A high yielding IFNAR1 ECD mammalian expression process for use in autoimmune disease drug development.

Interferon-alpha receptor 1 (IFNAR1) is a target of interest for recombinant biotherapeutics that block the JAK/STAT pathway. This pathway is believed to play a role in many diseases including Hepatitis B and C, Herpes Simplex, Multiple Sclerosis, and other autoimmune disorders. By using IFNAR1 as a target to block Type I IFN from binding to the JAK/STAT pathway and prevent activation of this target, autoimmune disease progression can be modulated. Current IFNAR1 extracellular domain (ECD) expression and purification protocols are labor intensive with low product yield and limited scalability. In this work, we evaluate three different expression systems (baculovirus, human embryonic kidney 293 (HEK293×), and Chinese hamster ovary (CHO)) to improve expression of IFNAR1 ECD. We demonstrate the benefits of utilizing mammalian CHO cell transient transfection to increase expression titer, as well as an improved two-step purification process performed using immobilized metal affinity chromatography (IMAC) as the capture step and Ceramic Hydroxyapatite (CHT) Type II for HMW impurity removal in flow through mode. This process showed an 20-fold increase in productivity compared to the baseline process as measured by grams purified per liter of cell culture fluid. Lastly, the improved process showed good scalability, enabling efficient purification of 3.6 g of product from a 30 L scale bioreactor.

Charge variants characterization and release assay development for co-formulated antibodies as a combination therapy.

Combination therapy is a fast-growing strategy to maximize therapeutic benefits to patients. Co-formulation of two or more therapeutic proteins has advantages over the administration of multiple medications, including reduced medication errors and convenience for patients. Characterization of co-formulated biologics can be challenging due to the high degree of similarity in the physicochemical properties of co-formulated proteins, especially at different concentrations of individual components. We present the results of a deamidation study of one monoclonal antibody component (mAb-B) in co-formulated combination antibodies (referred to as COMBO) that contain various ratios of mAb-A and mAb-B. A single deamidation site in the complementarity-determining region of mAb-B was identified as a critical quality attribute (CQA) due to its impact on biological activity. A conventional charge-based method of monitoring mAb-B deamidation presented specificity and robustness challenges, especially when mAb-B was a minor component in the COMBO, making it unsuitable for lot release and stability testing. We developed and qualified a new, quality-control-friendly, single quadrupole Dalton mass detector (QDa)-based method to monitor site-specific deamidation. Our approach can be also used as a multi-attribute method for monitoring other quality attributes in COMBO. This analytical paradigm is applicable to the identification of CQAs in combination therapeutic molecules, and to the subsequent development of a highly specific, highly sensitive, and sufficiently robust method for routine monitoring CQAs for lot release test and during stability studies.

Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping.

Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp isomerization in the complementarity-determining regions of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control method for routine monitoring is desirable. In this work, we utilized a liquid chromatography-mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the complementarity-determining regions of a therapeutic monoclonal antibody. To quantitate the site-specific Asp isomerization of the monoclonal antibody, a UV detection-based quantitation assay utilizing the same LC platform was developed. The assay was qualified and implemented for routine monitoring of this product-specific modification. Compared with existing methods, this analytical paradigm is applicable to identify Asp isomerization (or other modifications) and subsequently develop a rapid, sufficiently robust quality control method for routine site-specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product-related impurity (a critical quality attribute) caused by isomerization, deamidation, oxidation, or other post-translational modifications, and then utilizes synthetic peptides and MS to assist the development of a LC-UV-based chromatographic method that separates and quantifies the product-related impurities by UV peaks. The established LC-UV method has acceptable peak specificity, precision, linearity, and accuracy; it can be validated and used in a good manufacturing practice environment for lot release and stability testing. LAY ABSTRACT: Aspartate isomerization is a common post-translational modification of recombinant proteins during manufacture process and storage. Isomerization in the complementarity-determining regions (CDRs) of a monoclonal antibody A (mAb-A) has been detected and has been shown to have impact on the binding affinity to the antigen. In this work, we utilized a mass spectrometry-based peptide mapping approach to detect and quantitate the Asp isomerization in the CDRs of mAb-A. To routinely monitor the CDR isomerization of mAb-A, a focused peptide mapping method utilizing reversed phase chromatographic separation and UV detection has been developed and qualified. This approach is generally applicable to monitor isomerization and other post-translational modifications of proteins in a specific and high-throughput mode to ensure product quality.

LC/MS characterization of meningococcal depolymerized polysaccharide group C reducing endgroup and internal repeating unit.

Hydrogen peroxide has been used to cleave the native Neisseria meningiditis polysaccharide (PS) from mega-Dalton molecular weight to a smaller size (approximately 20 kDa) depolymerized polysaccharide. The polysaccharide was examined after partial peroxide depolymerization to verify the presence of the carboxyl group at position 1 and the intactness of the internal sialic acid repeating units. The reducing end group of meningococcal polysaccharide type C was also examined after derivatization by L-tyrosine hydrazide. Partial peroxide depolymerization did not result in loss of the position 1 carboxyl group at the reducing end of the polysaccharide. In addition, no loss of structural integrity was noted for the internal sialic repeat units.

Quantification of residual EDU (N-ethyl-N'-(dimethylaminopropyl) carbodiimide (EDC) hydrolyzed urea derivative) and other residual by LC-MS/MS.

An LC-MS/MS method for determination of the break down product of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) urea derivative, EDU, has been developed and validated for monitoring the residual coupling reagents. Results indicate that the method exhibits suitable specificity, sensitivity, precision, linearity and accuracy for quantification of residual EDU in the presence of meningococcal polysaccharide-diphtheria toxoid conjugate vaccine and other vaccine matrix compounds. The assay has been validated for a detection range of 10-100 ng/mL and then successfully transferred to quality control (QC) lab. This same method has also been applied to the determination of residual diaminohexane (DAH) in the presence of EDU. LC-MS/MS has proven to be useful as a quick and sensitive approach for simultaneous determination of multiple residual compounds in glycoconjugate vaccine samples.