Kimberly May

Advances in the assessment and control of the effector functions of therapeutic antibodies

The Fc (crystallizable fragment) region of therapeutic antibodies can have an important role in their safety and efficacy. Although much is known about the structure–activity relationship of antibodies and the factors that influence Fc effector functions, a process has not yet been defined to clearly delineate how Fc functionality should be assessed and controlled during antibody development and manufacturing. In this article, we summarize the current knowledge of antibody Fc functionality, provide a strategy for assessing the effector functions of different classes of therapeutic antibodies (including Fc fusion proteins) and propose a path for routine testing and controls for manufacturers of antibody products.

Disulfide bond structures of IgG molecules: Structural variations, chemical modifications and possible impacts to stability and biological function

The disulfide bond structures established decades ago for immunoglobulins have been challenged by findings from extensive characterization of recombinant and human monoclonal IgG antibodies. Non-classical disulfide bond structure was first identified in IgG4 and later in IgG2 antibodies. Although, cysteine residues should be in the disulfide bonded states, free sulfhydryls have been detected in all subclasses of IgG antibodies. In addition, disulfide bonds are susceptible to chemical modifications, which can further generate structural variants such as IgG antibodies with trisulfide bond or thioether linkages. Trisulfide bond formation has also been observed for IgG of all subclasses. Degradation of disulfide bond through β-elimination generates free sulfhydryls disulfide and dehydroalanine. Further reaction between free sulfhydryl and dehydroalanine leads to the formation of a non-reducible cross-linked species. Hydrolysis of the dehydroalanine residue contributes substantially to antibody hinge region fragmentation. The effect of these disulfide bond variations on antibody structure, stability and biological function are discussed in this review.

Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies.

The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.

Quantitation of asparagine deamidation by isotope labeling and liquid chromatography coupled with mass spectrometry analysis

Nonenzymatic asparagine (Asn) deamidation is one of the commonly observed posttranslational modifications of proteins. Recent development of several specific analytical methods has allowed for efficient identification and differentiation of the deamidation products (i.e., isoaspartate [isoAsp] and aspartate [Asp]). Isotope labeling of isoAsp and Asp that are generated during sample preparation by 18O has been developed and can differentiate isoAsp and Asp as analytical artifacts from those present in the samples prior to sample preparation for an accurate quantitation. However, the 18O labeling procedure has a limitation due to the additional incorporation of up to two 18O atoms into the peptide C-terminal carboxyl groups. Variability in the incorporation of 18O atoms into the peptide C-terminal carboxyl groups results in complicated mass spectra and hinders data interpretation. This limitation can be overcome by the dissection of the complicated mass spectra using a calculation method presented in the current study. The multiple-step calculation procedure has been successfully employed to determine the levels of isoAsp and Asp that are present in the sample prior to sample treatment.

Disulfide bond assignment of an IgG1 monoclonal antibody by LC–MS with post-column partial reduction

Confirmation of the correct disulfide linkage and demonstration of the lack of a significant level of scrambled disulfide bonds are critical to ensure the appropriate folding and structure of recombinant monoclonal antibodies. Currently these are typically achieved by carrying out multiple experiments, most commonly via the comparison of the samples before and after reduction by LC-MS and MS/MS. The data are then analyzed by searching across all the possible disulfide linkages manually or with the aid of computer algorithms. To eliminate the need of multiple experiments and complicated data analysis, a simple LC-MS-based method coupled with post-column partial reduction was developed. Using a recombinant monoclonal IgG1 antibody as an example, this method demonstrates the ability to confirm the correct disulfide linkage and the ability to detect scrambled disulfide bonds from a single experiment with a simple data analysis strategy.

Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

The presence of N-linked oligosaccharides in the CH2 domain has a significant impact on the structure, stability, and biological functions of recombinant monoclonal antibodies. The impact is also highly dependent on the specific oligosaccharide structures. The absence of core-fucose has been demonstrated to result in increased binding affinity to Fcγ receptors and, thus, enhanced antibody-dependent cellular cytotoxicity (ADCC). Therefore, a method that can specifically determine the level of oligosaccharides without the core-fucose (afucosylation) is highly desired. In the current study, recombinant monoclonal antibodies and tryptic peptides from the antibodies were digested using endoglycosidases F2 and H, which cleaves the glycosidic bond between the two primary GlcNAc residues. As a result, various oligosaccharides of either complex type or high mannose type that are commonly observed for recombinant monoclonal antibodies are converted to either GlcNAc residue only or GlcNAc with the core-fucose. The level of GlcNAc represents the sum of all afucosylated oligosaccharides, whereas the level of GlcNAc with the core-fucose represents the sum of all fucosylated oligosaccharides. LC-MS analysis of the enzymatically digested antibodies after reduction provided a quick estimate of the levels of afucosylation. An accurate determination of the level of afucosylation was obtained by LC-MS analysis of glycopeptides after trypsin digestion.

