- Email: [email protected]
Cytokines: from technology to therapeutics
FOCUS One of the strengths of microelectrophoresis devices is clearly the increase in assay speed. DNA-fingerprinting applications, such as STR analysis, are now possible in minutes or even seconds. This speed offers many new applications for genotyping. However, the advantages of highly multiplexed or automated systems remain to be demonstrated but should not really be a problem once resources are applied to the problem of scale up. Automating the microdevice format may well push the sequencing-throughput bottleneck back into sample preparation. Acknowledgments The authors’ work was supported by the US National Institutes of Health and the US Air Force Office of Scientific Research. We thank A. Adourian, L. Koutny and D. Schmalzing for extensive contributions to the Whitehead Institute results discussed here. References 1 Fodor, S. et al. (1996) Science 274, 610–614 2 Manz, A. et al. (1992) J. Chromatogr. 593, 253–258
3 Pace, S. J. (1990) US Patent 4 908 112 4 Effenhauser, C. S., Paulus, A., Manz, A. and Widmer, H. M. (1994) Anal. Chem. 66, 2949–2953 5 Woolley, A. T. and Mathies, R. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11348–11352 6 Jacobson, S. C. and Ramsey, J. M. (1996) Anal. Chem. 68, 720–723 7 McCormick, R. M., Nelson, R. J., Alonso-Amigo, M. G., Benvegnu, D. J. and Hooper, H. H. (1997) Anal. Chem. 69, 2626–2630 8 Woolley, A. T. and Mathies, R. A. (1995) Anal. Chem. 67, 3676–3680 9 Schmalzing, D., Koutny, L., Adourian, A., Belgrader, P., Matsudaira, P. and Ehrlich, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10273–10278 10 Woolley, A. T., Hadley, D., Landre, P., deMello, A. J., Mathies, R. A. and Northrup, M. A. (1996) Anal. Chem. 68, 4081–4086 11 Woolley, A. T., Sensabaugh, G. F. and Mathies, R. A. (1997) Anal. Chem. 69, 2181–2186 12 Schmalzing, D., Adourian, A., Koutny, L., Ziaugra, L., Matsudaira, P. and Ehrlich, D. J. (1998) Anal. Chem. 70, 2303–2310 13 Hjerten, S. J. (1985) J. Chromatogr. 347, 191–198 14 Mathies, R. A. and Huang, X. C. (1992) Nature 359, 167–169 15 Ueno, T. and Yeung, E. S. (1994) Anal. Chem. 66, 1424–1431
Cytokines: from technology to therapeutics Anthony R. Mire-Sluis Cytokines are playing an ever-increasing role in the treatment of human disease. The characterization of these proteins plays a vital role in their development as useful therapeutic agents. Physicochemical techniques can produce information about the structure and composition of cytokine therapeutics but cannot yet predict their biological activity, for which biological assays are required. Because of the large number of techniques available and the variety of products requiring analysis, the tests used to characterize cytokine products must be both appropriate for the product and adequately controlled if the information they provide is to be of value.
ytokines and growth factors mediate a wide range of physiological processes, including haematopoiesis, immune responses, wound healing and general tissue maintenance1; for the purposes of this review, growth factors will be included in the term ‘cytokine’. As cytokines are involved in many physiological processes, it is not surprising that they are also involved in the pathogenesis of many diseases. Associated with this is their vast potential in replacement or modulatory therapy2. Recombinant DNA technology has resulted in the discovery of increasing numbers of proteins that have been categorized as either cytokines or growth factors. There are over 150 well-established cytokines and growth factors3, and many other proteins have been discovered through random
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A. R. Mire-Sluis ([email protected]) is at the Division of Immunobiology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, UK EN6 3QG. TIBTECH AUGUST 1999 (VOL 17)
DNA screening that appear to have cytokine-like properties but whose true functions remain unidentified3. Widespread application of recombinant DNA technology within the biotechnology industry has dramatically increased the number of cytokines available for clinical evaluation (Table 1). New cytokines are being discovered, cloned and entered into clinical trials at such a rate that their structural properties and biological activities are often poorly understood during their development as therapeutic agents. Safety, efficacy and quality are major concerns for the success of any biological product. Safety, involving toxicity and possible infectious agents, is dealt with through well-documented procedures (regulatory guidelines available from the European Medicines Evaluation Agency website http://www.eudra.org/emea.html) but efficacy can only be evaluated through clinical trials4. Quality assessment needs to address a variety of issues including heterogeneity, consistency, potency, stability
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Table 1. Examples of cytokine and growth-factor therapeutics Agent
Product type(s)
Some therapeutic uses
Interferon a
Multicomponent, natural (leukocyte derived) Multicomponent, natural (lymphoblastoid derived) Recombinant (Escherichia coli-derived), nonglycosylated
Interferon b
Recombinant (E. coli-derived), nonglycosylated mutein (Cys-17-Ser) Recombinant (CHO-derived), glycosylated
Antiviral: Hepatitis B and C Immunomodulation: condyloma Antiangiogenic: Kaposi’s sarcoma Antitumour: hairy-cell leukaemia, chronic myelocytic leukaemia Immunomodulatory: multiple sclerosis Antiviral: post-interferon-a resistance in hepatitis
Insulin-like growth factor-1 Granulocyte-colonystimulating factor (G-CSF)
Recombinant (yeast-derived), nonglycosylated
Interleukin 2 Erythropoietin Platelet-derived growth factor BB
Recombinant (E. coli-derived), non-glycosylated, additional methionine Recombinant (CHO-derived), glycosylated Recombinant (E. coli-derived), nonglycosylated mutein (Cys-125-Ser) Recombinant (CHO-derived), glycosylated Recombinant (yeast-derived), glycosylated
