- Email: [email protected]
Purity analysis of protein pharmaceuticals produced by recombinant DNA technology
[ ; [
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DECEMBER 1989 [Vol. 7]
Purity analysis of protein pharmaceuticals produced
have played an important role in the approval process of rt-PA.
by recombinant DNA technology
Impurities (Table 1) are processrelated substances present in raw materials or the drug itself, which are not active material, additives or excipients. Impurities can be either innocuous (they do not pose a health concern) or deleterious. They can also be classified as major (~> 0.5%) or minor (< 0.5%). Deleterious impurities should be controlled at the lowest level possible and, in certain instances, it may be appropriate to perform toxicological, pharmacological, or immunological studies on the impurities themselves 23-25. Contaminants, as distinct from impurities, are adventitious biological or chemical agents which have been accidentally introduced into the product 23-25. Specific methods should be developed for impurities that are endogenous to a process and have a predictable presence in the product 26. The detection of contaminants and impurities of poorly-defined composition, however, must rely on broad spectrum methods. A common example of a major impurity is the undesirable product variant such as deamidated 27 or unintentionally amino-acid-substituted forms 28'29. The detection and elimination of variants is currently an area of intense effort within the industry. Human growth hormone has served as a model for the detection of aggregated, deamidated, oxidized and proteolytically modified variants 27'3°-33. Variants are also expected for rt-PA where there is potential for greater complexity because of the higher molecular mass, variation in the glycosylation pattern and the existence of a one-chain or two-chain configuration 34-36. Minor deleterious impurities such as host cell proteins, DNA or endotoxins pose potential health hazards and therefore must be reduced to part-per-million levels or less (Table 1) 26. Protein impurities can be derived from the host organism, the fermentation medium or from process raw materials (such as mono-
V. R. Anicetti, B. A. Keyt and W. S. Hancock The routine production of protein pharmaceuticals in large amounts has provided new challenges to the analytical chemist. In particular, the determination of protein purity is a complex problem dependent on the structural characteristics and spectrum of potential impurities of the product. The resulting purity determination is only as accurate and complete as the analytical methods used. Through the selection of appropriate methods, an analytical testing strategy to ensure product consistency and purity can be developed. This paper reviews strategies for the purity determination of protein pharmaceuticals using recombinant tissue plasminogen activator as an example. The protein pharmaceuticals produced by recombinant DNA technology and approved by the Food and Drug Administration (FDA) include human insulin 3, human growth hormone 2'3, alpha interferon 4'5, hepatitis vaccine 6, and the recently introduced tissue plasminogen activator (t-PA) 7. The accompanying advancement in analytical protein chemistry has been notable. The approval of any pharmaceutical by the FDA relies upon a convincing demonstration by the manufacturer of the safety and efficacy of the product s-12. The approval criteria will vary with the drug's inherent complexity, its intended use and the method and complexity of manufacture. Ultimately, the safety and efficacy of a pharmaceutical can only be established through appropriately designed human clinical trials 1~'12. However, a comprehensive analytical chemistry program for the characterization and control of the recombinant product often provides an important level of assurance in the
V. R, Anicetti, B. A. Keyt and W.S. Hancock are at Genentech, Inc., 460 Pt. San Bruno Blvd, South San Francisco, CA 94080, USA.
approval process 13-15. Through a close interaction between the regulatory agencies, industry and academia, guidelines for the testing of rDNA (recombinant DNA)-derived pharmaceuticals have been developed and are presented as 'Points to Consider' documents 8'1°'15-18. S o m e of these focus on methods and issues specific to rDNA production, such as plasmid stability or characterization of the clone containing the expression vector 15-18. Others have been directed towards the detection of alterations in the molecular structure of proteins 8. When compared with small-molecule drugs, a large, glycosylated protein with many disulfide bonds, such as recombinant tissue plasminogen activator (rt-PA), is extremely complex, particularly in the variety of chemical and conformational changes which may occur either during manufacture or storage. Furthermore, quantifying these changes can be especially difficult given the natural heterogeneity of many glycoproteins 8'19'2°. Fortunately, the routine production of multi-gram amounts of highly purified proteins has facilitated the development of improved preparative and analytical methods for assessing purity ~4'21-z~. The advancements in analytical biotechnology
@ 1989, Elsevier Science Publishers Ltd (UK) 0 1 6 7 9430/89/$2.00
Types of impurities in protein pharmaceuticals
TIBTECH- DECEMBER 1989 [Vol. 7]
--Table 1 Typical impurities in protein pharmaceuticals, their detection and target levels Impurity
Category
Detection m e t h o d s
Target levelsa
Endotoxin
Undefined
Rabbit pyrogen, LAL
Host cell and media proteins Monoclonal antibodies and defined production proteins DNA
Undefined Defined
SDS-PAGE, immunoassay SDS-PAGE, immunoassay
Dependent on dose, duration and route of administration 54 Parts-per-million 12,13 Parts-per-million 26,38,48
Undefined
Infectious agents
Undefined
Hybridization assays, antibody or binding protein sandwich assays Reverse transcriptase assay, cell culture cytopathic effects (CPE), electron microscopy
Product variants Deamidation products Oxidation products Amino acid substitutions
Deft ned Isoelectric focusing HPLC HPLC-tryptic mapping
< 10-100 pg per dose 2°'5° Eliminated or inactivated 1°'15'18'37
Dependent on the product complexity b
GPC SDS-PAGE, HPLC Monoclonal antibody-based immunoassays
Aggregated forms Proteolytic products Highly conserved or homologous host cell species proteins
~The values presented represent the range of established levels or values which have been published for particular rDNA pharmaceuticals. The references discuss these levels and related issues. bThe target levels for these variants must be examined on a case by case basis for each product. Generally variants greater than 0.5% should be characterized 11"14'25.
clonal antibodies used in affinity chromatography). Finally, infectious agents which may be pathogenic in man could, potentially, be present. These may be contaminants of the bioreactor system or be latent within the production cell. Microorganisms must be eliminated through a combination of process control steps, process validation and rigorous product testing 15,1~'37.
The relationship between purity and safety The potential consequences of impurities can be severe. They may have immunological or biological effects either directly on a drug recipient, or indirectly by affecting the quality or stability of the drug itself. Oncogenic DNA, endotoxin, infectious agents or materials which can function as immunological adjuvants all exert direct biological effects on the drug recipient. Product variants or non-human proteins, such as mouse monoclonal antibodies or a host-cell-derived version of the product, are impurities which might stimulate an immune response. The purity requirements for protein pharmaceuticals are an area of continual review and, on occasion, revision. This is because purity can only be defined by the resolution and sensitivity of available analytical methods. Also, purity requirements
cannot be universally established for all protein drugs, but need to be developed on a case-by-case basis 8,1°,38. The dose, route and frequency of administration, the nature of the impurities and the nature of the disease state are among the factors determining the risk-tobenefit ratio of the drug and, therefore, its purity requirements 8'12. General strategies for analytical methods In addressing analytical approaches, it makes sense to group impurities as either undefined or defined (Table 1). Defined impurities can be assayed specifically using electrophoretic, chromatographic, or monoclonalbased immunochemical methods.
Undefined impurities are unknown or exceedingly heterogeneous populations of complex materials which cannot be assayed specifically. Recombinant tissue plasminogen activator (rt-PA), a protein with complex physicochemical properties, shows h o w analytical chemistry can be used in the purity analysis of rDNA proteins. Tissue plasminogen activator is a serine protease with potent fibrinolytic activity. It is a single polypeptide chain of approximately 64 kDa containing 35 cysteine residues. The recombinant molecule is produced by large-scale cell culture of Chinese Hamster Ovary (CHO) cells which allows both the glycosylation and secretion of the protein.
