Chromatographic Analysis of Host-Cell Protein Impurities in

Chapter 10

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Chromatographic Analysis of Host-Cell Protein Impurities in Pharmaceuticals Derived from Recombinant DNA Donald O. O'Keefe and Mark L. Will Department of Analytical Research, Merck Sharp and Dohme Research Laboratories, Rahway, NJ 07065 The purity of recombinant protein therapeutics is of paramount concern. Impurities in these drugs can be potentially unsafe and can include endotoxin, DNA, and host-cell proteins. The ability to detect, identify, and quantitate these impurities is an ongoing challenge for the bioanalyst. In this chapter, we provide an overview of the current chromatographic methods used, including gel electrophoresis, to analyze host-cell protein impurities. The advantages and disadvantages of each method are presented and demonstrate that any technique by itself is insufficient, but several techniques together provide a more complete and reliable protein impurity analysis.

The arrival of recombinant DNA technology has provided the pharmaceutical industry with a unique source of therapeutic proteins. This advent in technology has also generated new problems for the pharmaceutical analyst. Among these problems are the detection, quantitation, and identification of drug impurities. There are four basic sources of impurities in protein pharmaceuticals: 1) the fermentation medium 2) the host cells, 3) the purification process, and 4) the protein drug itself. Medium-related impurities include additives used to supplement host-cell growth. Certain cell lines, notably those of mammalian origin, are routinely supplemented with protein additives. Host-related impurities include endotoxin, DNA, and proteins. Impurities derived from the process can include Protein A and any monoclonal antibodies used in affinity chromatography. Drug-related impurities include aggregates, dégradâtes, structural isomers, and compositional isomers. The limited spectrum of impurities presented here makes the complex analysis apparent. Our intention in this chapter is to focus on one type of impurity, that of host-cell proteins. The level of host-cell protein impurities in an unprocessed recombinant therapeutic depends on the cell line used for production. Currently, recombinant

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proteins are produced in either Escherichia coli, yeast or mammalian cells. Early process samples of recombinant products derived from either yeast or mammalian systems are often highly enriched in the drug because the protein is usually secreted into the extracellular medium. Proteins produced in E. coli are not secreted and are only obtained after the cell is lysed. Bacterial cells have been estimated to contain a minimum of 1500 different proteins. Any number of these cellular proteins are potential impurities. This underscores the difficulty faced in both the processing and bioanalytical areas. Guidelines for the analysis of recombinant protein therapeutics for hostcell protein impurities are provided by the Food and Drug Administration (FDA)(i). The tolerable impurity level is reflected by the drug dose and the mode of administration. Initial analysis focuses on detecting protein impurities. In contrast to more traditional drugs, proteins are large and often immunogenic in trace amounts. An immunogenic impurity could act as an adjuvant when administered along with the drug. This can lead to an immunological response towards the drug itself rendering it ineffective. Highly sensitive assays are required to determine both aggregate and individual levels of impurities. Often sensitivities down to ppm (parts per million by weight) or lower are required (2). The identity of protein impurities provides valuable information on several fronts. Firstly, identification of an impurity can lead to an understanding of its physicochemical properties. This, in turn, can provide information for process modifications and the subsequent elimination of the impurity from future drug lots. Secondly, an impurity's identity can provide assurance to the FDA and individuals conducting safety and clinical trials as to the possible toxicity, carcinogenicity, or teratogenicity of the impurity. Thirdly, any effect that impurities have on the drug, such as proteolytic degradation, can be examined after their identification. Finally, identification influences the development of specific assays to monitor an impurity for lot-to-lot drug consistency. Our objective in this chapter is to review the chromatographic procedures used by bioanalysts to assay recombinant protein therapeutics for host-cell protein impurities. Generally, these procedures involve either electrophoresis or high performance liquid chromatography (HPLC). We will also review current technology used in conjunction with these procedures to specifically identify protein impurities. A short discussion is also included that examines the applicability of capillary electrophoresis to the analysis of host cell-protein impurities. Gel Electrophoresis

Gel electrophoresis is an indispensable form of separation and analysis for the bioanalyst. The application of an electric current induces proteins to migrate according to their overall charge through a semisolid medium generally consisting of crossed-linked polyacrylamide. The polyacrylamide gel acts as a sieve that differentially impedes proteins based on their molecular size and shape. The electrical current provides the driving force similar to more traditional mobile phases while the gel's sieving effect acts as a retarding force. As in all forms of