and formulation. This involves critical scientific consideration of the many analytical techniques that can provide the necessary information to assure product quality. In general, there are three main groups of analytical tests for cytokines – biological assays, immunoassays and binding assays, and physicochemical techniques. Within each of these groups, there are several different assay systems. Individual assays have their own particular strengths and weaknesses, which are dependent on the type of material under test and the information that is required (Table 2). For example, in comparison with a non-glycosylated protein, a product that is glycosylated will require additional analytical tests to explore the sugar aspect of the protein. In addition, glycosylation causes proteins to perform differently on many of the standard analytical tests used, which have either to be adapted to take this into account or have the data they produce interpreted accordingly5. The purpose of this article is both to illustrate the various analytical techniques currently available to analyse biological products and to discuss the scientific rationales required to apply these tests to cytokines with different structural and biological characteristics. Biological assays Bioassays for cytokines can take several forms but they all rely on the ability of the protein to induce some measurable activity in cells or tissues6. It is now rare to measure the biological activity of a cytokine in vivo. The development of clonal cell lines that respond to cytokines is a significant improvement as a source of materials for bioassays. Cell lines (usually from murine or human cancers) can be dependent on a cytokine for growth and are often immortal, thus providing a single homogeneous source of cells that can be distributed from laboratory to laboratory7. A good example of this is the G-NFS-60 murine myeloid leukaemia cell line that is used to measure
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Neuroprotection: amyotrophic lateral sclerosis Immunostimulation: Immunoreconstitution after a bone-marrow transplant Reduce neutropaenia after chemotherapy Mobilization of peripheral-blood stem cells Antitumour: bladder carcinoma, melanoma Production of red cells for anaemia in haemodialysed patients Malignancy-associated anaemia Wound healing: diabetic foot ulcers
the cytokine granulocyte-colony-stimulating factor (G-CSF). G-CSF is used therapeutically to reconstitute the immune system after bone-marrow transplantation (Table 1) and has been highly successful as a biological product. However, prior to the development of G-CSF-responsive cell lines, the potency of G-CSF was determined using murine-bone-marrow-colony assays. These assays were time consuming (taking 5–7 days), technically laborious (often using colony counting as a read-out) and variable, owing to interanimal differences. Cell-line assays are much more rapid, with G-CSF bioassays taking only 24 h, and are much more reproducible. The advent of recombinant DNA technology has allowed the cloning of specific cytokine receptors and their expression on previously unresponsive cell lines. This can create a specific, responsive cell line for almost any cytokine without the need to screen a wide range of existing cell lines for cytokine responsiveness7. The receptor for the cytokine thrombopoietin (TPO) has been cloned into the TPO-unresponsive murinemyeloid-leukaemia cell line 32D to produce a cell line designated 32D/Mpl1, which proliferates in response to TPO and has been used to determine the potency of therapeutic products8. The use of murine cell lines to assay human cytokines is dependent on the species cross-reactivity of the cytokine to be tested, as not all cytokines can cross the species barrier1. The cellular response induced by cytokines that is utilized as a bioassay read-out can take a variety of forms but is most often the proliferation or inhibition of proliferation, the expression of cellular markers or enzymes, cytotoxicity or antiviral activity6. There has been some controversy amongst the regulatory agencies over whether the bioassay used to measure the potency of the product should be related to its clinical activity. For example, should interferon a (Table 1) be assayed for its antiviral activity or its antitumour activity, TIBTECH AUGUST 1999 (VOL 17)
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Table 2. Analytical techniques for the analysis of cytokines Procedure
Data provided/inferred Molecular size
Charge
Primary Secondary structure or tertiary structure
Quaternary structure
Purity
Potency
Timea (h)
Bioassays
2
2
111
1111
1111
1/2
11111
24–48
Binding assays Immunoassays Immunoblotting Receptor binding
2 111 2
2 1 2
1/2 11 11
1/2 111 111
1/2 111 111
2 2 2
2 2 1/2
8–24 24 5
Gel electrophoresis Isoelectric focusing Nonreducing Reducing
2 2 111
1111 2 2
111 11 111
1 111 2
1 111 2
111 111 111
2 2 2
36 24 24
HPLC Hydrophobic interaction Ion exchange Reverse phase Size exclusion
1 2 1 111
1 11111 1 2
111 111 1111 2
11 2 11 11
11 1 2 111
111 111 1111 111
2 2 2 2
4 4 4 4
2 11111 1
2 1 2
2 11111 11
1111 1/2 11111
111 2 2
11 2 1111 2 11 2
2 1 1
Structural assays Circular dichroism Mass spectrometry Nuclear-magnetic resonance
Abbreviations: 11111, extremely informative; 1, only a little useful information is provided; 1/2, can provide minimal information in an indirect manner (e.g. if an immunoassay can detect a protein, it suggests that the structural conformation of the antibody-recognition sites may be correct); 2, no useful data provided. aTime for the procedure from start to finish is approximate and depends on whether the various systems are already set up and, in the case of gel electrophoresis, on the gel size.
