A high-resolution multi-attribute method for product characterization, process characterization, and quality control of therapeutic proteins.

During the manufacturing of therapeutic proteins, Critical Quality Attributes (CQAs) have been monitored by conventional methods, such as cation exchange chromatography (CEX), reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS), and 1,2-diamino-4,5-methylenedioxybenzene (DMB) labelling method. The conventional methods often generate individual peaks that contain multiple components, which may obscure the detection and the quantification of individual critical quality attributes (CQAs). Alternatively, Multi-Attribute Method (MAM) enables detection and quantification of specific CQAs. A high resolution MAM has been developed and qualified to replace several conventional methods in monitoring product quality attributes, such as oxidation, deamidation, clipping, and glycosylation. The qualified MAM was implemented in process characterization, as well as release and stability assays in quality control (QC). In combination with a design-of-experiments (DoE), the MAM method identified multivariate process parameter ranges that yield acceptable CQA level, which provides operational flexibility for manufacturing.

Application of a Quantitative LC-MS Multiattribute Method for Monitoring Site-Specific Glycan Heterogeneity on a Monoclonal Antibody Containing Two N-Linked Glycosylation Sites.

A significant challenge of traditional glycan mapping techniques is that they do not provide site-specific glycosylation information. Therefore, for proteins containing multiple glycosylation sites, the individual glycan species present at a particular site cannot be differentiated from those species present at the other glycosylation sites on the molecule. Quantification of glycoform has previously been demonstrated using a multiattribute method (MAM), which can quantify multiple post-translational modifications including deamidation, oxidation, glycosylation variants, and fragmentation ( Rogers, R. S.; Nightlinger, N. S.; Livingston, B.; Campbell, P.; Bailey, R.; Balland, A. MAbs 2015 , 7 , 881 - 890 ; ref 1). In this paper we describe the application of an MAM based method for site specific quantification of N-linked glycan heterogeneity present on an IgG1 mAb molecule containing two distinct N-linked glycosylation sites: one present on the heavy chain (HC) variable region (Fab) and the other present on the conserved HC constant region (Fc). MAM is a peptide mapping method utilizing mass spectrometry to detect and quantify specific peptides of interest. The ionization properties of the glycopeptides with different classes of glycan structural variants, including high mannose, sialylated, and terminal galactosylated species were studied in detail. Our results demonstrate that MAM quantification of individual glycan species from both the Fab and Fc N-Linked glycosylation sites is consistent with quantification using the traditional hydrophilic interaction liquid chromatography (HILIC) analysis of enzymatically released and fluorescently labeled glycans. Furthermore, no significant impact from the glycoform on the ionization properties of the glycopeptide is observed. Our work demonstrates that the MAM method is a suitable approach for providing quantitative, site-specific glycan information for profiling of N-linked glycans on immunoglobulins.

Assessing Analytical Similarity of Proposed Amgen Biosimilar ABP 501 to Adalimumab.

BACKGROUND: ABP 501 is being developed as a biosimilar to adalimumab. Comprehensive comparative analytical characterization studies have been conducted and completed. OBJECTIVE: The objective of this study was to assess analytical similarity between ABP 501 and two adalimumab reference products (RPs), licensed by the United States Food and Drug Administration (adalimumab [US]) and authorized by the European Union (adalimumab [EU]), using state-of-the-art analytical methods. METHODS: Comprehensive analytical characterization incorporating orthogonal analytical techniques was used to compare products. Physicochemical property comparisons comprised the primary structure related to amino acid sequence and post-translational modifications including glycans; higher-order structure; primary biological properties mediated by target and receptor binding; product-related substances and impurities; host-cell impurities; general properties of the finished drug product, including strength and formulation; subvisible and submicron particles and aggregates; and forced thermal degradation. RESULTS: ABP 501 had the same amino acid sequence and similar post-translational modification profiles compared with adalimumab RPs. Primary structure, higher-order structure, and biological activities were similar for the three products. Product-related size and charge variants and aggregate and particle levels were also similar. ABP 501 had very low residual host-cell protein and DNA. The finished ABP 501 drug product has the same strength with regard to protein concentration and fill volume as adalimumab RPs. ABP 501 and the RPs had a similar stability profile both in normal storage and thermal stress conditions. CONCLUSION: Based on the comprehensive analytical similarity assessment, ABP 501 was found to be similar to adalimumab with respect to physicochemical and biological properties.