--Fig. 1 SH
CONH 2
CONH 2
I
117
Ls sJ
184
F S
s-ll 276
448 t
527
CONH 2
The essential structural features of recombinant-DNA-derived tissue plasminogen activator. The free sulfhydryl, disulfide and amide groups are illustrative (the molecule contains multiple disulfide and amide groups). *, site of attachment of carbohydrates.
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--Table 2 Molecular variants
masses
1-Chain Type I Type II
~Fig, 2
4-
pl 5.1
6.0
7.0
9.6
The isoelectric focusing of rt-PA with visualization by silver staining. + and - , in the presence or absence of neuraminidase, The lowered heterogeneity after treatment with neuraminidase indicates that most of the charge variability can be attributed to the presence of sialic acid residues.
Defined imp urities intrinsic to the product Impurities derived from the expected product are defined in this review as intrinsic to the product. Figure 1 shows the potential sites of molecular heterogeneity of rt-PA. Even a highly purified preparation will contain many variants either because of the inherent heterogeneity of the molecule (e.g. the carbohydrate side chains) or because of degradative reactions such as deamidation. To demonstrate consistency in manufacture, it is necessary to be able to monitor the level of different variants. Two 'natural' variants present in rt-PA provide excellent
Reduced, 2-chain A-chain Type I Type II B-chain
of
rt-PA
64-68 kDa 62-64 kDa
34-36 kDa 32-32.5 kDa 30-32 kDa
markers for this purpose; the Type I and Type II glycosylated forms and the one- and two-chain forms. The molecule has glycosylation sites at positions 117, 184 and 448, but the 184 site is not always glycosylated. Thus, some molecules contain three carbohydrate groups (Type I), and some, two groups (Type II). In addition, rt-PA can exist in a two-chain form as the result of proteolytic cleavage between residues 275 and 27623'34-36. Therefore, a preparation ofrt-PA will contain four populations (Type I, 1 chain; Type I, 2 chain; Type II, 1 chain; Type II, 2 chain), the relative proportions of which will be characteristic of the culture conditions and the recovery process. Other processes such as deamidation and proteolysis will introduce additional heterogeneity into each of these populations. Since all of these variants contain the essential features of the rt-PA molecule, polyclonal, antibody-based assays and biological assays cannot discriminate between these forms. Electrophoretic or chromatographic methods are required. With the recent determination of the carbohydrate structures of rt-PA 39, the molecular masses of the four variants can be estimated (Table 2). The variability in glycosylation is responsible not only for the range of molecular masses, but also for charge heterogeneity (due to terminal sialic acid residues present on many of the carbohydrate chains), which can be revealed by isoelectric focusing (IEF) gel electrophoresis (Fig. 2). The formation of the 2-chain variant has no significant effect on the molecular mass of the unreduced form as the A and B chain fragments are held together by a single disulfide bond. However, reduction releases two smaller fragments (Table 2). The 2chain form also could add to the charge heterogeneity. With this level of complexity, part of the analytical strategy must be to
select methods that are specific for only a particular property of the molecule. Gel permeation chromatography (GPC), for instance, is a relatively low resolution analytical technique that is insensitive to carbohydrate microheterogeneity and thus allows a facile and accurate estimation of the amount of the 2-chain form in a given rt-PA preparation (Fig. 3). Similarly, reversed phase HPLC (RP-HPLC) of tryptic fragments has become widely accepted as the best analytical technique for monitoring the primary sequence of a protein. The technique is primarily sensitive to differences in hydrophobicity. By using tryptic fragments rather than the intact protein as the analyte, sequence differences both on the surface and in the interior of the protein can be --Fig. 3 1-chain 73.8%
2-chain
7.0
8.0 9.0 Elution time (minutes)
10.0
The separation of a sample of 1chain and 2-chain rt-PA by gel permeation chromatography. The sample has been reduced so that the A- and B-chain fragments run as a single peak (labeled 2-chain) significantly slower than the 1chain species.
TIBTECH - DECEMBER 1989 [Vol. 7] --Fig.
4
A28
b'~ I--" o4 r,,.. b-
o 0
co
VI_.Fo9 t.-
A~4
~ g
I
~-v-
I--
I
CO
LO
}-~+
o4 I--
I--
~
if)
o3
I--
t.to
0 CO
%
II
04
I
~
I~. I--
I
i
I
0
10
20
30
I
I
40
5O
I
60 Elution time (minutes)
I
I
I
70
80
90
I
100
The primary sequence signature of rt-PA as shown by reverse-phase HPLC of tryptic fragments. The tryptic fragments are numbered from the N-terminus. Glycopeptides are highlighted in black.
revealed. Trypsin cleaves at frequent intervals producing peptides of 2-20 amino acid residues in length. Therefore, any amino acid substitution or cleavage of the polypeptide backbone would be expected to generate a shift in retention time. The tryptic map for rt-PA includes over 50 peptides that are separated in a single chromatographic analysis (Fig. 4). Carbohydrate heterogeneity results in poorly resolved multiplets. Removal of the carbohydrate with a glycosidase results in collapse of the multiplet and shift of the deglycosylated p e p t i d e to a longer retention time (Table 3). Because of the relative insensitivity of RP-HPLC to the presence of carbohydrate side-chains, it can be used to monitor the primary structure of rt-PA. The carbohydrates liberated by glyc0sidase digestion can be analysed in the absence of the peptide mixture by high pH anion exchange chromatography 39'4°. At present, quality control procedures focus on the amino acid sequence of a protein rather than the carbohydrate portion. This is partly because the methods available for monitoring protein sequence are excellent, while those for carbohydrate analysis have been much
more time consuming and less effective 14. Other barriers to routine carbohydrate analysis are the high site-dependent microheterogeneity of many native glycoproteins 19'4~ and the potential for greater structural complexity relative to proteins. Such an analysis, although necessarily complex, will become important because carbohydrate can serve as an effector of glycoprotein clearance 42. Furthermore, glycosylation will vary with host cell type, the transformation state, and establishment in cell culture 18 and carbohydrate mapping could be an indicator of the competence of the cell culture system. Although the tryptic map is highly reproducible and suitable for lot release purposes, the complexity of the map limits its usefulness in monitoring protein degradation reactions such as proteolysis and deamidation. IEF, however, can monitor charge variants of rt-PA and can thus be used to examine many degradative reactions. SDS-PAGE with silver stain, after reduction and S-carboxymethylation of rt-PA, can be used to develop a catalog of patterns of proteolytically generated forms. Silver stain does not yield a quantitative analysis and, therefore,
must be compared with reference material. The production, characterization and maintenance of a reference material are critical quality control program requirements and should be included on all final product tests 14'38. Other important degradation forms result from aggregation. Neither SDS-PAGE or reverse phase HPLC are suitable for the detection of aggregated rt-PA, since aggregates may irreversibly bind to the HPLC column or fail to penetrate the polyacrylamide gel or be dissociated by the analytical conditions. Size exclusion chromatography is better suited to this analysis. Defined imp urities extrinsic to th e product Defined impurities extrinsic to the product include components which are added to the culture medium or proteins which are used as raw materials in the purification process. Insulin, transferrin, antibiotics, and neoplastic agents are among the impurities that could arise from the fermentation medium. They may have significant biological or immunogenic activity and must be removed to part-per-million levels sJ°'2~. Demonstration of such purity levels
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requires a combination of highly sensitive assays and process validation studies 38. Clearance of each impurity can be determined by assay at each step in the process 21. The clearance of an impurity which may be present in very low levels is also calculable by adding known amounts of the impurity to the product pool before each process step and subsequent assay after each step. This approach has been used to validate the removal of media and host cell components 21 and virus 37. Purification processes which employ affinity chromatography are more common 43 and may introduce antibody-derived impurities 38'44. Antibodies and antibody fragments may possess significant immunogenic potential if they are non-human in origin and if the therapeutic dose is large enough 45-47. Immune complexes may be carried through the process making the analysis difficult. Also, the antibody preparation itself may contain significant impurities 26'38'48. Fetal calf serum, bovine immunoglobulins and Protein A or Protein G are some impurities associated with murine monoclonal antibodies. Their leakage rate from an immuno-affinity column and their clearance by the purification process in the presence of the product must be demonstrated as part of the validation effort 44. The specificity and sensitivity necessary to detect antibodies at ppm concentrations in the presence of their respective protein antigen (i.e. the product) is best provided by immunoassays 4a. Undefined impurities