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Standard curve Dilution of test sample Linear portion of response curve Relative activity
Concentration of sample (reciprocal dilution)
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or both? One can argue that, if a product has only one clinical activity, its bioassay should attempt to measure it. However, because potency measurements are a quality issue and should not to be used to relate to dosing in the clinic (clinical trials establish this), then any functional activity could, in theory, be used4. Although the different bioassay systems have disadvantages, most often lack of specificity or nonspecific interference of sample matrix with the cells, refinement of the assay technique and cell maintenance has resulted in bioassays that are as accurate (if not more so) than many physicochemical techniques; that is, a bioassay is only as good as its design and execution. The design of any bioassay must take into account factors that introduce variability, and the analysis of bioassays must test for variability if results are to be statistically valid. Although the design of in vitro bioassays can take many forms, a single point assay is definitely not valid. A titration of the test material has to be made and compared with a titration of a reference material, with particular attention paid to comparisons of the linear portion of the dose–response curve9 (Fig. 1). In order to reduce the effects of position within microtitre-plate assays, randomization of the position of sample titration curves within plates is recommended, as is the inclusion of a standard reference preparation on each plate10. Carefully controlled bioassays are technically demanding, relying heavily on the competence of the staff
Figure 1 A diagrammatic example of the design of a parallel-line bioassay. Samples and reference preparations are diluted in series to produce a dose–response curve. The displacement between the linear portions of the dose–response curves is representative of the relative activity of the two preparations and is calculated using parallel-line statistical analysis7.
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carrying out the assays to dilute and to pipette cytokine solutions accurately and reproducibly. However, attempts to automate biossays have been particularly successful when such tasks have been taken over by robots. The cost of bioassays varies, depending on the amount and sophistication of hardware used. The development of in vitro bioassay techniques has led to the increased use of microtitre-plate-based assays using continuous cell lines to bioassay the majority of cytokines. However, for some growth factors and cytokines (particularly the chemokines), there is no suitable cell-line-based bioassay that is appropriate for reproducible routine use. In this case, the development of receptor-based ‘biochemical bioassays’ are being developed: the binding of some cytokines to their receptor on cell surfaces induces the rapid phosphorylation of the receptor on tyrosine residues; the cells are then lysed and an immunoassay used to detect the level of phosphotyrosine being produced11. Although these assays are relatively rapid and reproducible, they are only of value for cytokines for which simple cell-line-based bioassays do not exist, because they still require the culture of cells or cell lines and involve an additional immunoassay step. How receptor phosphorylation relates to overall biological activity also requires further investigation. Current assays for chemokines involve the chemotaxis of fresh blood cells (usually neutrophils or T cells) through nitrocellulose membranes. These assays are very technically demanding and produce highly variable results. However, the binding of chemokines to their receptors induces Ca2+ fluxes, the magnitude of which correlates directly with the quantity of chemokine present12. These assays are currently being refined as alternatives to chemotaxis assays. As different manufacturers’ products, even the same product from the same cell source, can possess very different specific activities, mass cannot be used as a measure of the functional activity of a biological material13. In addition, using mass as an indicator of drug content is not helpful in itself, as one cannot possibly weigh a supplied drug, particularly if it contains excipients (as many biotherapeutics do). Therefore, a unit has to be defined for such activity, for without a single reference preparation, units with different definitions can occur. This causes confusion, leading to substantial interlaboratory variation in estimates of potency. The concept of a single internationally available potency standard has been developed under the auspices of the World Health Organization and shown to be extremely valuable in reducing the laboratory variability in potency estimates and enhancing the comparability of clinical and research studies14. International units and standards are vital if the biological potency of any preparation is to be assessed. Immunological assays Immunoassays are assays that use the binding of antibodies to a ligand to detect specific interactions, resulting in quantitation of that ligand. Immunoassays can be highly specific, quick and simple to carry out and require little expensive machinery. However, immunoassays do not measure a biological function15 and they cannot predict biological activity, because subtle changes in structure impacting greatly on biological
