Analytical Biotechnology of Recombinant Products The biotechnological revolution has created many new challenges and op portunities in analytical chemistry. A number of these challenges involve the monitoring of raw materials, in-process intermediates, and final products in the commercial production of pharma ceuticals. T h e opportunities arise from the creativity involved in developing analytical techniques for a new area that, until recently, was primarily the province of biochemists. Recombinant products are generally produced in cultures of transformed (genetically modified) bacterial or mammalian cells. T h e use of bacteria is advantageous in that production rates are frequently higher than in mamma lian cells, which makes the economics of production very favorable. Bacteria, however, do not secrete their products, which necessitates mechanical or chemical lysis of the cells. Moreover, bacteria are not capable of the posttranslational processing steps (such as glycosylation) that are required in the production of certain proteins, such as tissue plasminogen activator (TPA), a glycoprotein t h a t dissolves clots in myocardial infarcts. A further disad vantage of working with bacteria is the possible presence of impurities such as endotoxins and E. coli proteins in re combinant products derived from bac teria. In an attempt to overcome these problems and a number of other disad vantages of bacteria, mammalian cell culture has been adopted for many re combinant applications (J). Mammali an cells are capable of appropriate posttranslational processing (e.g., gly cosylation and the oxidation of sulfhydryl groups to disulfides) and are able to secrete recombinant products into the surrounding medium. According to Robert L. Garnick, director of quality control operations at Genentech, Inc., "I frankly predict t h a t there will be a tremendous increase in the number of products produced in mammalian cell culture in the next 5 to 10 years." Gar-
nick spoke at the Analytical Biotech nology Intensive Seminar held recently in Baltimore (see box). Analytical requirements for biologies To d a t e , four r e c o m b i n a n t sub stances—insulin, h u m a n growth hor mone, α-interferon, and hepatitis-B vaccine—have been licensed by the Food and Drug Administration (FDA) for commercial production, and many more have been submitted for approv al. Much of the challenge of modern analytical biotechnology involves the detection of protein impurities and contaminants in such products. Impor t a n t analytes found in recombinant products and the analytical techniques that correspond to them are shown in Table I. Recombinant products intended for the treatment of human diseases must be shown to be safe, effective, and pure (or, if heterogeneous, at least consis tent in composition). Their potency must be determined, and they must be
thoroughly tested for sterility and sta bility (the maintenance of structure and function). Multiple assays are used to assure the identity, purity, safety, and potency of recombinant products. These four testing categories are cov ered in the following sections. Identity and structural analysis Important techniques for identity de terminations and structural analyses of recombinant proteins include tryptic mapping, amino acid analysis, and pro tein sequencing. T r y p t i c m a p p i n g (peptide map ping) is the premier method available today for the quality control of recom binant protein products. This tech nique involves digestion of a protein with trypsin, an enzyme t h a t cleaves on the C-terminal side of arginine and ly sine residues to produce a set of pep tides that is then separated by highperformance liquid chromatography (HPLC). The chromatogram of these peptide fragments is the tryptic map.
The Analytical Biotechnology Intensive Seminar, held in Baltimore in May, was organ ized by Barry Karger of the Barnett Institute at Northeastern University and Fred Régnier of Purdue University's biochemistry department to bring analytical chemists, biochemists, and biotechnologists together for discussions on common interests and research needs. Six major speakers and session topics were featured at the seminar: Jack Dixon (Purdue University), on the scientific needs and problems of biotechnology; Kenneth Rinehart (University of Illinois), on potential applications of new mass spectrometry techniques to biotechnology; John Markley (University of Wisconsin), on the role of macromolecular nuclear magnetic resonance spectrometry in biotechnology; Edgar Haber (Harvard Medical School and Massachusetts General Hospital), on antibodies as analytical tools; Karger and Régnier, on high-performance liquid chromatography; and Pier Giorgio Righetti (University of Milan, Italy), on electrophoresis. In addition, lectures were presented by Frank A. Robey (National Institutes of Health, formerly of the Food and Drug Administration), on the evolving regulatory response to products of recombinant DNA technology, and by Robert L. Garnick (Genentech, Inc.), on the application of analytical biochemical methods to the quality control of recombinant DNA-derived pharmaceutical products. The present article covers only the presentations by Robey and Garnick. Another Analytical Biotechnology Intensive Seminar is planned for May 1988 in Baltimore. Information on next year's meeting can be obtained from either Barry Karger or Fred Régnier.
