for trace-level species. The robustness and selectivity of HILIC for N-glycan analyses allowed for method qualification. The method is suitable for bioprocess development activities, heightened characterization, and clinical drug substance release. Application of this HILIC/MS method to the detailed characterization of a marketed therapeutic mAb, Rituxan(®), is described.

Conjugated linoleic acid isomers reduce blood cholesterol levels but not aortic cholesterol accumulation in hypercholesterolemic hamsters.

The aim of the present study was to characterize plasma lipids and lipoprotein cholesterol and glucose concentrations in hamsters fed either cis-9,trans-11 CLA (9c,11 tCLA); trans-10,cis-12 CLA (10t,12c CLA); or linoleic acid (LA) on the accumulation of aortic cholesterol in hypercholesterolemic hamsters. One hundred male F1B strain Syrian Golden Hamsters (Mesocricetus auratus) (BioBreeders Inc., Watertown, MA) approximately 9 wk of age were housed in individual stainless steel hanging cages at room temperature with a 12-h light/dark cycle. Hamsters were given food and water ad libitum. Following a 1-wk period of acclimation, the hamsters were fed a chow-based (nonpurified) hypercholesterolemic diet (HCD) containing 10% coconut oil (92% saturated fat) and 0.1% cholesterol for 2 wk. After an overnight fast, the hamsters were bled and plasma cholesterol concentrations were measured. The hamsters were then divided into 4 groups of 25 based on similar mean plasma VLDL and LDL cholesterol (nonHDL-C) concentrations. Group 1 remained on the HCD (control). Group 2 was fed the HCD plus 0.5% 9c,11t CLA isomer. Group 3 was fed the HCD plus 0.5% 10t,12c CLA isomer. Group 4 was fed the HCD plus 0.5% LA. Compared with the control, both CLA isomers and LA had significantly lower plasma total cholesterol and HDL cholesterol concentrations (P < 0.001) after 12 but not 8 wk of treatment and were not significantly different from each other. Also, both CLA isomers had significantly lower plasma nonHDL-C concentrations (P < 0.01) compared with the control after 12 but not 8 wk of treatment and were not significantly different from each other or the LA-fed hamsters. Plasma TG concentrations were significantly higher (P < 0.004) with the 10t, 12c CLA isomer compared with the other treatments at 8 but not at 12 wk of treatment. Plasma TG concentrations were also significantly lower (P < 0.03) with the 9c,11t CLA isomer compared with the control at 12 wk of treatment. Also, the 10t,12c CLA isomer and LA had significantly higher plasma glucose concentrations compared with the control and 9c,11t CLA isomer (P < 0.008) at 12 wk of treatment, whereas at 8 wk, only the LA treatment had significantly higher plasma glucose concentrations (P < 0.001) compared with the 9c,11t CLA isomer. Although liver weights were significantly higher in 10t,12c CLA isomer-fed hamsters, liver total cholesterol, free cholesterol, cholesterol ester, and TG concentrations were significantly lower in these hamsters compared with hamsters fed the control, 9c,11t CLA isomer, and LA diets (P< 0.05). The 9c,11t CLA isomer and LA diets tended to reduce cholesterol accumulation in the aortic arch, whereas the 10t,12c CLA isomer diet tended to raise cholesterol accumulation compared with the control diet; however, neither was significant. In summary, no differences were observed between the CLA isomers for changes in plasma lipids or lipoprotein cholesterol concentrations. However, the 9c,11t CLA isomer did appear to lower plasma TG and glucose concentrations compared with the 10t,12c CLA isomer. Such differences may increase the risk of insulin resistance and type 2 diabetes in humans when the 10t,12c CLA isomer is fed separately.