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activity may not affect antibody binding. For example, the gross change in structural integrity caused by proteolytic cleavage would not be detected in an immunoassay if the antibody recognition sites were unaffected. Immunoassays can be extremely reproducible and are particularly useful for detecting cytokines in samples that contain other substances that might affect bioassay results, such as those found in serum. However, immunoassays can be affected by the constituents of biological samples (matrix effects), particularly when dealing with biological fluids such as serum, cerebrospinal fluid and urine, and immunoassays must be tested very carefully for such effects and any found must be taken into account. For example, the reference material used to calibrate an immunoassay should ideally be made up in the same type of fluid as the samples, unless it has been shown that the formulation of the standard and samples is not relevant to their comparison in the assay. Although immunoassays are highly specific, this can be a problem for their use16. The antibodies that make up an immunoassay are usually raised against a cytokine from a particular source [such as Escherichia coli or Chinese hamster ovary (CHO) cells] and are rarely raised against natural material. Therefore, it is almost impossible to predict how well such antibodies recognize naturally derived cytokines in biological samples. Few cytokines have been purified from natural sources in sufficient quantity to carry out comparative experiments, but there is evidence to show that some antibodies can recognize differences between E. coli, CHO and yeast-derived material, suggesting that differential recognition of natural materials might well occur17. Recent work has also illustrated the problem of cytokines measured in the presence of soluble cytokine receptors and binding proteins18. It is extremely difficult to assess the true levels of unbound and bound cytokine using an immunoassay because of its dependency on temperature, time and the relative concentrations of the cytokine and binding protein. As the antibodies used in immunoassays are effectively in competition for binding the cytokine, it appears that environmental factors that affect the affinity of any of the cytokine-binding molecules have the potential radically to alter the results of the immunoassay. Immunoassays are also often employed to detect the presence of host-cell proteins in products by raising antibodies against a lysate of whole host cell and using these in an immunoassay format. The validity of this practice is questionable because there is no guarantee that the antibodies produced this way will result in an immunoassay that recognizes any of the host-cell proteins that evade purification processes19. There have been several recent developments in immunoassay technology for the detection of cytokines, one example of which is an immunoassay format with improved sensitivity – dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)20. The DELFIA system is based on the theory of time-resolved fluoroimmunoassay using europium (Eu)-labelled antibodies. Eu emits fluoresecent light that is detected to a higher degree than the colour changes used in standard enzyme-linked immunosorbant assays (ELISAs). This results in an immunoassay that is 10–100 times more sensitive than current ELISAs. TIBTECH AUGUST 1999 (VOL 17)
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The use of electrochemiluminescence has allowed the sensitivity of immunoassays to be increased and has resulted in assays that appear to be less affected by matrix effects. Typically, these assays take the form of a cytokine bound to an antibody on a bead, which is then sandwiched to an antibody labelled with ruthenium (Ru). An electrical potential applied to this complex produces light, which can then be detected and quantified21. Recent biosensor developments allow the interaction of cytokines and cytokine-binding proteins (such as antibodies and soluble receptors) to be measured accurately, a process termed ‘biospecific interaction analysis’. A biosensor can be defined as an instrument that combines a biological recognition mechanism with a sensing device or transducer. One example of biosensor technology employs surface-plasmon resonance (SPR) to monitor biomolecular interactions. SPR is an optical technique that uses changes in the reflectance of light off a thin metal film to detect changes in the concentration of macromolecules in a surface layer of solution in contact with the metal film. The use of biosensors enables real time on and off rates to be calculated, in addition to much-more-detailed information on cytokine binding than can easily be produced using conventional techniques22. Physicochemical analysis There are many physicochemical techniques available for the analysis of cytokines (Table 2). The widespread use of mass spectrometry (MS) has had the most recent impact on protein characterization and produces extremely accurate data on the molecular mass (and hence amino acid content) of a protein. The technique involves ionizing a protein and measuring various parameters (e.g. time of flight and mass:charge ratios of the resulting ionized protein species) to estimate the mass of the original material. Two of the most popular methods to achieve ionization are matrix-assisted laser-desorption ionization (MALDI) and electrospray ionization (ESI)23. MS is claimed to have a mass resolution of 0.1% and an accuracy of 0.01%, and to be able to detect contaminants at 1%. However, ESI, in particular, requires sample desalting, which can affect the nature of the material being investigated, and both MALDI and ESI are not as yet particularly good at resolving complex mixtures of protein species24. In addition, not all protein species ionize equally and therefore might not be correctly represented. It must also be noted that, although MS provides valuable information about the amino acid content of proteins as a whole, it does not provide information about the actual sequence of the protein unless peptide mapping is used in addition. Other techniques, such as nuclear magnetic resonance (NMR) or circular dichroism (CD) can provide information about the higher-order structure of proteins25. NMR involves applying strong magnetic fields to proteins in solution and analysing the absorption or emission of electromagnetic radiation by the nuclei of the constituent atoms. NMR is an extremely powerful technique and can provide a highly complex ‘fingerprint’ of the secondary and tertiary structure of a protein but it requires milligram quantities of protein and is affected by the presence of sugars, organic solvents and detergents. TIBTECH AUGUST 1999 (VOL 17)