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"The position of arginines and ly sines throughout a protein is unique to that protein," said Frank A. Robey of the National Institutes of Health (for merly of FDA). "I don't know of any case of two different proteins with their lysines and arginines in the exact same place. But even if this were the case, the amino acids in each fragment would still be different, which would change the HPLC elution profile. So that's why, when we talk about a tryptic map, we're talking about a fingerprint in the true sense of the word." Application of the tryptic mapping technique is complicated by the fact that many large proteins contain inter nal disulfide linkages that tend to re strict the access of trypsin to portions of the molecule. "One gets to a certain point and the digestion stops," ex plained Garnick. "The only way we have found to get around that is to re duce all the disulfides with dithiothreitol and to then carboxymethylate the resulting sulfhydryl groups with iodoacetic acid." When a tryptic map is developed, the goal is to obtain as many distinct peaks as possible on the chromatogram. "What one strives to do," explained Garnick, "is to isolate the Ν and C ter-
Recombinant products intended for the treatment of human diseases must be shown to be safe, effective, and either pure or consistent. mini of the molecule and other points of concern to ascertain whether any changes have occurred in production. This [Figure 1] is a typical production map of TPA, in which the tryptic digest is chromatographed and compared to a reference material, shown bere in a mirror-image format. One can clearly see that each of the peaks present in the reference material is also present in the production lot. Beyond this, we also ratio corresponding peaks in some cases to pick up changes that might be hard to spot in a visual comparison." Possible problems with tryptic map ping include incomplete digestion, in complete reduction or carboxymethylation, chymotryptic cleavages (chymotrypsin and chymotrypsinogen are
Table I. Impurities and contaminants of concern in recombinant DNA technology3 Likely impurities or contaminants Endotoxin Host cell proteins Other protein impurities, introduced from mammalian cell culture media DNA Mutants, including single-point mutations Presence of formyl methionine residues Presence of oxidized methionine residues Proteolytic cleavages9 Deamidation' Microbiological contamination Mycoplasma' Virus
Analytical detection method Rabbit pyrogen test, LAL6 SDS-PAGEC, immunoassays such as ELISAd SDS-PAGE, HPLC
DNA dot-blot hybridization' Tryptic mapping Tryptic mapping Amino acid analysis, tryptic mapping, Edman degradation (protein sequencing) IEF", SDS-PAGE, HPLC IEF Sterility testing 21CFR [Code of Federal Regs.] method Viral susceptibility assay
a
Adapted with permission of Robert L. Garnick, Genentech, Inc. " Limulus amoebocyte lysate procedure. c Sodium dodecylsulfate polyacrylamide gel electrophoresis. " Enzyme-linked immunosorbent assay. • High-performance liquid chromatography. ' The identification of impurities in recombinant products through hybridization with 32P-labeled DNA probes on nitrocellulose. 9 Proteolytic clips can be a major problem in the isolation and stability testing of a protein product. " Isoelectric focusing. 1 Hydrolysis of asparagine and glutamine residues to form aspartate and glutamate, respectively. ' Gram-negative microorganisms that are intermediate in some respects between viruses and bacteria.
