Richard Ludwig

Isomerization and Oxidation in the Complementarity-Determining Regions of a Monoclonal Antibody: A Study of the Modification–Structure–Function Correlations by Hydrogen–Deuterium Exchange Mass Spectrometry

Chemical modifications can potentially change monoclonal antibodys (mAb) local or global conformation and therefore impact their efficacy as therapeutic drugs. Modifications in the complementarity-determining regions (CDRs) are especially important because they can impair the binding affinity of an antibody for its target and therefore drug potency as a result. In order to understand the impact on mAb attributes induced by specific chemical modifications within the CDR, hydrogen-deuterium exchange mass spectrometry (HDX MS) was used to interrogate the conformational impact of Asp isomerization and Met oxidation in the CDRs of a model monoclonal antibody (mAb1). Our results indicate that despite their proximity to each other, Asp54 isomerization and Met56 oxidation in CDR2 in the heavy chain of mAb1 result in opposing conformational impacts on the local and nearby regions, leading directly to different alterations on antibody-antigen binding affinity. This study revealed direct evidence of local and global conformational changes caused by two of the most common degradation pathways in the CDRs of a mAb and identified correlations between chemical modification, structure, and function of the therapeutic monoclonal antibody.

Glycine to glutamic acid misincorporation observed in a recombinant protein expressed by Escherichia coli cells

A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography–tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation.

Investigation of Color in a Fusion Protein Using Advanced Analytical Techniques: Delineating Contributions from Oxidation Products and Process Related Impurities

ABSTRACTPurposeDiscoloration of protein therapeutics has drawn increased attention recently due to concerns of potential impact on quality and safety. Investigation of discoloration in protein therapeutics for comparability is particularly challenging primarily for two reasons. First, the description of color or discoloration is to certain extent a subjective characteristic rather than a quantitative attribute. Secondly, the species contributing to discoloration may arise from multiple sources and are typically present at trace levels. Our purpose is to development a systematic approach that allows effective identification of the color generating species in protein therapeutics.MethodsA yellow-brown discoloration event observed in a therapeutic protein was investigated by optical spectroscopy, ultra-performance liquid chromatography, and mass spectrometry (MS).ResultsMajority of the color generating species were identified as oxidatively modified protein. The location of the oxidized amino acid residues were identified by MS/MS. In addition, the impact of process-related impurities co-purified from media on discoloration was also investigated. Finally a semi-quantitative scale to estimate the contribution of each color source is presented, which revealed oxidized peptides are the major contributors.ConclusionsA systematic approach was developed for identification of the color generating species in protein therapeutics and for estimation of the contribution of each color source.

Identification and quantification of signal peptide variants in an IgG1 monoclonal antibody produced in mammalian cell lines

Sequence variants of a monoclonal antibody resulting from incomplete processing of signal peptide were identified and characterized using multiple mass spectrometry platforms and reverse phase chromatography. Detection and quantification of these variants by three LC/MS platforms were assessed. Quantification was also performed by mass spectrometric analysis of the subunits of the antibody generated by reduction and IdeS proteolysis. Peptide mapping with LC/MS/MS detection was used to quantify and confirm the identities of signal peptide sequence variants. Although quantification of the signal peptide variants thru mass spectrometry approaches is system dependent, our data revealed the results are close to the values determined by chromatographic separation with UV detection. Each of the methods have proven effective in demonstrating the consistency of signal peptide variants levels across the manufacture history of the antibody.

Molecular Variants Characterization in Protein Therapeutics Development

The fervor for biologics in the pharmaceutical industry has been fueled by several successful launches in recent years and the potential of biologics for delivering novel therapeutics to patients and high financial returns for pharmaceutical companies. While more and more promising candidates are generated from different drug discovery platforms to fill the pipeline, many challenges remain in developing efficacious and safe products. Due to specific interactions between biomolecules, protein therapeutics are generally more specific toward therapeutic targets resulting in fewer side effects. However, the complex nature of biomolecules and the sophisticated manufacturing processes pose more challenges than those for small molecule drugs with regard to analytics for the control of quality, and ultimately efficacy and safety.

Enhanced Precision of Circular Dichroism Spectral Measurements Permits Detection of Subtle Higher Order Structural Changes in Therapeutic Proteins

Protein higher order structure (HOS) is an essential quality attribute to ensure protein stability and proper biological function. Protein HOS characterization is performed during comparability assessments for product consistency as well as during forced degradation studies for structural alteration upon stress. Circular dichroism (CD) spectroscopy is a widely used technique for measuring protein HOS, but it remains difficult to assess HOS with a high degree of accuracy and precision. Moreover, once spectral changes are detected, interpreting the differences in terms of specific structural attributes is challenging. Spectral normalization by the protein concentration remains one of the largest sources of error and reduces the ability to confidently detect differences in CD spectra. This work develops a simple method to enhance the precision of the CD spectral measurements through normalization of the CD spectra by the protein concentration determined directly from the CD measurement. This method is implemented to successfully detect small CD spectral changes in multiple forced degradation studies as well as comparability assessments during biologics drug development. Furthermore, the interpretation of CD spectral changes in terms of HOS differences are provided based on orthogonal data in conjunction with structural insights gained through in silico homology modeling of the protein structure.