Ahuja; Chromatography of Pharmaceuticals ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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chromatography, separation is the result of these two forces interacting on individual analytes. Numerous forms of gel electrophoresis exist for the analysis of proteins (5), but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), popularized by Laemmli (4\ predominates. SDS is an anionic detergent that denatures and binds proteins in a ratio of 1.4 g of SDS per gram of protein (5). SDS denaturation produces a similar shape and a constant charge density for all protein molecules. These changes make molecular size the sole basis of protein separation by this method. SDS-PAGE is often the first choice for analyzing recombinant proteins for purity (2). Electrophoretically separated proteins are detected by staining. The two most common stains are Coomassie Brilliant Blue R-250 and silver. The mechanism of Coomassie Blue staining is not fully understood. It is thought that the dye binds primarily to basic and aromatic residues in proteins (6). The major advantage of Coomassie Blue is the almost universal quantitative binding of the dye to proteins. In a study of Coomassie Blue binding to twenty-six proteins in solution, staining variability was less than two-fold (6). Protein impurities are detected and quantitated electrophoretically when known amounts of the drug product are subjected to SDS-PAGE followed by Coomassie Blue staining. Although slight variations are common, generally 2 14 μg of the drug product are within the linear range of the assay. We have found some impurities are detected at levels as low as 60 ng. This yields a detection limit of 0.6% for a 10 Mg sample (6000 ppm). Since the linear range of the assay must not be exceeded, analyzing more than 10 μg of protein only increases the detection limit slightly. Once stained, protein impurities are estimated by visually comparing their staining intensity to that of known amounts of standard proteins electrophoresed through the same gel. Quantitation of protein impurities is more accurately determined by scanning the stained gel with a laser densitometer to produce an electropherogram. The area percent of each peak in the electropherogram is determined by methods identical to those of HPLC chromatograms. Figure 1 demonstrates this process. Samples of recombinant acidic fibroblast growth factor (aFGF) were spiked with known amounts of a E. coli protein impurity (S3 ribosomal protein) and electrophoresed through a 15% SDS-polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Blue and scanned with a laser densitometer. The detection limit for this particular protein impurity was 200 ng or 0.7%. The mechanism of silver staining proteins is not entirely understood (7). In general, silver staining is more sensitive than Coomassie Blue staining. Protein levels as low as 0.2 ng have been detected (#). If comparable sensitivity was universal among proteins then a protein impurity as low as 0.002% (20 ppm) could be detected by assaying 10 μg of total protein. Unfortunately, silver staining is highly variable and thus does not facilitate accurate quantitation (8,9). In our lab, we have analyzed one protein that is marginally detected at 125 ng by silver and not detected at all at 50 ng. If a protein impurity showed this same sensitivity then it could not be detected at a level of 1.25% (>10 ppm). This level of detection is equivalent to Coomassie Blue staining. When the two staining procedures are used simultaneously for model proteins the sensitivity is reportedly 4



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Figure 1. Electropherograms and SDS-PAGE of recombinant aFGF spiked with an E. coli protein impurity. Left: Two electropherograms of an identical sample of 30 μg of aFGF spiked with 500 ng of £. coli S3. The peak representing the S3 impurity was observed when the absorbance scale was expanded 30-fold. The 30 μg of aFGF was within the linear range of this assay. Right: The Coomassie Blue-stained gel. Each lane contained 30 μg aFGF spiked with: 1) 500 ng of S3, 2) 200 ng of S3, and 3) no S3. The location of the S3 impurity is indicated by the arrows. The migration of molecular weight markers is indicated in kilodaltons.