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impurities in some batches of trypsin), nonspecific cleavages, and the deamidation (hydrolysis) of glutamine and asparagine residues (which changes them into glutamate and aspartate res idues and therefore changes the reten tion times of peptides containing these residues). "One thing we've discover ed," said Garnick, "is that people are very tuned in to using autosamplers these days for tryptic mapping. If the sample sits in the autosampler for a period of time, it tends to deamidate and one certainly gets new peaks in the peptide map." Amino acid analysis, a classical procedure used to establish the identi ty of proteins, involves the acid hydro lysis of a protein or peptide to separate all of the component amino acids. The resulting mixture is then separated ei ther by ion-exchange or reversed-phase chromatography. Amino acid analysis is valuable for small proteins, but the method becomes less powerful as pro teins increase in size. The classical technique for amino acid analysis is ion-exchange chroma tography with detection of primary and secondary amines at 440 and 570 nm after postcolumn reaction with ninhydrin. An increasingly popular alterna tive to ion-exchange involves precolumn derivatization with phenyl isothiocyanate, o-phthalaldehyde, or 9-fluorenylmethyl chloroformate, re agents that make it possible to separate amino acid mixtures on reversed-phase columns with detection by UV or fluo rescence spectrometry. Protein sequence analysis, which is frequently used to find amino acid deletions or substitutions in recombi nant proteins, is performed using an other classical procedure, the Edman degradation technique. This technique involves the coupling of phenyl isothiocyanate (the same reagent used in re versed-phase amino acid analysis) to the N-terminus of a peptide, cleavage of the product with trifluoroacetic acid, and subsequent rearrangement to a phenylthiohydantoin derivative, which is then determined by HPLC with UV detection. Sequence analysis can be used to detect the presence of homoge neous impurities, but it is not of much help for products that are intrinsically heterogeneous, such as Factor VIII (antihemophilia factor), which consists of a set of proteins. Purity Robey alluded to the importance of testing for purity when he pointed out that a drug such as insulin, which was the first recombinant product licensed by FDA, "must be injected into some patients three times a day for the rest of their lives. A low-level contaminant in such a product could eventually build up and cause health problems."
T^WWTO 1
0.0
10.0
20.0
30.0
40.0
50.0 Time (min)
60.0
70.0
80.0
I ' ' ' ' I
90.0 100.0
Figure 1 . Mirror-image comparison of the tryptic maps of a production lot of TPA (top) and a TPA reference material (bottom). Adapted with permission of Genentech, Inc.
Sodium dodecylsulfate polyacrylamide gel electrophoresis ( S D S P A G E ) , isoelectric focusing ( I E F ) , HPLC, and enzyme-linked immunosorbent assay (ELISA) tests are some of the more important techniques for determinations of purity and batch-tob a t c h consistency in r e c o m b i n a n t products. Although discussed here as tests of purity, electrophoresis and immunological techniques are also used as tests for identity and structure. S D S - P A G E . "FDA looks for lot-tolot consistency in the electrophoretogram of a new product as a minimum requirement," said Robey. "In fact, lotto-lot consistency is probably more important than purity per se, because the lot-to-lot material is what will actually be tested in human trials. Once a heterogeneous product has been shown to be safe, I have heard people say t h a t they would be afraid to see what would happen if the pure substance were tested. Consistency is the really important thing." S D S - P A G E separates individual proteins from a heterogeneous mixture based on their molecular weight or size. The proteins are separated in an electric field that induces their migration through a microporous acrylamide matrix that retards large proteins more than small ones. Association of the proteins with SDS eliminates the effect that protein charges would have on the separation because SDS coats the peptide chains and imparts a strong negative charge to each one. A disadvantage of the S D S - P A G E technique is that it is extremely labor intensive. "I have an entire shift of people at night who do virtually nothing else but pour, stain, and destain gels,"
said Garnick. "In addition, a lot of judgment and experience is required in interpreting these gels as to whether one is looking at an actual impurity in the product or just an artifact of the technique. However, we have developed the technique to a point where it has become a very reliable procedure." IEF is a mode of electrophoresis in which proteins are induced to migrate through a medium in which a continuous p H gradient has been established. An individual protein ceases migrating when it reaches the point where the p H of the gel equals the protein's isoelectric point (pi), at which the number of positive and negative charges on a protein are equal. T h u s I E F separates on the basis of charge. ELISA procedures are applicable to the detection of impurities t h a t are present at concentrations below the detection limits of either S D S - P A G E or IEF. According to Garnick, they are very reliable, reasonably precise, and quite accurate when properly validated. "ELISA procedures are extremely sensitive," he said, "but they are not generic methods. They have to be developed for each product and process. If t h e manufacturing process is changed during t h e development phase, the ELISA procedure must be re-evaluated. Often we have to go back and repeat the blank run or make sure t h a t the antibodies we have generated still meet the requirements of the assay." Regulatory r e q u i r e m e n t s specify t h a t the concentration of E. coli proteins in a recombinant product be less than 100 ppm. T o test for E. coli proteins at t h a t level with ELISA, one first needs to have samples of the E. coli