Vitamin B12 association with mAbs: Mechanism and potential mitigation strategies

Process control for manufacturing biologics is critical for ensuring product quality, safety, and lot to lot consistency of therapeutic proteins. In this study, we investigated the root cause of the pink coloration observed for various in‐process pools and drug substances in the antibody manufacturing process. Vitamin B12 is covalently bound to mAbs via a cobalt‐sulfur coordinate bond via the cysteine residues. The vitamin B12 was identified to attach to an IgG4 molecule at cysteine residues on light chain (Cys‐214), and heavy chain (Cys‐134, Cys‐321, Cys‐367, and Cys‐425). Prior to attachment to mAbs, the vitamin B12 needs to be in its active form of hydroxocobalamin. During culture media preparation, storage and cell culture processing, cyanocobalamin, the chemical form of vitamin B12 added to media, is converted to hydroxocobalamin by white fluorescence light (about 50% degradation in 11–14 days at room temperature and with room light intensity about 500–1,000 lux) and by short‐wavelength visible light (400–550 nm). However, cyanocobalamin is stable under red light (wavelength >600 nm) exposure and does not convert to hydroxocobalamin. Our findings suggests that the intensity of pink color depends on concentrations of both free sulfhydryl groups on reduced mAb and hydroxocobalamin, the active form of vitamin B12. Both reactants are necessary and neither one of them is sufficient to generate pink color, therefore process control strategy can consider limiting either one or both factors. A process control strategy to install red light (wavelength >600 nm) in culture media preparation, storage and culture processing areas is proposed to provide safe light for biologics and to prevent light‐induced color variations in final products.

Using hydrogen peroxide to prevent antibody disulfide bond reduction during manufacturing process

ABSTRACT During large-scale monoclonal antibody manufacturing, disulfide bond reduction of antibodies, which results in generation of low molecule weight species, is occasionally observed. When this happens, the drug substance does not meet specifications. Many investigations have been conducted across the biopharmaceutical industry to identify the root causes, and multiple strategies have been proposed to mitigate the problem. The reduction is correlated with the release of cellular reducing components and depletion of dissolved oxygen before, during, and after harvest. Consequently, these factors can lead to disulfide reduction over long-duration storage at room temperature prior to Protein A chromatography. Several strategies have been developed to minimize antibody reduction, including chemical inhibition of reducing components, maintaining aeration before and after harvest, and chilling clarified harvest during holding. Here, we explore the use of hydrogen peroxide in clarified harvest bulk or cell culture fluid as a strategy to prevent disulfide reduction. A lab-scale study was performed to demonstrate the effectiveness of hydrogen peroxide in preventing antibody reduction using multiple IgG molecules. Studies were done to define the optimal concentration of hydrogen peroxide needed to avoid unnecessary oxidization of the antibody products. We show that adding a controlled amount of hydrogen peroxide does not change product quality attributes of the protein. Since hydrogen peroxide is soluble in aqueous solutions and decomposes into water and oxygen, there is no additional burden involved in removing it during the downstream purification steps. Due to its ease of use and minimal product impact, we demonstrate that hydrogen peroxide treatment is a powerful, simple tool to quench reducing potential by simply mixing it with harvested cell culture fluid.

Subunit Mass Analysis for Monitoring Multiple Attributes of Monoclonal Antibodies

RATIONALE Multi-Attribute Methods (MAMs) are appealing due to their ability to provide data on multiple molecular attributes from a single assay. If fully realized, such tests could reduce the number of assays required to support a product control strategy while providing equivalent or greater product understanding relative to the conventional approach. In doing so, MAMs have the potential to decrease development and manufacturing costs by reducing the number of tests in a release panel. METHODS In this work, we report a MAM which is based on subunit mass analysis. RESULTS The MAM assay is shown to be suitable for use as a combined method for identity testing, glycan profiling, and protein ratio determination for co-formulated monoclonal antibody (mAb) drugs. This is achieved by taking advantage of the high mass accuracy and relative quantification capabilities of intact mass analysis using quadrupole time-of-flight mass spectrometry (Q-TOF MS). Protein identification is achieved by comparing the measured masses of light chain (LC) and heavy chain (HC) mAbs against their theoretical values. Specificity is based on instrument mass accuracy. Glycan profiling and relative protein ratios are determined by the relative peak intensities of the protein HC glycoforms and LC glycoforms, respectively. Results for these relative quantifications agree well with those obtained by the conventional hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC methods. CONCLUSIONS The suitability of this MAM for use in a quality control setting is demonstrated through assessment specificity for mAb identity, and accuracy, precision, linearity and robustness for glycan profiling and ratio determination. Results from this study indicate that a MAM with subunit mass analysis has the potential to replace three conventional methods widely used for mAb release testing including identification assay, glycosylation profiling, and ratio determination for co-formulated mAbs.

Codon-Directed Determination of the Biological Causes of Sequence Variants in Therapeutic Proteins

Recombinant monoclonal antibodies (mAbs) manufactured from immortalized mammalian cell lines are becoming increasingly important as therapies. Ensuring the quality of expressed proteins is critical when developing manufacturing processes. Protein sequence variants (PSVs) are a type of product-related variant in which errors in the protein sequence are present. Detecting PSVs and determining their origins, either by DNA mutation or mRNA mistranslation, is critical. Mutations cannot be remediated without developing new clones, which can be costly and time-consuming. In contrast, mistranslation can usually be mitigated by optimizing cell culture conditions. In this work, we first developed a new method to detect low-abundance PSVs with improved sensitivity. Then, a statistical metric was proposed to determine whether the observed PSVs originate from mutation or mistranslation by characterizing the distribution of PSVs. This method was applied to the evaluation of 50 clones from five mAbs programs, allowing for identification of five mutation and 139 mistranslation PSVs. The presence of even a few mutations demonstrates the necessity of clone screening during process development.