Knowing the precision, specificity and accuracy of the analytical method is of particular concern in the detection and quantitation of undefined impurities. Unlike a processderived impurity in a well-developed purification scheme, undefined impurities may vary in both quantity or composition from one production run to another. Broad spectrum assays which detect a general biological effect or an assay signal from any of a family of components are generally used. However, reference methods are rare (given the lack of specific analytes) and the methods used can only be validated by
rigorous control of assay reagents and careful design of the assay. In view of these difficulties, it is clear that flesh approaches are needed if one is to have assurance that a new drug is not at risk from contamination with undefined impurities. • Microbial c o n t a m i n a n t s . Microorganisms such as bacteria, mycoplasmas or viruses are important possible contaminants of pharmaceuticals isolated from biological materials. The presence of the AIDS virus in blood and blood products and the probable presence of the agent causing Creutzfeld Jakob disease in early preparations of pituitaryderived human growth hormone 49 demonstrate an advantage of rDNA technology, where the host cell, medium and isolation materials can be rigorously characterized and controlled ~4. Again, the development of rt-PA shows how undefined impurities can be controlled. While evidence of the presence of virus of potential pathogenicity has been found in primary cell culture or tissues, endogenous virus has not been isolated from any of the human diploid cells used for vaccine production 5° nor from CHO cells 37, the line used to produce rt-PA. Taking cells from a wellcharacterized cell bank allows them to be examined carefully for the presence of virus or virus-like bodies in the cells and the evaluation of a potential risk for humans 5°. An extensive cell characterization program contributed to the approval of CHO cells as a drug substrate 37'5°. Assays for a wide range of animal viruses were performed on the cell substrate medium 37. This cell substrate was also tested extensively for reverse transcriptase activity and mice were inoculated to identify endogenous murine viruses 37. Each of over 200 lots of harvested cell culture fluid has been assayed for adventitious virus by testing for cytopathic effects on three different cell lines and none has been found positive 37. Through this program of carefully selected, characterized and maintained host cell banks, controlled production systems with inactivation steps and product testing, the possibility of infectious
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DECEMBER 1989 [Vol. 7]
agents as impurities or contaminants is essentially eliminated. • Host D N A . Host cell DNA is another undefined impurity which must be measured in every lot of drug produced TM. The recommended limit for DNA impurities is 10 pg per dose 2°,5° and the purification process must contain steps to eliminate DNA 51. The accepted assays are dot blots using specific 32p-labeled DNA probes obtained from the DNA of the host ce1152; n e w assays have recently been introduced 53. Current manufacturing procedures can achieve very low levels of DNA contamination, often below the threshold for detection with current assays. • Pyrogens. Endotoxin and other pyrogenic materials are among the other poorly defined impurities of major concern 54'55. A combination of the USP rabbit pyrogen assay and the Limulus Amoebocyte Lysate (LAL) assay is considered the most conservative and appropriate approach for the testing of recombinant biologics 14. However, the rabbit pyrogen assay is highly variable and may result in false positives due to infection, antigenicity or handling practices 14,26. Some of the recombinant cytokines, such as tumor necrosis factor, are themselves pyrogens and will be cross-reactive in the whole animal model 14'26. The LAL method may not detect endotoxin after lyophilization and is subject to a number of interferences 14'56. Therefore, both tests are often required. Where conflicting results or suspected interference occur, a third method which relies on a different biological reaction can be useful. The pyrogen assay developed by Dinarello 56 relies on the production of interleukin I (IL-1) by tissue cultured monocytes in response to endotoxin. The IL-1 is then measured specifically by immunoassay. • H o s t proteins. The detection of host cell proteins requires both a ppm sensitivity and the detection of large numbers of proteins; this has been achieved through the development of process-specific immunoassays 57. The potential biological or immunological consequences of these impurities have
TIBTECH - DECEMBER 1989 [Vol. 7]
~Table 3
Retention time shifts on deglycosylation. Since residue 184 is not always glycosylated there are two peaks in the original tryptic map (peptide T17 with and without the carbohydrate side chain). Only one of them shifts its retention time after reaction with glycosidase.
RCM rt-PA peptide and carbohydrate type a T11-(High mannose) T17-(Complex) T17-(Nonglycosylated) T45-(Complex)
Residue no. (Asn) 117 184 184 448
Glycopeptidase F t r e a t m e n t (Asp) (-) 42.2 b 40.5 44.9 25.0
(+) 46.0 45.4 44.9 30.5
aRCM, reduced and s-carboxymethylated. bRetention time measured in minutes in a TFA mobile phase system 65.
demanded this extreme level of performance 3'58. The sensitivity and selectivity has been markedly improved by the development of Antigen Selected Immunoassays (ASIA) where a population of antibodies raised against process-specific host cell proteins are further selected by affinity chromatography before use in an ELISA method in which the host cell contaminants are quantified as a group 13'59. Technical problems occur in developing these assays involving the selection of the host cell protein reference material, the method for production of antibodies and the validation of the assay. The reference proteins could be isolated from in-process material by immunoabsorbing the product away from the other proteins. The immunoabsorption procedures, however, are very difficult to perform and validate: all of the product and product fragments must be removed without losing any of the impurities. Any residual product wilt produce antibodies during the immunization and render the assay nonspecific. An alternative way of producing the reference material is the 'blank run' approach; a fermentation and partial purification of the host are conducted using the host cell lacking the product gene 13'59. Two key assumptions are made. The first is that the lack of product expression does not significantly affect the population of in-process impurities. The second is that the lack of product does not significantly affect the chromatography of the impurities: for E. coil proteins purified by the growth hormone process, the presence and absence of a small amount of growth hormone did not
create any significant changes in the distribution of contaminants 6°. Similar studies could be performed for each product. Performing the blank run at manufacturing scale will minimize differences in both the growth conditions and chromatography from the actual r u n 13'21'59. Typical reference impurity mixtures can contain 100 or more components as shown by a silverstained 2D gel 60. The usefulness of the antisera produced against such mixtures is limited by the amount of antibody produced to the least immunogenic component. The use of multiple animals, extended immunization schedules and rigorous antisera characterization are necessary to ensure that antibodies have been produced to all of the minor components in the mixture 61. Finally, the design and development of these assays present a number of problems: the development lead times are very long, a rigid format must be used 62, and the only approach to validation of the assays is to demonstrate internal consistency of the method and the quality of the reagents 59'61. Furthermore, because each assay is process specific, purity values cannot be compared between manufacturers unless the processes are identical. Given that at least 1500 proteins are produced by E. COIi63, it is doubtful that a sufficiently sensitive generic assay could ever be developed 5u.
