A Robust Purity Method for Biotherapeutics Using New Peak Detection in an LC-MS-Based Multi-Attribute Method.

New peak detection (NPD), as part of the LC-MS-based multi-attribute method (MAM), allows for sensitive and unbiased detection of new or changing site-specific attributes between a sample and reference that is not possible with conventional UV or fluorescence detection-based methods. MAM with NPD can serve as a purity test that can establish whether a sample and the reference are similar. The broad implementation of NPD in the biopharmaceutical industry has been limited by the potential presence of false positives or artifacts, which increase the analysis time and can trigger unnecessary investigations of product quality. Our novel contributions to the success of NPD are the curation of false positives, use of the known peak list concept, pairwise analysis approach, and the development of a NPD system suitability control strategy. In this report, we also introduce a unique experimental design utilizing sequence variant co-mixes to measure NPD performance. We show that NPD has superior performance relative to conventional control system methods in the detection of an unexpected change as compared with the reference. NPD is a new frontier in purity testing that reduces subjectivity, need for analyst intervention, and potential for missing unexpected product quality changes.

Mass spectrometry-based multi-attribute method in protein therapeutics product quality monitoring and quality control.

The multi-attribute method (MAM), a liquid chromatography-mass spectrometry (LC-MS)-based peptide mapping method, has gained increased interest and applications in the biopharmaceutical industry. MAM can, in one method, provide targeted quantitation of multiple site-specific product quality attributes, as well as new peak detection. In this review, we focus on the scientific and regulatory considerations of using MAM in product quality attribute monitoring and quality control (QC) of therapeutic proteins. We highlight MAM implementation challenges and solutions with several case studies, and provide our perspective on the opportunities to use MS in QC for applications other than standard peptide mapping-based MAM.

Multi-attribute method performance profile for quality control of monoclonal antibody therapeutics.

Multi-attribute method (MAM) using peptide map analysis with high resolution mass spectrometry is increasingly common in product characterization and the identification of critical quality attributes (CQAs) of biotherapeutic proteins. Capable of providing structural information specific to amino acid residues, quantifying relative abundance of product variants or degradants, and detecting profile changes between product lots, a robust MAM can replace multiple traditional methods that generate profile-based information for product release and stability testing. In an effort to provide informative and efficient analytical monitoring for monoclonal antibody (mAb) products, from early development to manufacturing quality control, we describe the desired MAM performance profile and address the major scientific challenges in MAM method validation. Furthermore, to support fast speed investigational product development, we describe a platform method validation strategy and results of an optimized MAM workflow. This strategy is applied to support the use of MAM for multiple mAb products with similar structures and physicochemical properties, requiring minimal product-specific method validation activities. Three mAb products were used to demonstrate MAM performance for common and representative product quality attributes. Method validation design and acceptance criteria were guided by the Analytical Target Profile concept, as well as relevant regulatory guidelines to ensure the method is fit-for-purpose. A comprehensive system suitability control strategy was developed, and reported here, to ensure adequate performance of the method including sample preparation, instrument operation, and data analysis. Our results demonstrated sufficient method performance for the characteristics required for quantitative measurement of product variants and degradants.

The biosynthesis of methanobactin.

Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.

Copper-responsive gene expression in the methanotroph Methylosinus trichosporium OB3b.

Methanotrophic bacteria convert methane to methanol using methane monooxygenase (MMO) enzymes. In many strains, either an iron-containing soluble (sMMO) or a copper-containing particulate (pMMO) enzyme can be produced depending on copper availability; the mechanism of this copper switch has not been elucidated. A key player in methanotroph copper homeostasis is methanobactin (Mbn), a ribosomally produced, post-translationally modified natural product with a high affinity for copper. The Mbn precursor peptide is encoded within an operon that contains a range of putative transporters, regulators, and biosynthetic proteins, but the involvement of these genes in Mbn-related processes remains unclear. Extensive time-dependent qRT-PCR studies of Methylosinus trichosporium OB3b and the constitutive sMMO-producing mutant M. trichosporium OB3b PP358 show that the Mbn operon is indeed copper-regulated, providing experimental support for its bioinformatics-based identification. Moreover, the Mbn operon is co-regulated with the sMMO operon and reciprocally regulated with the pMMO operon. Within the Mbn and sMMO operons, a subset of regulatory genes exhibits a distinct and shared pattern of expression, consistent with their proposed functions as internal regulators. In addition, genome sequencing of the M. trichosporium OB3b PP358 mutant provides new evidence for the involvement of genes adjacent to the pMMO operon in methanotroph copper homeostasis.