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.
Common Analytical Techniques Used for Residual Impurity Analysis
Mass spectrometry (MS) is an analytical technique for the determination of the elemental composition of a sample or molecule. It is also used for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. MS instruments consist of three modules: an ion source, which converts molecules into ions; a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields; and a detector, which measures the ions thus, providing data for calculating the abundances of each ion present. In a typical MS procedure, the sample is introduced to the MS instrument after chromatographic separation (GC or LC). The components are ionized by one of a variety of methods (electron impact, electrospray, or chemical ionization). The ions are then directed by an electromagnetic field in an ion trap, quadrupole, or time-of-flight to a detector. Computation of the mass-to-charge ratio (m/z) is then performed to generate a mass spectrum.
MS has both qualitative and quantitative uses and is one of the primary tools for monitoring and identifying residual impurities. Extractables and leachables profiles are typically generated using a combination of GC/MS, LC/MS, and ICP/MS. Triple quad mass spectrometry is a powerful tool for monitoring known residual impurities. Tandem quadrupole mass spectrometers separate a compound, and further break it down to yield highly specific daughter ions. These daughter ions can be used for quantification and yield high sensitivity and selectivity. Residual antibiotics can be measured using LC/MS/MS. Internal standards are commonly used and in many cases, part-per-billion (ppb) levels can be accurately detected and quantitated in very complex sample matrices.
High performance liquid chromatography (HPLC) is one of the most common methods of separation and analysis. Separation of components of a mixture is achieved by taking advantage of their different solubility. Therefore, the partition of the solute between the mobile phase and the stationary phase provides the basis of the analysis. A mobile phase gradient is usually used to separate the components. This technique is best suited for nonvolatile organic compounds. Size-exclusion chromatography (SEC) is a form of HPLC that allows one to separate molecules based on size. The mechanism used is that the large molecules are excluded from the pores of the stationary phase. HPLC systems are configured with various detectors including ultraviolet (UV), refractive index, fluorescence, photodiode array, electrochemical, evaporative light scattering detector, charged aerosol, and mass spectrometer. Each of these detectors has advantages and disadvantages, and therefore, must be chosen based on the analyte of interest, the sample matrix, the sensitivity, and the selectivity required. Coupling of HPLC with the CAD detector has recently found many applications for monitoring impurities that do not contain chromaphores.
Gas chromatography (GC) is another one of the most common methods of separation and analysis. Separation of components of a mixture is achieved by taking advantage of their different solubility. Therefore, the partition of the solute between the gas phase and the liquid phase coating the chromatographic column provides the basis of the analysis. Samples are introduced into the GC column either by direct injection of the liquid or by sampling the headspace over a liquid sample. The most common detectors include flame-ionization and mass spectrometer. The technique is best suited for volatile and semivolatile organic compounds and is commonly used for residual solvent analysis.
Ion chromatography (IC) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. IC can be used for almost any kind of charged molecule including large proteins, small nucleotides, amino acids, and small molecules. It is a powerful technique best suited for determining low concentrations of ions.
Inductively coupled plasma (ICP) is a technique where samples are vaporized in an argon plasma. These instruments can be coupled to an optical emission or mass spectral detector. They are used to determine the level of various metals.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is a denaturing gel electrophoresis technique used in the development and characterization of proteins. SDS is a detergent that dissociates and unfolds oligomeric proteins into its subunits. The SDS binds to the polypeptides to form complexes with fairly constant charge to mass ratios. The electrophoretic migration rate through a gel is therefore determined only by the size of the complexes. Molecular weights are determined by simultaneously running marker proteins of known molecular weight. The gel can be stained by silver staining or colloidal blue. The gels are scanned and analyzed using a densitometer to identify the sample bands. This approach can be used to monitor HCP.
Western blot is an analytical technique used to detect specific proteins in a given sample. Blotting is the transfer of large molecules on to the surface of an immobilizing membrane. This blotting technique is used to establish protein identity and purity. Native or denatured proteins are separated by gel electrophoresis and then transferred to a membrane. They are then identified using antibodies specific to the target protein, which develops color at the site of the protein-antibody complex on the membrane. This approach also can be used to monitor HCP.