advances in methodology further refine the definition of purity, the regulatory guidelines must be flexible enough to anticipate the observation of previously undetected impurities 38. In the production of hGH, approximately 750 separate tests are performed during the manufacturing and release processes 64, a result of the greater sophistication and complexity of recombinant manufacturing processes. These tests are most effective if their respective strengths and weaknesses are complementary. For instance, immunoassays provide very specific and sensitive detection of host cell protein impurities but will only detect proteins which elicit an antibody response. Therefore, a sensitive, non-antibody based method such as silver-stained SDSPAGE should also be used. Silver stain SDS-PAGE, however, is highly variable and impurities might be concealed due to co-migration of different proteins. Thus both methods should be used with a rigorous process validation program 15'21'38. The value of complementary methods is also apparent in the detection of rt-PA product variants (Figs 2-4). A combination of complementary analytical methods and process validation provide the best assurance that impurities are minimal in the final product. The safety of protein pharmaceuticals can only be determined through appropriate preclinical and clinical testing. The approval to enter a clinical .testing program and the confidence that a safe product can be consistently manufactured relies on a comprehensive analytical chemistry program.
Acknowledgements The authors would like to acknowledge the following experimenters working on rt-PA: IEF (Robert Bridenbaugh, Mary Sliwkowski, Andrew Guzzetta). GPC (Charles DuMee). tryptic mapping (Rosanne Chloupek).
An integrated approach to purity analysis It is clear from the variety of methods discussed here and the spectrum of possible impurities that exact protocols for purity analysis cannot be universally applied. As
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Biol. Chem. 256, 7035-7041 36 Pennica, D., Holmes, W.E., Kohr, W.J. et al. (1983) Nature 301, 214-221 37 Wiebe, M. E., Becker, F., Lazar, R. et a]. in Advances in Animal Cell Biology and Technology for Bioprocesses (Spier, R. E., Griffiths, J. B., Stephenne, J. and Crooy, P. J., eds), pp. 68-71, Butterworth 38 Committee for Proprietary Medicinal Products A d Hoc Working Party on Biotechnology/Pharmacy (1987) Trends Biotechnol. 5, G1-G4 39 Spellman, M. W., Basa, L. J., Leonard, C.K. et ai.(1989) J. Biol. Chem. 264, 14100-14111 40 Hardy, M. R. and Townsend, R. R. (1988) Proc. Nat1 Acad. Sci. USA 85, 3289-3293 41 Montgomery, R. (1972) in Glycoproteins: Their Composition, Structure and Function. Part A (Gottschalk, A., ed.), pp. 518-528, Elsevier 42 Hotchkiss, A., Refino, C. J., Leonard, C.K. et al. (1988) Throm. Haemostasis 60, 255-261 43 Moks, T., Abrahm~en, L., ()terltf, B. et al. (1987) Bio/Technology 5, 379-382 44 WHO Consultation (1983) Bull. WHO 61,987-911 45 Sears, H. F., Herlyn, D., Steplewski, Z. and Koprowski, H. (1984) J. Biol. Response Mod. 3, 138-150 46 Shawler, D. L., Bartholomew, R. M., Smith, L.M. and Dillman, R.O. (1985) J. Immunol. 135, 1530-1535 47 Ehrlich, P. H., Harfeldt, K. E., Justice, J. C., Moustafa, Z. A. and Ostberg, L. (1987) Hybridoma 6, 151-160 48 Lucas, C., Nelson, C., Peterson, M. L. et al. (1988) J. Immunol. Methods 113, 113-122 49 Lazarus, L. (1985) Med. ]. Aust. 143, 57-59 50 Petricciani, J. C. (1988) in Biotechnologically Derived Medical Agents. The Scientific Basis of Their Regulation (Gueriguian, J. L., Fattorusso, V. and Poggiolini, D., eds), pp. 106-121, Raven Press 51 Petricciani, J. C. (1985) in Standardization and Control of Biologicals Produced by Recombinant DATA Technology; Developments in Biological Standardization Vol. 59, pp. 149-153, S. Karger 52 Kafatos, F. C., Jones, C. W. and Efstratiadis, A. (1979) Nucleic Acid Res. 7, 1541-1552 53 Briggs, J., Kung, V. T., Pomtis, G. et al. (1989) Amer. Biotechnoi. Laboratory 7, 34-38 54 Guideline on the Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for
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55 56 57 58
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Human and Animal Parenteral Drugs, Biological Products, and Medical Devices (1987) Center for Drug Evaluation and Research, FDA Weary, M. and Pearson, F. (1988) Biochem. Pharmacol. 1, 22-29 Dinarello, C. A., O'Connor, J. V., LoPreste, G. and Swift, R. L. (1984) J. Clin. Microb. 20, 323-329 Baker, R. S., Schmidtke, J. R., Ross, J. W. and Smith, W. C. (1981) Lancet ii, 1139-1192 Frykland, L., Brandt, J., Hagerman, M. et al. (1986) in Human Growth []
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59 60
61
62
Hormone (Raiti, S. and Tolman, R. A., eds), pp. 257-266, Plenum Anicetti, V. R., Fehskens, E. F., Reed, B.R. et al. (1986) 1. Immunol. Methods 91, 213-224 Anicetti, V. R. (1988) in Abstracts of Papers 196th ACS National Meeting, Div. of Anal. Chem., No. 63, American Chemical Society Anicetti, V. R., Simonetti, M.A., Blackwood, L. L., Jones, A. J. S. and Chen, A.B. (1989) Appl. Biochem. Biotech. 22, 1-10 Jones, A. J. S. (1988) in Abstracts of
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Thermophilic organisms as sources of thermostable enzymes Jakob K. Kristjansson Thermostable enzymes are receiving considerable attention and there are many industrial applications already. Most thermostable enzymes on the market have, however, been derived from mesophiles. Further research, especially into the possibilities of cloning enzymes from thermophiles into mesophilic hosts, will greatly increase the exploitation of thermophiles in biotechnology. Temperature is probably the most important environmental factor affecting the activity and evolution of living organisms. Most organisms that have been studied in depth are adapted to 'moderate' temperatures and operate within a relatively narrow span of temperature (generally less than 30°C). In these organisms, the upper temperature at which life is possible (Tmax) is usually reached quite abruptly and is only a few degrees above the optimum growth temperature (Topt). In recent years, there has been increasing interest in microorganisms which are adapted
lakob K. Kristjansson is at the Department of Biotechnology, Technological Institute of Iceland, Keldnaholt, IS-112 Reykjavik and the Institute of Biology, University of Iceland, Grensasvegur 12, IS-108 Reykjavik, Iceland.
to environments of extreme temperature, such as hot springs 1,2.
What is a thermophile? Organisms have been classified according to growth temperature Topt and/or Tmax1'3-6. These classifications will not be discussed here beyond saying that the term 'thermophile' is not very precise and its meaning depends on the group of organisms being considered. The upper temperature limit for growth of different groups of organisms varies widely, from 38°C for fish, 55-60°C for the eukaryotic algae and fungi, to 85°C for eubacteria and up to 110°C for certain archaebacteria 1,6'7. Here I will use the term 'thermophile' ('thermophilic') to describe those bacteria with Topt of 65-85°C and 'extreme thermophile' to describe those with Topt >85°C. Organisms with Topt <60°C ('slight thermophiles') will not be discussed.