CD is an adsorption technique that detects changes in left and right circularly polarized ultraviolet (UV) light passing through a protein solution. Near-UV CD detects absorption by aromatic residues (chromophores) and disulphide linkages, as well as changes in tertiary structure. Far-UV CD shows changes in secondary structures (a helices and b-pleated sheets). CD provides a less complex profile of protein structure than NMR and is essentially a comparative technique, able to illustrate differences in protein structures, although the exact nature of those changes at a molecular level are more difficult to establish. Chromatographic techniques [e.g. high-performance liquid chromatography (HPLC)] are, taken together, probably the most informative of the various physicochemical techniques, as they are often able to resolve the majority of protein species in the sample, allowing further detailed characterization in association with other techniques26. Reverse-phase HPLC can detect 0.1% impurities under optimal conditions and works in the microgram range, but is denaturing to the protein. Subtle changes might not necessarily be detected within large proteins and unequal denaturation can cause problems in resolving power. Size-exclusion HPLC is not as resolving as reverse-phase HPLC, being able to detect only 0.5% aggregates, but is nondenaturing. Ion-exchange HPLC is able to detect the most subtle of changes, even single amino acid changes if that change alters the charge of the protein. HPLC techniques are most useful when used in a panel of tests, particularly in association with peptide mapping, when they allow most known differences between protein species to be detected27. In general, it appears that the more informative the physicochemical technique, the more complex the hardware (or, possibly, the exclusivity of use to a few laboratories) and therefore the greater the costs involved. The latest developments in physicochemical analysis involve the use of existing techniques in an automated sequence. For example, tandem MS involves the use of two mass spectrometers in sequence and gives a much greater analytical potential, with products produced by the first being selected and reanalysed by the second. HPLC systems can also be linked directly to mass spectrometers so that peptides separated by chromatography can be further characterized by MS in one automated process28. One of the most advanced developments in MS is the combination of ionization technology with Fouriertransform ion-cyclotron resonance (FTICR) mass spectrometry29. ESI has been combined with FTICR to produce a technique so sensitive that it can detect mass changes of 1 Da (the equivalent of a single hydrogen atom). Although this technology is far beyond that available for the majority of laboratories, it indicates the future trends and possibilities within this field. Which test to use and why? The primary aim of characterizing a protein product during its development as a therapeutic agent is to gain as much insight into its composition and activity as possible, as this information is essential to ensure the quality of the product. The quality of a biological product involves batch-to-batch consistency, appropriate potency and stability, and other factors4. As described in the previous sections, no single analytical procedure
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a
b
CytokineÐHSA aggregates
HSA
CytokineÐHSA adducts
Cytokine 10 mg
1.0 mg 10 mg 0.5 mg 5 mg 25 mg
Figure 2 Examples of how the choice of technique and sample handling affect the results of physicochemical analysis. (a) A silver stain of an SDS-PAGE gel of a cytokine product formulated in the presence of albumin, illustrating a wide range of protein bands that does not discriminate between species related to the cytokine, albumin, adducts or other contaminants present. (b) An immunoblot of a SDS-PAGE gel of the same product as in (a), illustrating only those protein species containing the cytokine, including cytokine–albumin adducts. The quantity of the product loaded is shown at the bottom of the blot and shows the dose-related detection of various bands.
can provide all the data required to ensure the quality of a biological product and so a range of tests is required to form the specifications for the product on its release. Although a panel of physicochemical tests can provide a great deal of information about the structure of protein components, impurities and so on, the biological assay is the only assay that can provide a measure of biological activity30. A bioassay is defined as a ‘functional’ assay, and no physicochemical test can measure function. However, for some peptide hormones that are less complex in structure than the majority of cytokines, physicochemical tests characterized over many years can now be used as surrogate correlates for biological activity. Examples of the type of information that can be produced by the various analytical techniques are provided in Table 2, but the following points must be considered if any technique used to characterize a protein is to provide useful data: assay selection; assay design; sample handling; validity testing; interpreting results; and presenting the findings. For example, sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) can provide data about the proteins within a product, separated nominally by mass. It must be noted, however, that not only do different proteins bind SDS to different extents but they do not all pass through a gel matrix similarly (with or without reducing agents), as differential retention of higher structure occurs. The use of stains to detect proteins within the gel is equally important. Silver stains are highly sensitive and are the most-often used in the characterization of protein products. However, not only is the technique used for silver staining highly laboratory specific but it also tends to vary in sensitivity from day to day31. The development of such staining patterns is dependent on factors including time, temperature, the nature of the gel and the age of materials used. The effects of these must be explored and controlled if such a technique is to provide valid information.
