Process-related impurities chromatography

Process Residuals A source of potential impurities can be process residuals as well as host cells. Process residuals include fermentation media components as well as raw materials used in chromatography (immunoglobulin affinity ligands), etc. According to the ICH Guidelines,2 process-related impurities are impurities that are derived from the manufacturing process. They may be derived from cell substrates, cell culture, or downstream processing. 

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. 

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. 

J. Xu, R. Thompson, B. Li, and Z. Ge, Application of packed column supercritical fluid chromatography for separation of bromosulfone from process related impurities, /. Liq. Chrom. Relat. Technol. 25 (2002), 1007-1018. 

Experimental campaign bulk or in-process control samples (e.g., prior to recrystallization) are excellent sources of process-related impurities and are a vital component of the KPSS. Isolated fractions collected from a preparative or semi-preparative liquid chromatography (LC) system are also excellent KPSS samples. Sometimes small-scale synthesis of the impurity or degradant is possible and less time-consuming than the isolation techniques. 

The separation tasks for ion-exchange chromatography differ mainly by the type of expression system used. The most abundant systems are mammalian cells, microbial cells (yeast), and the bacterial organism E. coli. All expression systems have a common feature that they not only generate the target protein but also process related impurities, such as host cell proteins, DNA, viruses, endotoxins. In addition the downstream process has to isolate the active form of the target molecule from all its derivatives, for example, oxidated, deamidated, acetylated forms, dimers, aggregates, and unfolded proteins. 

Lorenz, LJ. Bashore, F.N. Olsen, B.A. Determination of process-related impurities and d adation products in cefaclor by hi -performance liquid chromatography. J.Chromatogr.ScL, 1992,30,211— 216 determination, [simultaneous impurities, degradation products bulk gradient coliunn temp 25] Nahata, M.C. Jackson, D.S. Liquid chromatographic method for the determination of cefadroxil in its suspension and in serum. J.Liq.Chromatogr., 1990,13,1651-1656 [simultaneous cefadroxil sui n-sions cefaclor is IS]... 

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. 

Zeolite adsorbents (mostly A-lype and X-type, LTA and FAU, respectively, and to a lesser extent Mordenite, MOR and Y, FAU) are used in a number of processes related to bulk gas separations, dilute impurity removal, drying and chromatography (Table 3). 

Purification processes require several steps in order to obtain a commercially pure product. Affinity chromatography is therefore extremely useful, but until a few years ago, was limited to antibodies produced by the immune systems of laboratory animals. However, antibodies often cannot discriminate between closely related impurities. In addition, the drastic sanitation conditions used in the production of therapeutic products may denature the antibodies. Phage display technology allows the isolation of affinity ligands with the required physical and chemical properties. This technique can also discriminate between the target and closely related impurities. Small peptides bound to resins are well suited for use in purification of proteins that are normally used as drugs. Once a peptide with... 

To facilitate the identification of compounds by spectroscopic or MS-related analyses, enriched process streams may be prepared for analyses. Aqueous extracts and mother liquors can provide convenient enrichment. In some cases a solid product may be slurried in a solvent in order to leach out an impurity that has slightly different solubility ( swish purification [19]). Such enrichment eases isolation of an impurity by preparative chromatography. 

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