© 1989, Elsevier Science Publishers Ltd (UK) 0167 - 9430/89/$2.00
Papers 196th ACS National Meeting, Div. of Anal. Chem., No. 62, American Chemical Society 63 O'Farre]], P. H. (1975) J. Biol. Chem. 350, 40074021 64 Ross, M. J. (1988) in Biotechnologically Derived Medical Agents. The Scientific Basis of Their Regulation (Gueriguian, J. L., Fottorusso, V. and Poggiolini, D., eds), pp. 168-174, Raven Press 65 Chloupek, R. C., Harris, R. J., Leonard, C.K. et al. (1989) J. Chrom. 463, 375-396 []
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Finding thermophiles Organisms which grow optimally at 45-55°C are ubiquitous, but those with an optimum temperature above 60°C are, in general, associated with permanently hot places such as areas of geothermal activity (although they can usually be isolated from many non-geothermal areas). Thermophilic sporeforming organisms are also abundant in natural environments only heated for a short time, such as composts and solar-heated soils and ponds 1. Many thermophiles, including Thermus spp. and spore-forming organisms, have colonized manmade hot places, such as domestic and industrial pipes 8. Before 1970, several thermophiles had been isolated which could grow up to 70°C (see Table 3.3 in Ref. 1) (e.g. Bacillus stearothermophi]us strains with Topt of 50-60°C). Much research has been performed on strains of B. stearotbermophilus including the isolation and study of thermostable enzymes 4. Thomas Brock's isolation of Thermus aquaticus with its Topt of 70-75°C, however, was a major discovery 9. During his work in Yellowstone National Park from 1968-1978, Brock also isolated Su]folobus acidoca]darius and Thermoplasma acidophilum, the first representatives of the thermoacidophilic archaebacteria. Brock and his colleagues also isolated enzymes from T. aquaticus and showed them to be more thermostable than any other previously known enzymes 4"1°. tn 1981, Stetter and Zillig isolated
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DECEMBER 1989 [Vol. 7]
Purity analysis of protein pharmaceuticals produced
have played an important role in the approval process of rt-PA.
by recombinant DNA technology
Impurities (Table 1) are processrelated substances present in raw materials or the drug itself, which are not active material, additives or excipients. Impurities can be either innocuous (they do not pose a health concern) or deleterious. They can also be classified as major (~> 0.5%) or minor (< 0.5%). Deleterious impurities should be controlled at the lowest level possible and, in certain instances, it may be appropriate to perform toxicological, pharmacological, or immunological studies on the impurities themselves 23-25. Contaminants, as distinct from impurities, are adventitious biological or chemical agents which have been accidentally introduced into the product 23-25. Specific methods should be developed for impurities that are endogenous to a process and have a predictable presence in the product 26. The detection of contaminants and impurities of poorly-defined composition, however, must rely on broad spectrum methods. A common example of a major impurity is the undesirable product variant such as deamidated 27 or unintentionally amino-acid-substituted forms 28'29. The detection and elimination of variants is currently an area of intense effort within the industry. Human growth hormone has served as a model for the detection of aggregated, deamidated, oxidized and proteolytically modified variants 27'3°-33. Variants are also expected for rt-PA where there is potential for greater complexity because of the higher molecular mass, variation in the glycosylation pattern and the existence of a one-chain or two-chain configuration 34-36. Minor deleterious impurities such as host cell proteins, DNA or endotoxins pose potential health hazards and therefore must be reduced to part-per-million levels or less (Table 1) 26. Protein impurities can be derived from the host organism, the fermentation medium or from process raw materials (such as mono-
V. R. Anicetti, B. A. Keyt and W. S. Hancock The routine production of protein pharmaceuticals in large amounts has provided new challenges to the analytical chemist. In particular, the determination of protein purity is a complex problem dependent on the structural characteristics and spectrum of potential impurities of the product. The resulting purity determination is only as accurate and complete as the analytical methods used. Through the selection of appropriate methods, an analytical testing strategy to ensure product consistency and purity can be developed. This paper reviews strategies for the purity determination of protein pharmaceuticals using recombinant tissue plasminogen activator as an example. The protein pharmaceuticals produced by recombinant DNA technology and approved by the Food and Drug Administration (FDA) include human insulin 3, human growth hormone 2'3, alpha interferon 4'5, hepatitis vaccine 6, and the recently introduced tissue plasminogen activator (t-PA) 7. The accompanying advancement in analytical protein chemistry has been notable. The approval of any pharmaceutical by the FDA relies upon a convincing demonstration by the manufacturer of the safety and efficacy of the product s-12. The approval criteria will vary with the drug's inherent complexity, its intended use and the method and complexity of manufacture. Ultimately, the safety and efficacy of a pharmaceutical can only be established through appropriately designed human clinical trials 1~'12. However, a comprehensive analytical chemistry program for the characterization and control of the recombinant product often provides an important level of assurance in the
V. R, Anicetti, B. A. Keyt and W.S. Hancock are at Genentech, Inc., 460 Pt. San Bruno Blvd, South San Francisco, CA 94080, USA.
approval process 13-15. Through a close interaction between the regulatory agencies, industry and academia, guidelines for the testing of rDNA (recombinant DNA)-derived pharmaceuticals have been developed and are presented as 'Points to Consider' documents 8'1°'15-18. S o m e of these focus on methods and issues specific to rDNA production, such as plasmid stability or characterization of the clone containing the expression vector 15-18. Others have been directed towards the detection of alterations in the molecular structure of proteins 8. When compared with small-molecule drugs, a large, glycosylated protein with many disulfide bonds, such as recombinant tissue plasminogen activator (rt-PA), is extremely complex, particularly in the variety of chemical and conformational changes which may occur either during manufacture or storage. Furthermore, quantifying these changes can be especially difficult given the natural heterogeneity of many glycoproteins 8'19'2°. Fortunately, the routine production of multi-gram amounts of highly purified proteins has facilitated the development of improved preparative and analytical methods for assessing purity ~4'21-z~. The advancements in analytical biotechnology
@ 1989, Elsevier Science Publishers Ltd (UK) 0 1 6 7 9430/89/$2.00
Types of impurities in protein pharmaceuticals
TIBTECH- DECEMBER 1989 [Vol. 7]
--Table 1 Typical impurities in protein pharmaceuticals, their detection and target levels Impurity
Category
Detection m e t h o d s
Target levelsa
Endotoxin
Undefined
Rabbit pyrogen, LAL
Host cell and media proteins Monoclonal antibodies and defined production proteins DNA
Undefined Defined
SDS-PAGE, immunoassay SDS-PAGE, immunoassay
Dependent on dose, duration and route of administration 54 Parts-per-million 12,13 Parts-per-million 26,38,48
Undefined
Infectious agents
Undefined
Hybridization assays, antibody or binding protein sandwich assays Reverse transcriptase assay, cell culture cytopathic effects (CPE), electron microscopy
Product variants Deamidation products Oxidation products Amino acid substitutions
Deft ned Isoelectric focusing HPLC HPLC-tryptic mapping
< 10-100 pg per dose 2°'5° Eliminated or inactivated 1°'15'18'37
Dependent on the product complexity b
GPC SDS-PAGE, HPLC Monoclonal antibody-based immunoassays
Aggregated forms Proteolytic products Highly conserved or homologous host cell species proteins
~The values presented represent the range of established levels or values which have been published for particular rDNA pharmaceuticals. The references discuss these levels and related issues. bThe target levels for these variants must be examined on a case by case basis for each product. Generally variants greater than 0.5% should be characterized 11"14'25.
clonal antibodies used in affinity chromatography). Finally, infectious agents which may be pathogenic in man could, potentially, be present. These may be contaminants of the bioreactor system or be latent within the production cell. Microorganisms must be eliminated through a combination of process control steps, process validation and rigorous product testing 15,1~'37.