proteins (antigens) being sought. Antibodies to those antigens, which will be used as ELISA test reagents, can then be generated by animal immunization. The traditional way to obtain the antigens is to attempt to isolate E. coli proteins from the recombinant product. However, one problem with this technique is that the isolated antigens may then contain traces of t h a t product. The presence of traces of product can induce the generation of additional antibodies in the animal immunization step, and these additional antibodies can then cause false positives in the ELISA test. An example of the ingenuity required in the development of analytical techniques for biotechnology is the procedure developed at Genentech to address this problem. " W h a t we've done," explained Garnick, "is to develop a blank run procedure in which we use a plasmid t h a t contains the promoter region for the recombinant product, such as human growth hormone, but which does not contain the gene for that product. We run a full-scale fermentation after transforming the cells and then isolate the impurities t h a t would contaminate the product. There are a couple of assumptions built into this, such as t h a t the absence of product expression in the blank run has little or no effect on the distribution and expression of host cell protein impurities, but these assumptions are largely experimentally verifiable." After the blank-run procedure, antibodies to the E. coli impurities are developed, as usual, by animal immunization. In t h e double-antibody-sandwich ELISA technique (Figure 2), a 96-well microtiter plate is coated with the E. coli protein antibodies. When the sample is added and the plate incubated, antigens in the sample bind to these antibodies. To be valid, this procedure must be performed in a state of antibody excess at all times. T h e plate is then washed, and a set of antibodies conjugated with horseradish peroxidase (HRP) is added. A substrate to the H R P enzyme (o-phenylenediamine in this case) is added, and the reaction proceeds wherever conjugated antibody is found, which corresponds to locations where antigen had bound with the first set of antibodies. T h e oxidation product of the substrate is monitored at 492 nm, and the concentration is evaluated using a previously prepared standard curve of absorbance vs. antigen concentration. Safety
Recombinant pharmaceuticals must also be tested for components t h a t could affect the safety of a product. One of these is endotoxin, pyrogenic lipopolysaccharides from the cell walls of gram-negative bacteria. "This is a
ANALYTICAL CHEMISTRY, VOL. 59, NO. 15, AUGUST 1, 1987 · 971A
major concern to us," explained Garnick. "We strive to eliminate any detectable trace of endotoxin in all the products we manufacture. Raw materials, in-process products, a n d final products are all monitored for endotoxin." Two procedures used in endotoxin testing are the rabbit test and the limulus amoebocyte lysate (LAL) test. T h e rabbit test is a time-honored U . S . Pharmacopeia procedure in which injection of a sample a t an appropriate level into rabbits generates a fever response (temperature elevation) if endotoxin is present. T h e LAL procedure, a newer test t h a t is more sensitive and more quantitative than the rabbit test, may eventually replace the rabbit pyrogen test for many applications. A typical LAL test involves mixing LAL reagent with sample and monitoring the resulting solution for turbidity in a turbidimetric kinetic analyzer. As the concentration of endotoxin increases, turbidity is produced more quickly. Nonturbidimetric forms of detection can also be used. According to Garnick, "We took a vote in the laboratory when we began a robotics project of which assay every analyst in the lab wanted never to do again, and the endotoxin assay won hands down. Those of you who have done this assay know exactly what I mean. It's time-consuming, laborious, and not very stimulating. So we decided to couple a robot for sample preparation to the turbidimetric kinetic analyzer. We were interested in having the analyst simply place samples in t h e sample block, after which t h e robot would prepare those samples, analyze them, and print out a report. At this date, we're very close to being able to do just that. This project has been going along nicely for six or eight months now, and we hope to present more data on it in the very near future." Potency There are three types of assay for determining the potency or activity of a product. Whole-animal assays are expensive and imprecise, and they require a lot of statistical support. An improvement on the whole-animal assay is the cell culture-derived bioassay, which is less expensive, more precise, and capable of being automated. T h e third type is t h e in vitro biochemical m e t h o d for potency evaluation, in which an attempt is made to mimic the biological functioning of the drug. An example of the in vitro type of assay is a procedure developed at Genentech in which the biological function (blood clot lysis) of T P A is approximated by t h e addition of T P A a n d plasminogen to a synthetic fibrin clot in a modified microcentrifugal analyzer. The assay is performed by spectro-
(a)
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t+**tt É k ÉÉ É (e)
NH2 Antibody/HRP conjugate
H 0
N = N
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NH, Absorbs at 492 nm
Figure 2. Broad-spectrum ELISA assay for low-level impurities. (a) Coat 96-well microtiter plate with process-specific polyclonal antibodies, (b) Add sample containing product (P) and impurities (antigens) of various types, (c) Incubation, followed by removal of product, (d) Coupling of antibody-HRP conjugate, completing double-antibody sandwich, (e) Color development. Adapted with permission of Genentech, Inc.