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increased two to eight-fold compared to silver alone (7). This variable enhancement might also extend to protein impurities. The high sensitivity of silver staining is itself problematic. To detect a protein impurity at 20 ppm requires analyzing at least 10 μg of the recombinant protein. Large protein loads obscure portions of the gel and possibly conceal impurities migrating close to the therapeutic. In addition to silver and Coomassie Blue detection, other stains exist, but their use is less widespread (3). Coomassie Blue and silver staining of protein gels are the standard electrophoretic methods used to detect and often quantitate protein impurities in recombinant therapeutics. Both these techniques suffer from an identical drawback, that is they lack specificity. An impurity detected by either approach is uncharacterized. The impurity can be a host-cell protein or another protein impurity such as an aggregate or degradate of the drug product. Host-cell impurities are specifically identified by employing an electrophoretic immunoassay. The most commonly used method is western blotting first popularized by Burnette a decade ago (10). The basic methodology of western blot analysis starts with SDS-PAGE. After electrophoresis, proteins are electrotransferred (blotted) from the gel to an immobilizing matrix (77). The two most common matrices are nitrocellulose paper and polyvinylidene fluoride (PVDF). Proteins are irreversibly bound to the matrix and then detected immunochemically. In direct analysis, immobilized proteins are probed with either radiolabeled antibodies, or antibodies conjugated to a fluorophore or an enzyme. If the protein is recognized by the antibody, the antibody binds the protein and is detected according to the appropriate signal (72). Often this approach is not practical and indirect techniques are required. Here, the immobilized proteins are first probed with specific unlabeled antibodies. The bound antibodies are then detected by a secondary probe consisting of either labeled antibodies or labeled Protein A. Western blot analysis is the most specific and sensitive assay available to the bioanalyst. As little as 30 pg of protein have been detected (75). With a total protein load of 10 μg this yields a sensitivity of 3 ppm. This level of sensitivity is adequate by most criteria. Unfortunately, this sensitivity does not prevail for all host-cell protein impurities. The antibodies used in these westerns are generally raised against a lysate derived from host cells that do not produce the recombinant therapeutic. A portion of the proteins in these lysates are only weakly reactive to the antiserum. Some proteins may not elicit an antibody response at all because their concentration is too low or they are poorly immunogenic. Hence, this technique may not be able to detect all protein impurities. Gooding and Bristow used western blot analysis to detect trace protein impurities in preparations of recombinant human growth hormone (14). They found that highly antigenic E. coli proteins were detected at a greater sensitivity in western blots than by silver staining, but the latter technique detected more protein impurities. Western blots are also poorly quantitative, but using antiserum that specifically recognizes a particular protein impurity provides limited quantitation. Recent work has led to the development of nonchromatographic immunoassays that quantitate protein impurities using host-cell



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antiserum (8,15). Although lacking quantitation, westerns are valuable tools for showing lot-to-lot drug consistency. Western blot analysis has additional shortcomings. Proteins are electrotransferred from polyacrylamide gels with variable efficiency. Transfer to the immobilizing matrix is inversely related to the protein's size; large molecules transfer less efficiently than small molecules. The inclusion of 0.1% SDS in the transfer buffer or limited proteolysis within gels enhance the transfer of large proteins (72). Small proteins bind less effectively to the immobilizing matrix than do large proteins. This drawback is reportedly overcome by using chemical crosslinkers to covalently bind the protein to the transfer matrix (16). These problems, as well as the general non-quantitative nature of westerns, demonstrate that this technique by itself cannot provide a complete impurity profile. This underscores the need to use both silver and Coomassie Blue staining in conjunction with western blot analysis (1,17). We have taken this multi-pronged approach for analyzing host-cell protein impurities in our laboratory. Recombinant aFGF produced at Merck is >99% pure when analyzed by SDS-PAGE. Protein impurities were undetected by Coomassie Blue staining (Figure 1, lane 3), but occasionally in some early production lots a low level impurity of ~ 25 kD was detected by silver staining. When antiserum raised against an E. coli cell lysate was used in a western blot of aFGF the source of this impurity proved to be the host cells (Figure 2). Through a combination of E. coli western blot analysis and HPLC, samples enriched in the ~ 25 kD impurity were prepared. The N-terminal sequence of the impurity was determined in an automated protein sequencer. The result was compared to the National Biomedical Research Foundation (NBRF) protein database and was found to match that of the S3 ribosomal protein of E. coli. We confirmed the identity of the impurity as the S3 protein using S3 monoclonal antibodies in a western blot of aFGF. The level of the S3 protein was estimated by the same technique using the enriched S3 sample as a standard. The amount of S3 in a number of aFGF preparations was shown to be less than .01% (

Chromatographic Analysis of Host-Cell Protein Impurities in

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