The relationship between purity and safety The potential consequences of impurities can be severe. They may have immunological or biological effects either directly on a drug recipient, or indirectly by affecting the quality or stability of the drug itself. Oncogenic DNA, endotoxin, infectious agents or materials which can function as immunological adjuvants all exert direct biological effects on the drug recipient. Product variants or non-human proteins, such as mouse monoclonal antibodies or a host-cell-derived version of the product, are impurities which might stimulate an immune response. The purity requirements for protein pharmaceuticals are an area of continual review and, on occasion, revision. This is because purity can only be defined by the resolution and sensitivity of available analytical methods. Also, purity requirements
cannot be universally established for all protein drugs, but need to be developed on a case-by-case basis 8,1°,38. The dose, route and frequency of administration, the nature of the impurities and the nature of the disease state are among the factors determining the risk-tobenefit ratio of the drug and, therefore, its purity requirements 8'12. General strategies for analytical methods In addressing analytical approaches, it makes sense to group impurities as either undefined or defined (Table 1). Defined impurities can be assayed specifically using electrophoretic, chromatographic, or monoclonalbased immunochemical methods.
Undefined impurities are unknown or exceedingly heterogeneous populations of complex materials which cannot be assayed specifically. Recombinant tissue plasminogen activator (rt-PA), a protein with complex physicochemical properties, shows h o w analytical chemistry can be used in the purity analysis of rDNA proteins. Tissue plasminogen activator is a serine protease with potent fibrinolytic activity. It is a single polypeptide chain of approximately 64 kDa containing 35 cysteine residues. The recombinant molecule is produced by large-scale cell culture of Chinese Hamster Ovary (CHO) cells which allows both the glycosylation and secretion of the protein.
--Fig. 1 SH
CONH 2
CONH 2
I
117
Ls sJ
184
F S
s-ll 276
448 t
527
CONH 2
The essential structural features of recombinant-DNA-derived tissue plasminogen activator. The free sulfhydryl, disulfide and amide groups are illustrative (the molecule contains multiple disulfide and amide groups). *, site of attachment of carbohydrates.
TIBTECH - DECEMBER 1989 [Vol. 7]
--Table 2 Molecular variants
masses
1-Chain Type I Type II
~Fig, 2
4-
pl 5.1
6.0
7.0
9.6
The isoelectric focusing of rt-PA with visualization by silver staining. + and - , in the presence or absence of neuraminidase, The lowered heterogeneity after treatment with neuraminidase indicates that most of the charge variability can be attributed to the presence of sialic acid residues.
Defined imp urities intrinsic to the product Impurities derived from the expected product are defined in this review as intrinsic to the product. Figure 1 shows the potential sites of molecular heterogeneity of rt-PA. Even a highly purified preparation will contain many variants either because of the inherent heterogeneity of the molecule (e.g. the carbohydrate side chains) or because of degradative reactions such as deamidation. To demonstrate consistency in manufacture, it is necessary to be able to monitor the level of different variants. Two 'natural' variants present in rt-PA provide excellent
Reduced, 2-chain A-chain Type I Type II B-chain
of
rt-PA
64-68 kDa 62-64 kDa
34-36 kDa 32-32.5 kDa 30-32 kDa
markers for this purpose; the Type I and Type II glycosylated forms and the one- and two-chain forms. The molecule has glycosylation sites at positions 117, 184 and 448, but the 184 site is not always glycosylated. Thus, some molecules contain three carbohydrate groups (Type I), and some, two groups (Type II). In addition, rt-PA can exist in a two-chain form as the result of proteolytic cleavage between residues 275 and 27623'34-36. Therefore, a preparation ofrt-PA will contain four populations (Type I, 1 chain; Type I, 2 chain; Type II, 1 chain; Type II, 2 chain), the relative proportions of which will be characteristic of the culture conditions and the recovery process. Other processes such as deamidation and proteolysis will introduce additional heterogeneity into each of these populations. Since all of these variants contain the essential features of the rt-PA molecule, polyclonal, antibody-based assays and biological assays cannot discriminate between these forms. Electrophoretic or chromatographic methods are required. With the recent determination of the carbohydrate structures of rt-PA 39, the molecular masses of the four variants can be estimated (Table 2). The variability in glycosylation is responsible not only for the range of molecular masses, but also for charge heterogeneity (due to terminal sialic acid residues present on many of the carbohydrate chains), which can be revealed by isoelectric focusing (IEF) gel electrophoresis (Fig. 2). The formation of the 2-chain variant has no significant effect on the molecular mass of the unreduced form as the A and B chain fragments are held together by a single disulfide bond. However, reduction releases two smaller fragments (Table 2). The 2chain form also could add to the charge heterogeneity. With this level of complexity, part of the analytical strategy must be to
select methods that are specific for only a particular property of the molecule. Gel permeation chromatography (GPC), for instance, is a relatively low resolution analytical technique that is insensitive to carbohydrate microheterogeneity and thus allows a facile and accurate estimation of the amount of the 2-chain form in a given rt-PA preparation (Fig. 3). Similarly, reversed phase HPLC (RP-HPLC) of tryptic fragments has become widely accepted as the best analytical technique for monitoring the primary sequence of a protein. The technique is primarily sensitive to differences in hydrophobicity. By using tryptic fragments rather than the intact protein as the analyte, sequence differences both on the surface and in the interior of the protein can be --Fig. 3 1-chain 73.8%
2-chain
7.0
8.0 9.0 Elution time (minutes)
10.0
The separation of a sample of 1chain and 2-chain rt-PA by gel permeation chromatography. The sample has been reduced so that the A- and B-chain fragments run as a single peak (labeled 2-chain) significantly slower than the 1chain species.
TIBTECH - DECEMBER 1989 [Vol. 7] --Fig.
4
A28
b'~ I--" o4 r,,.. b-
o 0
co
VI_.Fo9 t.-
A~4
~ g
I
~-v-
I--
I
CO
LO
}-~+
o4 I--
I--
~
if)
o3
I--
t.to
0 CO
%
II
04
I
~
I~. I--
I
i
I
0
10
20
30
I
I
40
5O
I
60 Elution time (minutes)
I
I
I
70
80
90
I
100
The primary sequence signature of rt-PA as shown by reverse-phase HPLC of tryptic fragments. The tryptic fragments are numbered from the N-terminus. Glycopeptides are highlighted in black.
