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.

Challenges and new frontiers in analytical characterization of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a growing class of biotherapeutics in which a potent small molecule is linked to an antibody. ADCs are highly complex and structurally heterogeneous, typically containing numerous product-related species. One of the most impactful steps in ADC development is the identification of critical quality attributes to determine product characteristics that may affect safety and efficacy.

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.

Identification of a host cell protein impurity in therapeutic protein, P1.

Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can pose safety health risks to patients. An adequate control of HCP levels in the final product, and demonstration of HCP clearance throughout a product manufacturing process is critical for all biotherapeutic products. Developing effective downstream purification processes can be challenging as HCPs and product proteins may possess an affinity for each other or have similar physicochemical properties, resulting in co-purification.

Drug-to-antibody determination for an antibody-drug-conjugate utilizing cathepsin B digestion coupled with reversed-phase high-pressure liquid chromatography analysis.

Antibody drug conjugates or ADCs are currently being evaluated for their effectiveness as targeted chemotherapeutic agents across the pharmaceutical industry. Due to the complexity arising from the choice of antibody, drug and linker; analytical methods for release and stability testing are required to provide a detailed understanding of both the antibody and the drug during manufacturing and storage. The ADC analyzed in this work consists of a tubulysin drug analogue that is randomly conjugated to lysine residues in a human IgG1 antibody.

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

Purpose: Discoloration 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.

Electron detachment dissociation and negative ion infrared multiphoton dissociation of electrosprayed intact proteins.

In top-down proteomics, intact gaseous proteins are fragmented in a mass spectrometer by, e.g., electron capture dissociation (ECD) to obtain structural information.

Negative-ion electron capture dissociation: radical-driven fragmentation of charge-increased gaseous peptide anions.

The generation of gaseous polyanions with a Coulomb barrier has attracted attention as exemplified by previous studies of fullerene dianions. However, this phenomenon has not been reported for biological anions. By contrast, electron attachment to multiply charged peptide and protein cations has seen a surge of interest due to the high utility for tandem mass spectrometry (MS/MS).

Hydrogen tunneling in adenosylcobalamin-dependent glutamate mutase: evidence from intrinsic kinetic isotope effects measured by intramolecular competition.

Hydrogen atom transfer reactions between the substrate and coenzyme are key mechanistic features of all adenosylcobalamin-dependent enzymes. For one of these enzymes, glutamate mutase, we have investigated whether hydrogen tunneling makes a significant contribution to the mechanism by examining the temperature dependence of the deuterium kinetic isotope effect associated with the transfer of a hydrogen atom from methylaspartate to the coenzyme. To do this, we designed a novel intramolecular competition experiment that allowed us to measure the intrinsic kinetic isotope effect, even though hydrogen transfer may not be rate-determining.

One-step immobilization of glucose oxidase in a silica matrix on a Pt electrode by an electrochemically induced sol-gel process.

We demonstrate here that the electrochemical generation of hydroxyl ions and hydrogen bubbles can be used to induce the synthesis of enzyme- or protein-encapsulated 3D porous silica structure on the surface of noble metal electrodes. In the present work, the one-step synthesis of a glucose oxidase (GOD)-encapsulated silica matrix on a platinum electrode is presented. In this process, glucose oxidase was mixed with ethanol and TEOS to form a doped precursory sol solution.