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New in vitro assays are needed to replace whole-animal methods and other laborious and imprecise techniques for the testing of recombinant products. photometric monitoring of the time required for lysis of the synthetic clot by plasmin produced by TPA's activation of plasminogen. According to Garnick, this assay was demonstrated to be far more accurate, precise, and stabilityindicating than typical in vivo bioassays. Analytical wish list It is hoped t h a t the future of analytical biotechnology will include other in vitro assays capable of replacing wholeanimal methods and other laborious and imprecise techniques for the testing of recombinant products. One of the first items on Frank Robey's wish list, for example, is the development of in vitro assays for the prediction of immunogenicity. "For example," said Robey, "if a product is not exactly identical to a host protein, the body can recognize t h a t and raise antibodies against it. These antibodies can neutralize the activity of a drug, they can cause its removal from the circulation, or they can induce anaphylactic shock resulting in death. Today, the only way we can determine the immunogenicity of a product is by clinical trials in humans. If we had in vitro assays to replace these trials, it would save everybody a lot of money and a lot of time." Robert Garnick favors the development of cloned receptors to proteins for use in cell-based or biochemical in vitro assays and the use of H P L C assays to replace in vivo bioassays for potency determinations. According to Garnick, it is important to remove as many in vivo bioassays as possible from the repertoire because of the cost, imprecision, and laboriousness of these techniques. Both Robey and Garnick would like to see proteins being profiled by tryptic mapping/mass spectrometry (MS) (2) or tryptic mapping/HPLC/MS. At the present time, H P L C alone is generally used to create tryptic maps after protein digestion. Although H P L C provides a fingerprint pattern of the protein, it doesn't provide information (aside from retention time) t h a t helps in the identification of specific peaks in the map. "Using mass spectrometry," said Robey, "the N-terminus and Cterminus could both be positively identified. I predict t h a t in the next 5-10
years LC/MS will be a standard technique in laboratories doing quality control of recombinant products." Biotechnologists would also like to see better C-terminal protein sequencing techniques to complement the Edm a n degradation, which begins its work at the N-terminus. "Caroboxyterminal sequencing is just as important as amino-terminal sequencing to verify the homogeneity of a protein product," said Robey. "Today's methods for carboxy-terminal sequencing are antiquated, generally requiring enzymatic digestion and kinetics to show the disappearance of one peak and the appearance of another peak as the enzyme eats into the C-terminal portion. That's not very helpful." According to Robey, the tryptic m a p p i n g / H P L C / MS technique mentioned above would be one way to effectively satisfy this need. Robey is also looking forward to the a p p e a r a n c e of a u t o m a t e d carbohyd r a t e compositional and sequence analysis. "We have a tremendous fear," he said, " t h a t these glycosylated proteins are going to be a problem. Considering t h a t in a single hexose you have six different positions a t which you can link another hexose, the number of isomers can explode very quickly as you link on each sugar. As a result, many of the recombinantly produced products may not have the carbohydrate composition and sequence of the host protein, and t h a t could be trouble. If the carbohydrate isn't on there as it's supposed to be in the native protein, then you're dealing with a case where you're affecting the composition, the configuration, the activity, the immunogenicity, and other properties." Other items on biotechnologists' wish lists include • more sensitive nuclear magnetic resonance instruments capable of verifying the purity of proteins at pg/mL levels, • increased commercialization of new forms of M S (e.g., 252 Cf plasma desorption) t h a t are capable of handling higher molecular weight species, • improved microanalysis for contaminants, and • improved automation for all of the labor-intensive techniques currently used in molecular biology, such as plasmid preparations, blotting, and sequencing, in addition to endotoxin, protein content, and electrophoretic assays. Stu Borman
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Suggested reading (1) An interesting and informative article on mammalian cell culture appeared recently: Lewis, R. High Technol. J u n e 1987, pp. 30-37. (2) See "Mass Spectrometry Methods for Protein Sequencing," by K. Biemann, Anal. Chem. 1986,58[13], 1288-1300 A.
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 15, AUGUST 1, 1987 · 973 A