revealed. Trypsin cleaves at frequent intervals producing peptides of 2-20 amino acid residues in length. Therefore, any amino acid substitution or cleavage of the polypeptide backbone would be expected to generate a shift in retention time. The tryptic map for rt-PA includes over 50 peptides that are separated in a single chromatographic analysis (Fig. 4). Carbohydrate heterogeneity results in poorly resolved multiplets. Removal of the carbohydrate with a glycosidase results in collapse of the multiplet and shift of the deglycosylated p e p t i d e to a longer retention time (Table 3). Because of the relative insensitivity of RP-HPLC to the presence of carbohydrate side-chains, it can be used to monitor the primary structure of rt-PA. The carbohydrates liberated by glyc0sidase digestion can be analysed in the absence of the peptide mixture by high pH anion exchange chromatography 39'4°. At present, quality control procedures focus on the amino acid sequence of a protein rather than the carbohydrate portion. This is partly because the methods available for monitoring protein sequence are excellent, while those for carbohydrate analysis have been much
more time consuming and less effective 14. Other barriers to routine carbohydrate analysis are the high site-dependent microheterogeneity of many native glycoproteins 19'4~ and the potential for greater structural complexity relative to proteins. Such an analysis, although necessarily complex, will become important because carbohydrate can serve as an effector of glycoprotein clearance 42. Furthermore, glycosylation will vary with host cell type, the transformation state, and establishment in cell culture 18 and carbohydrate mapping could be an indicator of the competence of the cell culture system. Although the tryptic map is highly reproducible and suitable for lot release purposes, the complexity of the map limits its usefulness in monitoring protein degradation reactions such as proteolysis and deamidation. IEF, however, can monitor charge variants of rt-PA and can thus be used to examine many degradative reactions. SDS-PAGE with silver stain, after reduction and S-carboxymethylation of rt-PA, can be used to develop a catalog of patterns of proteolytically generated forms. Silver stain does not yield a quantitative analysis and, therefore,
must be compared with reference material. The production, characterization and maintenance of a reference material are critical quality control program requirements and should be included on all final product tests 14'38. Other important degradation forms result from aggregation. Neither SDS-PAGE or reverse phase HPLC are suitable for the detection of aggregated rt-PA, since aggregates may irreversibly bind to the HPLC column or fail to penetrate the polyacrylamide gel or be dissociated by the analytical conditions. Size exclusion chromatography is better suited to this analysis. Defined imp urities extrinsic to th e product Defined impurities extrinsic to the product include components which are added to the culture medium or proteins which are used as raw materials in the purification process. Insulin, transferrin, antibiotics, and neoplastic agents are among the impurities that could arise from the fermentation medium. They may have significant biological or immunogenic activity and must be removed to part-per-million levels sJ°'2~. Demonstration of such purity levels
TIBTECH
requires a combination of highly sensitive assays and process validation studies 38. Clearance of each impurity can be determined by assay at each step in the process 21. The clearance of an impurity which may be present in very low levels is also calculable by adding known amounts of the impurity to the product pool before each process step and subsequent assay after each step. This approach has been used to validate the removal of media and host cell components 21 and virus 37. Purification processes which employ affinity chromatography are more common 43 and may introduce antibody-derived impurities 38'44. Antibodies and antibody fragments may possess significant immunogenic potential if they are non-human in origin and if the therapeutic dose is large enough 45-47. Immune complexes may be carried through the process making the analysis difficult. Also, the antibody preparation itself may contain significant impurities 26'38'48. Fetal calf serum, bovine immunoglobulins and Protein A or Protein G are some impurities associated with murine monoclonal antibodies. Their leakage rate from an immuno-affinity column and their clearance by the purification process in the presence of the product must be demonstrated as part of the validation effort 44. The specificity and sensitivity necessary to detect antibodies at ppm concentrations in the presence of their respective protein antigen (i.e. the product) is best provided by immunoassays 4a. Undefined impurities
Knowing the precision, specificity and accuracy of the analytical method is of particular concern in the detection and quantitation of undefined impurities. Unlike a processderived impurity in a well-developed purification scheme, undefined impurities may vary in both quantity or composition from one production run to another. Broad spectrum assays which detect a general biological effect or an assay signal from any of a family of components are generally used. However, reference methods are rare (given the lack of specific analytes) and the methods used can only be validated by
rigorous control of assay reagents and careful design of the assay. In view of these difficulties, it is clear that flesh approaches are needed if one is to have assurance that a new drug is not at risk from contamination with undefined impurities. • Microbial c o n t a m i n a n t s . Microorganisms such as bacteria, mycoplasmas or viruses are important possible contaminants of pharmaceuticals isolated from biological materials. The presence of the AIDS virus in blood and blood products and the probable presence of the agent causing Creutzfeld Jakob disease in early preparations of pituitaryderived human growth hormone 49 demonstrate an advantage of rDNA technology, where the host cell, medium and isolation materials can be rigorously characterized and controlled ~4. Again, the development of rt-PA shows how undefined impurities can be controlled. While evidence of the presence of virus of potential pathogenicity has been found in primary cell culture or tissues, endogenous virus has not been isolated from any of the human diploid cells used for vaccine production 5° nor from CHO cells 37, the line used to produce rt-PA. Taking cells from a wellcharacterized cell bank allows them to be examined carefully for the presence of virus or virus-like bodies in the cells and the evaluation of a potential risk for humans 5°. An extensive cell characterization program contributed to the approval of CHO cells as a drug substrate 37'5°. Assays for a wide range of animal viruses were performed on the cell substrate medium 37. This cell substrate was also tested extensively for reverse transcriptase activity and mice were inoculated to identify endogenous murine viruses 37. Each of over 200 lots of harvested cell culture fluid has been assayed for adventitious virus by testing for cytopathic effects on three different cell lines and none has been found positive 37. Through this program of carefully selected, characterized and maintained host cell banks, controlled production systems with inactivation steps and product testing, the possibility of infectious
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DECEMBER 1989 [Vol. 7]
agents as impurities or contaminants is essentially eliminated. • Host D N A . Host cell DNA is another undefined impurity which must be measured in every lot of drug produced TM. The recommended limit for DNA impurities is 10 pg per dose 2°,5° and the purification process must contain steps to eliminate DNA 51. The accepted assays are dot blots using specific 32p-labeled DNA probes obtained from the DNA of the host ce1152; n e w assays have recently been introduced 53. Current manufacturing procedures can achieve very low levels of DNA contamination, often below the threshold for detection with current assays. • Pyrogens. Endotoxin and other pyrogenic materials are among the other poorly defined impurities of major concern 54'55. A combination of the USP rabbit pyrogen assay and the Limulus Amoebocyte Lysate (LAL) assay is considered the most conservative and appropriate approach for the testing of recombinant biologics 14. However, the rabbit pyrogen assay is highly variable and may result in false positives due to infection, antigenicity or handling practices 14,26. Some of the recombinant cytokines, such as tumor necrosis factor, are themselves pyrogens and will be cross-reactive in the whole animal model 14'26. The LAL method may not detect endotoxin after lyophilization and is subject to a number of interferences 14'56. Therefore, both tests are often required. Where conflicting results or suspected interference occur, a third method which relies on a different biological reaction can be useful. The pyrogen assay developed by Dinarello 56 relies on the production of interleukin I (IL-1) by tissue cultured monocytes in response to endotoxin. The IL-1 is then measured specifically by immunoassay. • H o s t proteins. The detection of host cell proteins requires both a ppm sensitivity and the detection of large numbers of proteins; this has been achieved through the development of process-specific immunoassays 57. The potential biological or immunological consequences of these impurities have
TIBTECH - DECEMBER 1989 [Vol. 7]
~Table 3
Retention time shifts on deglycosylation. Since residue 184 is not always glycosylated there are two peaks in the original tryptic map (peptide T17 with and without the carbohydrate side chain). Only one of them shifts its retention time after reaction with glycosidase.
