Process related impurities

Protein impurities are either process- or product-related impurities. Process-related impurities include proteins added to the culture medium, proteins used during purification, such as nucleases and chromatography ligands, and proteins from the host organism. Product-related impurities include degra-dates, aggregates, and conformational isomers of the recombinant drug product. 

Nuclear wastes are classified according to the level of radioactivity. Low level wastes (LLW) from reactors arise primarily from the cooling water, either because of leakage from fuel or activation of impurities by neutron absorption. Most LLW will be disposed of in near-surface faciHties at various locations around the United States. Mixed wastes are those having both a ha2ardous and a radioactive component. Transuranic (TRU) waste containing plutonium comes from chemical processes related to nuclear weapons production. These are to be placed in underground salt deposits in New Mexico (see... 

Figure 13.17 gives an example of some process-related impurities that can be resolved by TLC. 

Industrial solutions invariably contain dissolved impurities that can increase or decrease the solubility of the prime solute considerably, and it is important that the solubility data used to design crystallisation processes relate to the actual system used. Impurities can also have profound effects on other characteristics, such as nucleation and growth. 

Process-related impurities Impurities that are derived from the manufacturing process. They may be derived from cell substrates (e.g., HCPs, host cell DNA), cell culture (e.g., inducers, antibiotics, or media components), or downstream processing (e.g., processing reagents or column leachables). 

NMR is a remarkably flexible technique that can be effectively used to address many analytical issues in the development of biopharmaceutical products. Although it is already more than 50 years old, NMR is still underutilized in the biopharmaceutical industry for solving process-related analytical problems. In this chapter, we have described many simple and useful NMR applications for biopharmaceutical process development and validation. In particular, quantitative NMR analysis is perhaps the most important application. It is suitable for quantitating small organic molecules with a detection limit of 1 to 10 p.g/ml. In general, only simple one-dimensional NMR experiments are required for quantitative analysis. The other important application of NMR in biopharmaceutical development is the structural characterization of molecules that are product related (e.g., carbohydrates and peptide fragments) or process related (e.g., impurities and buffer components). However, structural studies typically require sophisticated multidimensional NMR experiments. 

As mentioned previously, process related impurities are typically starting materials, by-products of side reactions, intermediates, or reagents. Starting materials are easy to identify as their structure is known, and their retention times can be quickly compared to known standards during method development. Reagents that are organic in nature could fall into... 

Once an assessment on a particular impurity has been made all process-related compounds will be examined to confirm that the impurity of interest is indeed an unknown. An easy way of doing this is to compare the retention times of known process-related compounds to that in question. If this analysis confirms that the compound is an unknown, the next step would be to obtain an LC-MS on the compound. Mass spectrometry provides structural information which aids in determining structure. In some cases, mass spectrometry will be enough to identify the compound. In other cases, more complicated methods like LC-NMR are needed or the impurity will need to be isolated in order to obtain additional information. Compounds that are not purified often contain high levels of by-products and can be used for this purpose. Alternatively, mother liquors from crystallizations also contain levels of by-products. Other ways of obtaining larger quantities of impurities include flash chromatography which is typically used for normal phase separations or preparative HPLC which is more common for reversed phase methods. Once a suitable quantity of the compound in question has been obtained a full characterization can be carried out to identify it. 

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

Table 5. Impurity formation related to process conditions...

Impurities in API. Treatment of the impurities in the API is similar to that for the new drug product. Impurities in the API include organic impurities (process and drug related), inorganic impurities, and residual solvents. Quality control analytical procedures are developed and validated to ensure appropriate detection and quantitation of the impurities. Specification limits for impurities are set based on data from stability studies and chemical development studies. A rationale for the inclusion or exclusion of impurities is set at this stage. The limits set should not be above the safety level or below the limit of the manufacturing process and analytical capability. 

Bulk and Intermediate Purification primarily for removal of process-related impurities, e.g. reagents, host cell proteins, DNA, endotoxins some product-related impurities common methods ... 

By phase II, stability assays should be validated and a good faith effort made to validate all in-process tests. Release assays should also be validated. Removal of product and process-related impurities should be demonstrated. Stability of cells during growth should be validated. 

Olsen BA, Baertschi SW. Strategies for investigation and control of process-related and degradation-related impurities in Pharmaceuticals. In Ahuja S, Alsante KM, eds. Handbook of Isolation and Characterization of Impurities in Pharmaceuticals, Volume 5 of Separation Science and Technology. San Diego, CA Academic Press, 2003. 

Lorenz LJ, Bashore FN, Olsen BA. Determination of process-related impurities and degradation products in cefaclor by high-performance liquid chromatography. J Chrom Sci 1992 30 211-216. 

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