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As discussed earlier, not only must we be cautious when using any assay to make sure that the data are relevant but the assay must also be designed in light of the material it is testing. Some cytokine therapeutics are formulated with albumin and this impacts heavily on the types of test required to analyse the product – assays must be altered to accommodate the presence of albumin, and the problem of the existence of drug–albumin adducts also occurs. Silver staining of SDS-PAGE gels of albumin-containing products is virtually useless as it produces a wealth of bands that require immunoblotting to distinguish between the drug and albumin, and to pick up any adduct formation (Fig. 2). In fact, special tests have to be designed to measure adduct formation, so that suitable limits can be applied. The quantity of sample loaded plays a vital role in the results: one can load enough sample to see tens of protein bands or amounts so small as to see only one or two (Fig. 2b). This problem relates to almost any physicochemical test and seriously compromises their validity if one considers that any physicochemical test can be designed to provide as little or as much information as is required. Therefore, the decision of which test to use when analysing a product depends heavily on the nature of the product itself, and can involve the adaptation of standard tests to provide information specific to that product. For example, one must consider the analysis of heterodimeric protein products differently from monomeric products, in addition to other issues such as N-terminal heterogeneity, oxidation, methylation and aggregation. Thus, the current regulatory approach of case-by-case consideration of biologicals must also apply to the analysis of cytokine products, where no single set of rules would be appropriate for all products. When analysing cytokines, one must also consider the stage of manufacture at which a material is being tested, and for what purpose. For example, during pharmacokinetic analysis, the range of useful techniques is limited in many cases by the low amount of material to be measured and by the problems of detecting cytokines in serum. Physicochemical analysis is not appropriate for serum samples because of the amount of interfering substances, and one is limited to the immunological and biological detection methods already discussed. Such issues are also involved in the choice of test during the manufacturing process, where the product is becoming purer and contaminating materials are being removed, allowing for more sophisticated analysis as the process continues. The storage and handling of the material influences physicochemical analysis and has to be carefully considered (Fig. 3). Conclusion There are now many methods for the characterization of cytokines and growth factors that together can provide a great deal of information about the structure and composition of cytokine therapeutic products. No single test is able wholly to characterize a protein and so a range of tests is required. The selection of which tests to use depends heavily on the type of product and the information required. Once a test has been selected, it must be designed and validated for the test material. Even though increasingly sophisticated analytical techniques are continually being developed, it is important TIBTECH AUGUST 1999 (VOL 17)
Dimer
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Monomer
mV Ö A214
0.10
0h
0.05
5h 16 h 24 h
0 10
Time (min)
12
Figure 3 A size-exclusion HPLC separation of a cytokine drug substance that is stored frozen. Thawing the material at time zero and testing it after storage at 48C for the times indicated illustrates the formation of dimer on freezing and its gradual dissociation on thawing. The storage and handling of this material can be seen to influence the physicochemical analysis and so this has to be considered carefully. Abbreviation: A214 , absorbance at 214 nm.
for those concerned with the manufacture of clinical products to consider the true importance of any characterization in terms of clinical use and necessity. Requirements for highly extensive analysis could significantly slow the development of products and yet have no real impact on the value of a material as a therapeutic agent. It is thus extremely important that both industry and regulators have experience of handling biological products in a variety of tests using different conditions, not only to explore how a technique can characterize a protein but also to show how that protein can be used to validate the technique. Acknowledgments I wish to thank the participants in the The Biological Characterization and Assay of Cytokines and Growth Factors meeting, held at National Institute for Biological Standards and Control, 10–12 September 1997, for discussions that led to this article. Proceedings of this
