Physicochemical and biological impact of metal-catalyzed oxidation of IgG1 monoclonal antibodies and antibody-drug conjugates via reactive oxygen species.

Biotherapeutics are exposed to common transition metal ions such as Cu(II) and Fe(II) during manufacturing processes and storage. IgG1 biotherapeutics are vulnerable to reactive oxygen species (ROS) generated via the metal-catalyzed oxidation reactions. Exposure to these metal ions can lead to potential changes to structure and function, ultimately influencing efficacy, potency, and potential immunogenicity of the molecules. Here, we stress four biotherapeutics of the IgG1 subclass (trastuzumab, trastuzumab emtansine, anti-NaPi2b, and anti-NaPi2b-vc-MMAE) with two common pharmaceutically relevant metal-induced oxidizing systems, Cu(II)/ ascorbic acid and Fe(II)/ H2O2, and evaluated oxidation, size distribution, carbonylation, Fc effector functions, antibody-dependent cellular cytotoxicity (ADCC) activity, cell anti-proliferation and autophaghic flux. Our study demonstrates that the extent of oxidation was metal ion-dependent and site-specific, leading to decreased FcγRIIIa and FcRn receptor binding and subsequently potentially reduced bioactivity, though antigen binding was not affected to a great extent. In general, the monoclonal antibody (mAb) and corresponding antibody-drug conjugate (ADC) showed similar impacts to product quality when exposed to the same metal ion, either Cu(II) or Fe(II). Our study clearly demonstrates that transition metal ion binding to therapeutic IgG1 mAbs and ADCs is not random and that oxidation products show unique structural and functional ramifications. A critical outcome from this study is our highlighting of key process parameters, route of degradation, especially oxidation (metal catalyzed or via ROS), on the CH1 and Fc region of full-length mAbs and ADCs.Abbreviations: DNPH 2,4-dinitrophenylhydrazine; ADC Antibody drug conjugate; ADCC Antibody-dependent cellular cytotoxicity; CDR Complementary determining region; DTT Dithiothreitol; HMWF high molecular weight form; LC-MS Liquid chromatography-mass spectrometry; LMWF low molecular weight forms; MOA Mechanism of action; MCO Metal-catalyzed oxidation; MetO Methionine sulfoxide; mAbs Monoclonal antibodies; MyBPC Myosin binding protein C; ROS Reactive oxygen species; SEC Size exclusion chromatography.

Assessing the Utility of Circular Dichroism and FTIR Spectroscopy in Monoclonal-Antibody Comparability Studies.

Protein characterization is a necessary activity during development, technical transfers, and licensure. One important aspect of protein characterization is higher order structure assessment, which can be accomplished in a variety of ways. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies provide global higher order structure and are routinely used to measure the overall structure for product characterization; however, their use as comparability tools is uncertain because of their insensitivity to local or small structure changes. We use a monoclonal antibody (mAb) to explore the usefulness of CD and FTIR compared with other indirect methods of structure characterization such as size-exclusion and ion-exchange chromatographies (SEC and IEC). A panel of degraded samples of a mAb was generated; their higher order structure evaluated using CD and FTIR and was found to be largely unchanged. However, the SEC and IEC chromatograms of certain degraded samples were found to have measurable changes. Based on these studies, we conclude that the application of CD and FTIR should be reserved for global higher order structure identification or product characterization only. The use of CD or FTIR comparability of mAbs should be carefully evaluated, as comparability can be sensitively determined using indirect methods based on chromatography.

Metal ion interactions with mAbs: Part 1.

Fragmentation in the hinge region of an IgG1 monoclonal antibody (mAb) can affect product stability, potentially causing changes in potency and efficacy. Metals ions, such as Cu(2+), can bind to the mAb and undergo hydrolysis or oxidation, which can lead to cleavage of the molecule. To better understand the mechanism of Cu(2+)-mediated mAb fragmentation, hinge region cleavage products and their rates of formation were studied as a function of pH with and without Cu(2+). More detailed analysis of the chemical changes was investigated using model linear and cyclic peptides (with the sequence of SCDKTHTC) derived from the upper hinge region of the mAb. Cu(2+) mediated fragmentation was determined to be predominantly via a hydrolytic pathway in solution. The sites and products of hydrolytic cleavage are pH and strain dependent. In more acidic environments, rates of Cu(2+) induced hinge fragmentation are significantly slower than at higher pH. Although the degradation reaction rates between the linear and cyclic peptides are not significantly different, the products of degradation vary. mAb fragmentation can be reduced by modifying His, which is a potential metal binding site and a known ligand in other metalloproteins. These results suggest that a charge may contribute to stabilization of a specific molecular structure involved in hydrolysis, leading to the possible formation of a copper binding pocket that causes increased susceptibility of the hinge region to degradation.

Stability of IgG1 monoclonal antibodies in intravenous infusion bags under clinical in-use conditions.

Compounding pharmacists are expected to prepare safe and efficacious doses of medication under time and economical constraints while protecting pharmacy staff and caregivers from inadvertent exposure to the drug. The pharmacist has the additional responsibility to ensure that the product is stable in the final-administrated form as the time between drug preparation and administration is considerable. Pharmacists are responsible for setting a "beyond-use" date based on United States Pharmacopeia 797, wherein the beyond-use date for the compounded sterile preparation (CSP) is defined as the time by which the compounded preparation must be used to avoid risks for product degradation, contamination, and so on. Physical and chemical stability of the CSP can be difficult to maintain over extended storage, especially since the formulation components are diluted within the intravenous (i.v.) bag contents. Recent published reports have suggested the use of extended time, beyond that recommended by the manufacturer, for the storage and administration of CSP. These recommendations were based on inadequate analytical testing of the CSP. Herein, we demonstrate that setting of the beyond-use date should be carefully assessed using the appropriate analytical methods and testing. Results from our studies clearly indicate that many of the tested IgG(1) monoclonal antibodies should not be diluted and stored in i.v. bags over extended period of time, and particularly should not be transported after dilution in the infusion bags without consulting the manufacturer. Results from this study also indicate that i.v. bag agitation studies should be performed during pharmaceutical development of protein therapeutics under clinical in-use conditions, especially when storage and transportation of i.v. bags are possible in global clinical trials and post-licensure usage.