Pharmaceutical NEWS - Fall 2009

Monitoring of Residual Impurities Encountered in Bioprocessing

by Dr. Jon S. Kauffman, director of Biopharmaceutical Services and Analytical Method Development & Validation

Profiling of impurities in biopharmaceutical products is a regulatory expectation. Since residuals are typically present at low levels in difficult sample matrices, development and qualification of assays can be quite challenging. Matrix types can vary greatly due to the fact that sampling at a variety of steps is required to accurately confirm clearance throughout the production process.

Some residual impurities are introduced in the upstream steps as essential ingredients of the fermentation or cell-culture media. Various impurities result from the culture growth and harvest. Nucleic Acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and Host Cell Proteins (HCPs) are some of the unwanted cell components that accompany the protein of interest after cell lysis. Antibiotics are added to the cell-culture media to control bacterial contamination. Common antibiotics used include kanamycin, ampicillin, penicillin, amphotericin B, tetracyline, gentamicin sulfate, hygromycin B and plasmocin to control mycoplasma.

Additional residual impurities are introduced throughout the process. Process-enhancing agents and catalysts are added 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 and Isopropyl â-D-1-thiogalactopyranoside (IPTG) is used to induce gene expression and to aid in the refold process.

Finally, there are other residual impurities introduced to aid in the purification of the product downstream. Chromatographic purification of target proteins may require the use of alcohols and glycols, which must be cleared from the process. Surfactants are added to aid in separating the protein, peptide and nucleic acids from the process stream. Examples include Triton-X, Pluronic, Antifoam- A, B, C, Tween or Polysorbate.

The variety of residual impurities requires the employment of a wide array of analytical techniques to characterize and quantify residuals. Lancaster Laboratories has added capacity to the following areas.

Mass spectrometry (MS) yields both qualitative and quantitative information and is one of the primary tools for monitoring and identifying residual impurities. Residual antibiotics can be measured accurately at part-per-billion (ppb) levels using LC/MS/MS in very complex sample matrices.

High Performance Liquid Chromatography (HPLC) is a common method of separation and can be configured with various detectors, including ultraviolet (UV), refractive index (RI), fluorescence, electrochemical, evaporative light scattering detector (ELSD). Detectors are chosen based on the residual of interest, the sample matrix, and the sensitivity and selectivity required. Lancaster Laboratories has had success using charged aerosol detectors (CADs) in detecting residuals without chromaphores that are not amenable to UV.

Gas Chromatography (GC) is another common method of separation and analysis that utilizes flame-ionization detectors (FID) and mass spectrometric detection (MSD). This technique is best suited for volatile and semivolatile organic compounds and is commonly used for residual solvent analysis. Metals analyses can be performed using inductively coupled plasma with optical emission spectroscopy or mass spectrometric detection (ICP-OES, ICP-MS). Polymerase Chain Reaction (PCR) is a technique used to amplify a single or few copies of a DNA fragment by several orders of magnitude. Therefore, it is a great technique for confirming clearance of residual DNA.

Also, Enzyme-Linked Immunosorbent Assay (ELISA) is used to detect an antibody or antigen in a sample. There are kits available for host cell proteins specific to a given cell line.

The first step is to determine how to handle the sample. The protein may first need to be precipitated out of solution and then the supernatant can be obtained by centrifugation or filtration. Care must be taken to ensure that the residual impurity is not co-precipitated and/or removed with the protein. The next step may involve further sample preparation, including extraction, distillation and/or cleanup. Extraction approaches may include liquid-liquid extraction with appropriate solvent. Some methods may employ derivatization, which is an approach that modifies the impurity of interest to make it more amenable to a specific detector. Again these steps would need to be evaluated in method development. 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, HPLC with Charged Aerosol Detection (CAD) 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 due to its selectivity and sensitivity.

Once method conditions are established, the method is evaluated for potential interferences and limit of detection within the particular matrix. Also, the method will be tested to ensure acceptable levels of precision, accuracy, and linearity for the intended application. The method then can be used as a qualified method or a protocol could be drafted to perform a formal method validation.

For more information, call Dr. Kauffman at 717-656-2300, ext. 1377.