Excerpted from Drug Development & Delivery - January/February 2021 Issue.
The global pharmaceutical analytical testing outsourcing market was estimated at $6.1 billion in 2019 and is anticipated to register a CAGR of 8.3% through 2027, according to Grand View Research, Inc.1 Increasing demand for analytical drugs, biosimilars, and biopharmaceuticals are contributing to market growth. Other factors such as increasing investments in R&D for pharmaceuticals, rising demand for product safety and quality, and changing regulations for in vivo and in vitro tests are also expected to drive the demand for pharmaceutical analytical testing outsourcing services. Additionally, the development of combination products, biosimilars, and other innovative medicines has led to an increase in demand for specific types of pharmaceutical analytical tests, such as bioanalytical testing, method development and validation, active pharmaceutical ingredient testing, and stability testing.
Demonstrate Control Over Biologics Manufacturing
Analytical testing of biologics presents unique challenges relative to small-molecule APIs and drug products. Manufacturing biologics using cellular systems results in a mixture of molecules from the expression of the biologic within the cells. This mixture makes it critical to demonstrate control over the manufacturing process. As a result of the size and complexity of biologics, multiple methods are required to detect the different types of potential in-process and degradation products. Control over the process can be demonstrated using both chromatographic and electrophoretic techniques as well as assays demonstrating that the desired activity of the biologic is maintained.
William Boomershine, PhD, Senior Manager, Biologics, Alcami, explains Alcami’s chromatographic/ electrophoretic techniques, such as in the case of charge state variants, which can arise during the fermentation process in the form of various glycans containing differing sialic acids. Charge state variants can also arise as degradation products on stability. Deamidation of aspartic acid or glutamic acid residues to yield asparagine or glutamine residues change the overall charge of the protein. Charge state variants can be separated chromatographically using ion-exchange chromatography (IEX). The decision to use cation-exchange or anion-exchange chromatography will depend on the isoelectric point of the protein and the overall charge of the protein in the formulation buffer. Charge state variants can also be separated using electrophoresis. Traditional iso-electric focusing and capillary iso-electric focusing can provide an orthogonal method to IEX for separation of charge state variants.
Dr. Boomershine goes on to explain how molecular weight variants can be in-process impurities formed during the purification or refolding steps of drug substance manufacturing, and can be degradation products, forming in the drug product on stability. Size exclusion chromatography can monitor both dimers and higher order aggregates, as well as smaller impurities in buffers that can closely mimic physiological buffers. Electrophoresis – both traditional SDS-PAGE and capillary – can separate impurities based on molecular weight. Non-reduced SDS is utilized to monitor molecular weight variants that use disulfide bonds to form dimers and other higher order aggregates while reduced SDS can detect molecular weight variants of the light and heavy chains from monoclonal antibodies.
“While the above chromatographic and electrophoretic techniques can be used to look at charge state and molecular weight variants of the biologic as a whole, these techniques may not easily see specific, individual changes that can impact activity,” he says.
Detecting and quantitating local changes require techniques with higher resolution, such as a peptide map. The individual peptides from a peptide map can be more easily separated from each other using reversed-phase HPLC. “Chromatography for a peptide map can be optimized to target the separation and quantitation of a specific process impurity or degradation product, such as methionine oxidation, N-terminal variants, or deamidation of a specific residue,” he says.