Polymerase chain reaction (PCR) is a technique used to amplify a single or few copies of a DNA fragment by several orders of magnitude when the ends of the sequence are known. Genomic DNA is digested into large fragments using a restriction enzyme and then is heat-denatured into single strands. Two synthetic oligonucleotides complementary to the 3' ends of the target DNA segment of interest are added in great excess to the denatured DNA, and the temperature is lowered to 50–60 °C. The genomic DNA remains denatured because the complementary strands are at too low a concentration to encounter each other during the period of incubation but the specific oligonucleotides, which are at a very high concentration, hybridize with their complementary sequences in the genomic DNA. The hybridized oligonucleotides then serve as primers for DNA chain synthesis, which begins after addition of a supply of deoxynucleotides and a temperature-resistant enzyme DNA polymerase (Taq polymerase). Taq polymerase can extend the primers at temperatures up to 72 °C. When synthesis is complete, the whole mixture is heated up further (to 95 °C) to melt the newly formed DNA duplexes. When the temperature is lowered again, another round of synthesis takes place because excess primer is still present. Repeated cycles of synthesis (cooling) and melting (heating) quickly amplify the sequence of interest. At each round, the number of copies of the sequences between the primer sites is doubled, and therefore, the desired sequence increases exponentially. PCR is a great technique for confirming residual DNA clearance. Another form of PCR called reverse-transcriptase (RT-PCR) can be used for residual RNA.
Enzyme-linked immunosorbent assay (ELISA) is used to detect an antibody or antigen in a sample. The sample with an unknown amount of antigen is immobilized on a solid support either by adsorption to the surface or by capture methodology, known as "sandwich" ELISA. After the antigen is immobilized on the support surface, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody, which is linked to an enzyme through bioconjugation. After washing, the plate is developed by adding an enzymatic substrate to produce a visible signal, indicating the quantity of antigen in the sample. There are kits available for HCP specific to a given cell line.
Approaches for Developing Residual Impurity Methods
The first step is to determine how to handle the sample and may involve removing the protein. Acetonitrile or sodium chloride can be added to precipitate out the protein, and then the supernatant can be obtained by centrifugation or filtration. Care must be taken to ensure that the residual impurity of interest is not co-precipitated or removed with the protein through thorough validation.
The next step may involve further sample preparation including extraction, distillation, and cleanup. Extraction approaches may include liquid–liquid extraction with appropriate solvent or solid phase extraction. Some methods may require derivatization, which is an approach that modifies the impurity of interest to make it more amenable to a specific detector. These steps must be evaluated through validation.
Next, the determinative approach must be investigated. For example, a volatile organic compound will most likely be best suited for GC, whereas a nonvolatile compound by HPLC. Also, the detector must be chosen based on the analyte of interest, the sample matrix, the sensitivity, and the selectivity required. As mentioned above, HPLC with charged aerosol detection may be a good approach for compounds that do not respond to UV. Also, if the compound can be ionized, MS/MS is usually a good approach because of selectivity and sensitivity.
After these conditions are established, the method is evaluated for potential interferences and limit of detection and limit of quantitation within its particular matrix. Also, the method must be tested to ensure acceptable levels of precision, accuracy, and linearity for the intended application. It then can be used as a qualified method, or a protocol could be drafted to perform a formal method validation.
Monitoring of residual impurities seen in bioprocessing can be quite challenging. Because of the range of potential impurities, many different analytical approaches may need to be used. After developed, these methods must be qualified or validated for the intended use.
Jon S. Kauffman, PhD, is the director of method development & validation and biopharmaceutical services at Lancaster Laboratories, Lancaster, PA, JKauffman@lancasterlabs.com.
1. US Food and Drug Administration. Guidance for industry. Genotoxic and carcinogenic impurities in drug substances and products. Rockville, MD; 2008 Dec.
Available from: http://www.fda.gov/
2. European Commission. EMEA Guideline on the limits of genotoxic impurities. Brussels, Belgium; 2006 Jun. Available from: http://www.emea.europa.eu/pdfs/human/swp/519902en.pdf .