meeting will appear in the Developments in Biological Standardisation series, Vol. 97. In addition, I am grateful to C. Dolman in the Immunobiology division at NIBSC for the HPLC data included in Fig. 3. References 1 Mire-Sluis, A. R. and Thorpe, R. (1998) Cytokines (1st edn), Academic Press 2 Oppenheim, J. J., Rossio, J. L. and Gearing, A. J. H. (1993) Clinical Applications of Cytokines: Role in Pathogenesis, Diagnosis and Therapy (1st edn), Oxford University Press 3 Thomson, A. (1998) The Cytokine Handbook (3rd edn), Academic Press 4 Mire-Sluis, A. R., Gaines-Das, R., Gerrard, T., Padilla, A. and Thorpe, R. (1996) Biologicals 24, 351–361 5 Townsend, R. R. and Hotchkiss, A. T. (1997) Techniques in Glycobiology (1st edn), Marcel Dekker 6 Wadhwa, M., Bird, C., Page, L., Mire-Sluis, A. R. and Thorpe, R. (1995) in Cytokines: A Practical Approach (Balkwill, F. R., ed.), pp. 357–391, IRL Press 7 Mire-Sluis, A. R. and Thorpe, R. (1998) J. Immunol. Methods 211, 199–210 8 Bartley, T. D. et al. (1994) Cell 77, 1117–1124 9 Thorpe, R., Wadhwa, M. and Mire-Sluis, A. R. (1997) Dev. Biol. Stand. 91, 79–88 10 Gaines Das, R. E. and Meager, A. (1995) Biologicals 23, 285–297 11 Sadick, M. D. et al. (1996) Anal. Biochem. 235, 207–214 12 Monteclaro, F. S., Hidenor, A. and Charo, I. F. (1997) Methods Enzymol. 288, 70–83 13 Mire-Sluis, A. R. (1997) Pharm. Sci. 3, 15–18 14 Mire-Sluis A. R., Gaines Das, R. and Padilla, A. (1998) J. Immunol. Methods 216, 103–116 15 Mire-Sluis, A. R., Gaines-Das, R. and Thorpe, R. (1995) J. Immunol. Methods 186, 161–164 16 Tsang, M. L. and Weatherbee, J. A. (1996) Pharmacol. Ther. 10, 55–62 17 Bird, C., Wadhwa, M. and Thorpe, R. (1991) Cytokine 3, 562–567 18 Jung, T. et al. (1998) J. Immunol. Methods 217, 41–50 19 Mire-Sluis, A. R. (1999) Biodrugs 11, 367–376 20 Ogata, A. et al. (1992) J. Immunol. Methods 148, 15–22 21 Grimshaw, C., Gleason, C., Chojnicki, E. and Young, J. (1997) J. Pharm. Biomed. Anal. 16, 605–612 22 Wong, R. L., Mytych, D., Jacobs, S., Bordens, R. and Swanson, S. J. (1997) J. Immunol. Methods 209, 1–15 23 Chapman, J. R. (1997) Protein and Peptide Analysis by Mass Spectrometry (1st edn), Humana Press 24 Jones, C., Mulloy, B. and Thomas, A. H. (1993) Spectroscopic Methods and Analyses (1st edn), Humana Press 25 Creighton, T. E. (1997) Protein Structure: A Practical Approach (2nd edn), IRL Press 26 Swadesh, J. (1997) HPLC: Practical and Industrial Applications (1st edn), CRC Press 27 Hancock, W. S. (1996) New Methods in Peptide Mapping for the Characterization of Proteins (1st edn), CRC Press 28 Burlinghame, A. L. and Carr, S. A. (1996) Mass Spectrometry in the Biological Sciences (1st edn), Humana Press 29 Green, M. K., Vestling, M. M., Johnston, M. V. and Larsen, B. S. (1998) Anal. Biochem. 260, 204–211 30 Mire-Sluis, A. R. (1993) Biologicals 21, 131–144 31 Gersten, D. M. (1996) Gel Electrophoresis: Proteins (1st edn), Wiley
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3 Pace, S. J. (1990) US Patent 4 908 112 4 Effenhauser, C. S., Paulus, A., Manz, A. and Widmer, H. M. (1994) Anal. Chem. 66, 2949–2953 5 Woolley, A. T. and Mathies, R. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11348–11352 6 Jacobson, S. C. and Ramsey, J. M. (1996) Anal. Chem. 68, 720–723 7 McCormick, R. M., Nelson, R. J., Alonso-Amigo, M. G., Benvegnu, D. J. and Hooper, H. H. (1997) Anal. Chem. 69, 2626–2630 8 Woolley, A. T. and Mathies, R. A. (1995) Anal. Chem. 67, 3676–3680 9 Schmalzing, D., Koutny, L., Adourian, A., Belgrader, P., Matsudaira, P. and Ehrlich, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10273–10278 10 Woolley, A. T., Hadley, D., Landre, P., deMello, A. J., Mathies, R. A. and Northrup, M. A. (1996) Anal. Chem. 68, 4081–4086 11 Woolley, A. T., Sensabaugh, G. F. and Mathies, R. A. (1997) Anal. Chem. 69, 2181–2186 12 Schmalzing, D., Adourian, A., Koutny, L., Ziaugra, L., Matsudaira, P. and Ehrlich, D. J. (1998) Anal. Chem. 70, 2303–2310 13 Hjerten, S. J. (1985) J. Chromatogr. 347, 191–198 14 Mathies, R. A. and Huang, X. C. (1992) Nature 359, 167–169 15 Ueno, T. and Yeung, E. S. (1994) Anal. Chem. 66, 1424–1431
Cytokines: from technology to therapeutics Anthony R. Mire-Sluis Cytokines are playing an ever-increasing role in the treatment of human disease. The characterization of these proteins plays a vital role in their development as useful therapeutic agents. Physicochemical techniques can produce information about the structure and composition of cytokine therapeutics but cannot yet predict their biological activity, for which biological assays are required. Because of the large number of techniques available and the variety of products requiring analysis, the tests used to characterize cytokine products must be both appropriate for the product and adequately controlled if the information they provide is to be of value.