Analysis of human antibody IgG2 domains by reversed-phase liquid chromatography and mass spectrometry.

It has been well documented that papain cleaves an IgG1 molecule to release Fab and Fc domains; however, papain was found unable to release such domains from an IgG2. Here we present a new combinatory strategy to analyze the heterogeneity of the light chain (LC), single chain Fc (sFc), and Fab portion of the heavy chain (Fd) of an IgG2 molecule released by papain cleavage under mild reducing conditions. These domains were well separated on reversed-phase high performance liquid chromatography (RP-HPLC) and analyzed by in-line liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS). In addition, some modifications of these domains were revealed by in-line mass spectrometry, and confirmed by the peptide mapping on LC-MS/MS analysis. This same strategy was proven suitable for IgG1 molecules as well. This procedure provides a simplified approach for the characterization of antibody biomolecules by facilitating the detection of low-level modifications in a domain. In addition, the technique offers a new strategy as an identification assay to distinguish IgG2 molecules on RP-HPLC, by which highly conserved Fc domains remain at a constant retention time (RT) unique to its subisotype, while varying RTs of the light chain and the Fd distinguish the monoclonal antibody from other molecules of the same isotype based on the underlying characteristics of each antibody.

Solid state fluorescence of lyophilized proteins.

Fluorescence spectroscopy has been used to measure changes in the tertiary structure of proteins in the solution state. The sensitivity of fluorescence to the protein tryptophan environment has made it a useful tool for studying protein conformation and stability. Using fluorescence spectroscopy to probe structural alterations in lyophilized proteins has been limited due to technical challenges and overwhelming background light scattering. We have investigated the possibility of analyzing lyophilized proteins using the Cary-Eclipse spectrofluorometer by monitoring the fluorescence of the protein therapeutic after subjecting the lyophilized cake to heat-induced accelerated degradation. We have been able to obtain reproducible fluorescence spectra, detecting possible structural changes under these conditions. Fluorescence and circular dichroism spectroscopic analyses of the reconstituted proteins indicated that changes in fluorescence intensities observed in the solid state could be correlated to that in solution and to possible tertiary structural changes. Size exclusion chromatography analysis of protein Y subject to accelerated degradation showed a correlation between decreasing fluorescence intensity and increasing protein Y tetramer in solution, consistent with long-term stability. This suggests that solid state, intrinsic protein fluorescence measurements using the Cary-Eclipse holder may be feasible for long-term stability studies and formulation development.

Structural requirements for additional N-linked carbohydrate on recombinant human erythropoietin.

N-Linked glycosylation is a post-translational event whereby carbohydrates are added to secreted proteins at the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline. Some consensus sequences in secreted proteins are not glycosylated, indicating that consensus sequences are necessary but not sufficient for glycosylation. In order to understand the structural rules for N-linked glycosylation, we introduced N-linked consensus sequences by site-directed mutagenesis into the polypeptide chain of the recombinant human erythropoietin molecule. Some regions of the polypeptide chain supported N-linked glycosylation more effectively than others. N-Linked glycosylation was inhibited by an adjacent proline suggesting that sequence context of a consensus sequence could affect glycosylation. One N-linked consensus sequence (Asn123-Thr125) introduced into a position close to the existing O-glycosylation site (Ser126) had an additional O-linked carbohydrate chain and not an additional N-linked carbohydrate chain suggesting that structural requirements in this region favored O-glycosylation over N-glycosylation. The presence of a consensus sequence on the protein surface of the folded molecule did not appear to be a prerequisite for oligosaccharide addition. However, it was noted that recombinant human erythropoietin analogs that were hyperglycosylated at sites that were normally buried had altered protein structures. This suggests that carbohydrate addition precedes polypeptide folding.