LC-MS analysis of glycopeptides of recombinant monoclonal antibodies by a rapid digestion procedure.

N-glycan analysis of recombinant monoclonal antibodies (mAbs) usually requires the removal of oligosaccharides by PNGase F followed by 2-AB labeling, normal phase high performance liquid chromatography (NP-HPLC) separation and fluorescence detection. Alternatively antibodies can be completely digested by trypsin to generate glycopeptides for analysis by liquid chromatography-mass spectrometry (LC-MS). Here, we report the development of a rapid digestion procedure to generate glycopeptides for quantitative LC-MS analysis. Recombinant monoclonal antibodies were digested using a combination of Lys-C and trypsin at 37°C for 15 min. The glycan profiles from this rapid digestion procedure are in good agreement with those from LC-MS analysis of glycopeptides from completely digested antibodies and those from NP-HPLC analysis of 2-AB labeled PNGase F released oligosaccharides. This rapid digestion procedure was applied to the comparison of oligosaccharides of two different antibodies. Glycopeptides from the two antibodies were differentially labeled with stable isotopes and analyzed simultaneously after a 1:1 mixing. The combination of the rapid digestion procedure and differential stable isotope labeling significantly reduced the turnaround time.

Examining Manufacturing Readiness for Breakthrough Drug Development.

In July 2012, Congress passed the Advancing Breakthrough Therapies for Patients Act as part of the Food and Drug Administration Safety and Innovation Act (FDASIA). Section 902 of FDASIA provides for designation of a drug as a breakthrough therapy Bif the drug is intended alone or in combination with one or more other drugs, to treat serious or life-threatening diseases or conditions and preliminary clinical evidence indicates that the drug may demonstrate substantial improvement over existing therapies (1).^ Breakthrough designation is a mechanism that the U.S. Food and Drug Administration (FDA) can grant to sponsors to expedite the development of these promising therapies. As part of the program, the FDA and sponsor collaborate in a dynamic, multi-disciplinary, resource-intensive process to determine the most efficient path using an Ball hands on deck approach^ involving senior managers and experienced review staff and more frequent and interactive communications (2,3). The objective is to expedite design and review of the clinical development program so that trials are as efficient as possible, and the number of patients exposed to potentially less efficacious treatment is minimized. As a consequence, clinical development timelines involving the traditional three distinct phases could be reduced from 7–10 to 3–5 years. The shorter clinical development programs will have significant impact on product and process development timelines requiring the manufacturing organization to reconsider traditional approaches to product and process development and undertake their own resource-intensive, cross-functional team approach to ensure a sustained supply of safe and efficacious product at the time of approval. To ensure success, the manufacturing organization should have good communications with the clinical organization to facilitate identification of potential candidates for breakthrough designation early and help gate or accelerate the appropriate Chemistry, Manufacturing, and Controls (CMC) and current Good Manufacturing Practice (cGMP) development activities. It is important to understand that breakthrough drug development programs are resource intensive; sponsors need to be selective about which programs to take forward and ensure management support. Moreover, a collaborative, cross-functional approach between development, commercial, and regulatory operations, with early and robust discussions, is essential to ensure successful development and launch of a breakthrough drug product. In March of 2015, Friends of Cancer Research (Friends) convened a group of industry and FDA stakeholders familiar with developing breakthrough drugs to explore options, The opinions expressed in this manuscript are those of Earl Dye, Annie Sturgess, Gargi Maheshwari, Kimberly May, Colleen Ruegger, Usha Ramesh, Heow Tan, Keith Cockerill, John Groskoph, Emanuela Lacana, Sau Lee, and Sarah Pope Miksinski and do not necessarily reflect the views or policies of the FDA.