Profiling of impurities in biopharmaceutical products and their associated intermediates and excipients is a regulatory expectation. The US Food and Drug Administration has recently made available a guidance for industry, Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches, which is intended to inform pharmaceutical manufacturers of the agency's current views with respect to genotoxic and carcinogenic impurities in drug substances and drug products, including biologics.1 This guidance provides recommendations on how to evaluate the safety of these impurities and exposure thresholds. The European Medicines Agency's (EMEA committee for Medicinal Products for Human Use (CHMP) also published the Guideline on the Limits of Genotoxic Impurities, which is being applied by European authorities for new drug products and in some cases also to drug substances in drug development.2 These guidelines augment the International Conference on Harmonization (ICH) guidances for industry: Q3A(R2) Impurities in New Drug Substances, Q3B(R2) Impurities in New Drug Products, and Q3C(R3) Impurities: Residual Solvents that address impurities in a more general approach.
Although some impurities are related to the drug product, others are added during synthesis, processing, and manufacturing. Because residuals typically are present at low levels in difficult sample matrices, development and validation of assays and ongoing testing can be quite challenging. Biomanufacturing is a complex process involving many steps from upstream fermentation, cell lysis, and solubilization to downstream refolding, purification, polishing, and formulation. The sample matrix types can vary greatly because sampling at a variety of process steps is required to accurately monitor the target throughout the production process. This article will discuss these challenges and some strategies used to overcome them.
Classes of Product-Related Impurities
Product-related impurities fall into several broad classes. Some are introduced in the upstream steps as required components of the fermentation or cell-culture media. Some impurities result from culture growth and harvest. With the increase in use of disposables for bioprocessing such as bags, filters, and tubing, other residuals can be introduced throughout the process. Some examples are listed below.
Product-related impurities and product-related substances introduced upstream
Nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are some of the unwanted cell components found in the protein of interest after cell lysis.
- Host cell proteins (HCP), like nucleic acids, are also unwanted cell components that are seen with the protein of interest after cell lysis.
- Antibiotics are added upstream to the cell-culture media to control bacterial contamination and maintain selective pressure on the host organisms. The common antibiotics used include kanamycin, ampicillin, penicillin, amphotericin B, tetracyline, gentamicin sulfate, hygromycin B, and plasmocin to control mycoplasma.
Residual impurities throughout the process
- Process enhancing agents or catalysts are added throughout the process to make some of the steps more efficient and increase yield of the product. Guanidine and urea are added for solubilization of the fermentation output. Glutathione and dithiothreitol (DTT) are used during reduction and refolding of proteins. DTT is used to reduce the disulfide bonds of proteins and prevent them from forming between cysteine residues of proteins. Glutathione is a reducing agent. Isopropyl -D-1-thiogalactopyranoside (IPTG) is used to induce gene expression and to aid in the refold process.
Residual impurities introduced downstream
- Chromatographic purification of target proteins may require the use of chemicals that must be cleared from the process. Examples of such chemicals are certain alcohols and glycols.
- Surfactants are lipid molecules that contain both hydrophilic and hydrophobic (lipophilic) moieties. They are added during downstream processing to aid in separating the protein, peptide, and nucleic acids from the process stream by lowering the interfacial tension by adsorbing at the liquid–liquid interface. Examples include Triton-X, Pluronic, Antifoam- A, B, C, Tween, or Polysorbate.
Residual impurities introduced from disposables
- Compounds that can be extracted from a component under exaggerated conditions, such as in the presence of harsh solvents or at elevated temperatures, and have the potential to contaminate the drug product are referred to as extractables. Compounds that leach into the drug product formulation from the component as a result of direct contact with the formulation under normal conditions or sometimes at accelerated conditions are referred to as leachables. Leachables are typically a subset of extractables. Extractables must be controlled to the extent that components used are appropriate. Leachables must be controlled so that the drug products are not adulterated. Disposables can greatly reduce the risk of cross-contamination between batches, do not require steam sterilization, minimize turnaround time, and cleaning requirements, and eliminate costly capital expenditures for stainless steel tanks and piping. With an increase in the use of disposables during bioprocessing, validation must be performed to ensure that appropriate filters, bags, fittings, and tubing are chosen. Some examples of potential extractables and leachables compounds from disposables include phthalates, nitrosamines, polynuclear aromatic hydrocarbons, and metals.