RCM rt-PA peptide and carbohydrate type a T11-(High mannose) T17-(Complex) T17-(Nonglycosylated) T45-(Complex)
Residue no. (Asn) 117 184 184 448
Glycopeptidase F t r e a t m e n t (Asp) (-) 42.2 b 40.5 44.9 25.0
(+) 46.0 45.4 44.9 30.5
aRCM, reduced and s-carboxymethylated. bRetention time measured in minutes in a TFA mobile phase system 65.
demanded this extreme level of performance 3'58. The sensitivity and selectivity has been markedly improved by the development of Antigen Selected Immunoassays (ASIA) where a population of antibodies raised against process-specific host cell proteins are further selected by affinity chromatography before use in an ELISA method in which the host cell contaminants are quantified as a group 13'59. Technical problems occur in developing these assays involving the selection of the host cell protein reference material, the method for production of antibodies and the validation of the assay. The reference proteins could be isolated from in-process material by immunoabsorbing the product away from the other proteins. The immunoabsorption procedures, however, are very difficult to perform and validate: all of the product and product fragments must be removed without losing any of the impurities. Any residual product wilt produce antibodies during the immunization and render the assay nonspecific. An alternative way of producing the reference material is the 'blank run' approach; a fermentation and partial purification of the host are conducted using the host cell lacking the product gene 13'59. Two key assumptions are made. The first is that the lack of product expression does not significantly affect the population of in-process impurities. The second is that the lack of product does not significantly affect the chromatography of the impurities: for E. coil proteins purified by the growth hormone process, the presence and absence of a small amount of growth hormone did not
create any significant changes in the distribution of contaminants 6°. Similar studies could be performed for each product. Performing the blank run at manufacturing scale will minimize differences in both the growth conditions and chromatography from the actual r u n 13'21'59. Typical reference impurity mixtures can contain 100 or more components as shown by a silverstained 2D gel 60. The usefulness of the antisera produced against such mixtures is limited by the amount of antibody produced to the least immunogenic component. The use of multiple animals, extended immunization schedules and rigorous antisera characterization are necessary to ensure that antibodies have been produced to all of the minor components in the mixture 61. Finally, the design and development of these assays present a number of problems: the development lead times are very long, a rigid format must be used 62, and the only approach to validation of the assays is to demonstrate internal consistency of the method and the quality of the reagents 59'61. Furthermore, because each assay is process specific, purity values cannot be compared between manufacturers unless the processes are identical. Given that at least 1500 proteins are produced by E. COIi63, it is doubtful that a sufficiently sensitive generic assay could ever be developed 5u.
advances in methodology further refine the definition of purity, the regulatory guidelines must be flexible enough to anticipate the observation of previously undetected impurities 38. In the production of hGH, approximately 750 separate tests are performed during the manufacturing and release processes 64, a result of the greater sophistication and complexity of recombinant manufacturing processes. These tests are most effective if their respective strengths and weaknesses are complementary. For instance, immunoassays provide very specific and sensitive detection of host cell protein impurities but will only detect proteins which elicit an antibody response. Therefore, a sensitive, non-antibody based method such as silver-stained SDSPAGE should also be used. Silver stain SDS-PAGE, however, is highly variable and impurities might be concealed due to co-migration of different proteins. Thus both methods should be used with a rigorous process validation program 15'21'38. The value of complementary methods is also apparent in the detection of rt-PA product variants (Figs 2-4). A combination of complementary analytical methods and process validation provide the best assurance that impurities are minimal in the final product. The safety of protein pharmaceuticals can only be determined through appropriate preclinical and clinical testing. The approval to enter a clinical .testing program and the confidence that a safe product can be consistently manufactured relies on a comprehensive analytical chemistry program.
Acknowledgements The authors would like to acknowledge the following experimenters working on rt-PA: IEF (Robert Bridenbaugh, Mary Sliwkowski, Andrew Guzzetta). GPC (Charles DuMee). tryptic mapping (Rosanne Chloupek).
An integrated approach to purity analysis It is clear from the variety of methods discussed here and the spectrum of possible impurities that exact protocols for purity analysis cannot be universally applied. As
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Thermophilic organisms as sources of thermostable enzymes Jakob K. Kristjansson Thermostable enzymes are receiving considerable attention and there are many industrial applications already. Most thermostable enzymes on the market have, however, been derived from mesophiles. Further research, especially into the possibilities of cloning enzymes from thermophiles into mesophilic hosts, will greatly increase the exploitation of thermophiles in biotechnology. Temperature is probably the most important environmental factor affecting the activity and evolution of living organisms. Most organisms that have been studied in depth are adapted to 'moderate' temperatures and operate within a relatively narrow span of temperature (generally less than 30°C). In these organisms, the upper temperature at which life is possible (Tmax) is usually reached quite abruptly and is only a few degrees above the optimum growth temperature (Topt). In recent years, there has been increasing interest in microorganisms which are adapted
lakob K. Kristjansson is at the Department of Biotechnology, Technological Institute of Iceland, Keldnaholt, IS-112 Reykjavik and the Institute of Biology, University of Iceland, Grensasvegur 12, IS-108 Reykjavik, Iceland.
to environments of extreme temperature, such as hot springs 1,2.
What is a thermophile? Organisms have been classified according to growth temperature Topt and/or Tmax1'3-6. These classifications will not be discussed here beyond saying that the term 'thermophile' is not very precise and its meaning depends on the group of organisms being considered. The upper temperature limit for growth of different groups of organisms varies widely, from 38°C for fish, 55-60°C for the eukaryotic algae and fungi, to 85°C for eubacteria and up to 110°C for certain archaebacteria 1,6'7. Here I will use the term 'thermophile' ('thermophilic') to describe those bacteria with Topt of 65-85°C and 'extreme thermophile' to describe those with Topt >85°C. Organisms with Topt <60°C ('slight thermophiles') will not be discussed.
© 1989, Elsevier Science Publishers Ltd (UK) 0167 - 9430/89/$2.00
Papers 196th ACS National Meeting, Div. of Anal. Chem., No. 62, American Chemical Society 63 O'Farre]], P. H. (1975) J. Biol. Chem. 350, 40074021 64 Ross, M. J. (1988) in Biotechnologically Derived Medical Agents. The Scientific Basis of Their Regulation (Gueriguian, J. L., Fottorusso, V. and Poggiolini, D., eds), pp. 168-174, Raven Press 65 Chloupek, R. C., Harris, R. J., Leonard, C.K. et al. (1989) J. Chrom. 463, 375-396 []
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Finding thermophiles Organisms which grow optimally at 45-55°C are ubiquitous, but those with an optimum temperature above 60°C are, in general, associated with permanently hot places such as areas of geothermal activity (although they can usually be isolated from many non-geothermal areas). Thermophilic sporeforming organisms are also abundant in natural environments only heated for a short time, such as composts and solar-heated soils and ponds 1. Many thermophiles, including Thermus spp. and spore-forming organisms, have colonized manmade hot places, such as domestic and industrial pipes 8. Before 1970, several thermophiles had been isolated which could grow up to 70°C (see Table 3.3 in Ref. 1) (e.g. Bacillus stearothermophi]us strains with Topt of 50-60°C). Much research has been performed on strains of B. stearotbermophilus including the isolation and study of thermostable enzymes 4. Thomas Brock's isolation of Thermus aquaticus with its Topt of 70-75°C, however, was a major discovery 9. During his work in Yellowstone National Park from 1968-1978, Brock also isolated Su]folobus acidoca]darius and Thermoplasma acidophilum, the first representatives of the thermoacidophilic archaebacteria. Brock and his colleagues also isolated enzymes from T. aquaticus and showed them to be more thermostable than any other previously known enzymes 4"1°. tn 1981, Stetter and Zillig isolated