ytokines and growth factors mediate a wide range of physiological processes, including haematopoiesis, immune responses, wound healing and general tissue maintenance1; for the purposes of this review, growth factors will be included in the term ‘cytokine’. As cytokines are involved in many physiological processes, it is not surprising that they are also involved in the pathogenesis of many diseases. Associated with this is their vast potential in replacement or modulatory therapy2. Recombinant DNA technology has resulted in the discovery of increasing numbers of proteins that have been categorized as either cytokines or growth factors. There are over 150 well-established cytokines and growth factors3, and many other proteins have been discovered through random
C
A. R. Mire-Sluis ([email protected]) is at the Division of Immunobiology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, UK EN6 3QG. TIBTECH AUGUST 1999 (VOL 17)
DNA screening that appear to have cytokine-like properties but whose true functions remain unidentified3. Widespread application of recombinant DNA technology within the biotechnology industry has dramatically increased the number of cytokines available for clinical evaluation (Table 1). New cytokines are being discovered, cloned and entered into clinical trials at such a rate that their structural properties and biological activities are often poorly understood during their development as therapeutic agents. Safety, efficacy and quality are major concerns for the success of any biological product. Safety, involving toxicity and possible infectious agents, is dealt with through well-documented procedures (regulatory guidelines available from the European Medicines Evaluation Agency website http://www.eudra.org/emea.html) but efficacy can only be evaluated through clinical trials4. Quality assessment needs to address a variety of issues including heterogeneity, consistency, potency, stability
0167-7799/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0167-7799(99)01330-X
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Table 1. Examples of cytokine and growth-factor therapeutics Agent
Product type(s)
Some therapeutic uses
Interferon a
Multicomponent, natural (leukocyte derived) Multicomponent, natural (lymphoblastoid derived) Recombinant (Escherichia coli-derived), nonglycosylated
Interferon b
Recombinant (E. coli-derived), nonglycosylated mutein (Cys-17-Ser) Recombinant (CHO-derived), glycosylated
Antiviral: Hepatitis B and C Immunomodulation: condyloma Antiangiogenic: Kaposi’s sarcoma Antitumour: hairy-cell leukaemia, chronic myelocytic leukaemia Immunomodulatory: multiple sclerosis Antiviral: post-interferon-a resistance in hepatitis
Insulin-like growth factor-1 Granulocyte-colonystimulating factor (G-CSF)
Recombinant (yeast-derived), nonglycosylated
Interleukin 2 Erythropoietin Platelet-derived growth factor BB
Recombinant (E. coli-derived), non-glycosylated, additional methionine Recombinant (CHO-derived), glycosylated Recombinant (E. coli-derived), nonglycosylated mutein (Cys-125-Ser) Recombinant (CHO-derived), glycosylated Recombinant (yeast-derived), glycosylated
and formulation. This involves critical scientific consideration of the many analytical techniques that can provide the necessary information to assure product quality. In general, there are three main groups of analytical tests for cytokines – biological assays, immunoassays and binding assays, and physicochemical techniques. Within each of these groups, there are several different assay systems. Individual assays have their own particular strengths and weaknesses, which are dependent on the type of material under test and the information that is required (Table 2). For example, in comparison with a non-glycosylated protein, a product that is glycosylated will require additional analytical tests to explore the sugar aspect of the protein. In addition, glycosylation causes proteins to perform differently on many of the standard analytical tests used, which have either to be adapted to take this into account or have the data they produce interpreted accordingly5. The purpose of this article is both to illustrate the various analytical techniques currently available to analyse biological products and to discuss the scientific rationales required to apply these tests to cytokines with different structural and biological characteristics. Biological assays Bioassays for cytokines can take several forms but they all rely on the ability of the protein to induce some measurable activity in cells or tissues6. It is now rare to measure the biological activity of a cytokine in vivo. The development of clonal cell lines that respond to cytokines is a significant improvement as a source of materials for bioassays. Cell lines (usually from murine or human cancers) can be dependent on a cytokine for growth and are often immortal, thus providing a single homogeneous source of cells that can be distributed from laboratory to laboratory7. A good example of this is the G-NFS-60 murine myeloid leukaemia cell line that is used to measure
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Neuroprotection: amyotrophic lateral sclerosis Immunostimulation: Immunoreconstitution after a bone-marrow transplant Reduce neutropaenia after chemotherapy Mobilization of peripheral-blood stem cells Antitumour: bladder carcinoma, melanoma Production of red cells for anaemia in haemodialysed patients Malignancy-associated anaemia Wound healing: diabetic foot ulcers
the cytokine granulocyte-colony-stimulating factor (G-CSF). G-CSF is used therapeutically to reconstitute the immune system after bone-marrow transplantation (Table 1) and has been highly successful as a biological product. However, prior to the development of G-CSF-responsive cell lines, the potency of G-CSF was determined using murine-bone-marrow-colony assays. These assays were time consuming (taking 5–7 days), technically laborious (often using colony counting as a read-out) and variable, owing to interanimal differences. Cell-line assays are much more rapid, with G-CSF bioassays taking only 24 h, and are much more reproducible. The advent of recombinant DNA technology has allowed the cloning of specific cytokine receptors and their expression on previously unresponsive cell lines. This can create a specific, responsive cell line for almost any cytokine without the need to screen a wide range of existing cell lines for cytokine responsiveness7. The receptor for the cytokine thrombopoietin (TPO) has been cloned into the TPO-unresponsive murinemyeloid-leukaemia cell line 32D to produce a cell line designated 32D/Mpl1, which proliferates in response to TPO and has been used to determine the potency of therapeutic products8. The use of murine cell lines to assay human cytokines is dependent on the species cross-reactivity of the cytokine to be tested, as not all cytokines can cross the species barrier1. The cellular response induced by cytokines that is utilized as a bioassay read-out can take a variety of forms but is most often the proliferation or inhibition of proliferation, the expression of cellular markers or enzymes, cytotoxicity or antiviral activity6. There has been some controversy amongst the regulatory agencies over whether the bioassay used to measure the potency of the product should be related to its clinical activity. For example, should interferon a (Table 1) be assayed for its antiviral activity or its antitumour activity, TIBTECH AUGUST 1999 (VOL 17)
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Table 2. Analytical techniques for the analysis of cytokines Procedure
Data provided/inferred Molecular size
Charge
Primary Secondary structure or tertiary structure
Quaternary structure
Purity
Potency
Timea (h)
Bioassays
2
2
111
1111
1111
1/2
11111
24–48
Binding assays Immunoassays Immunoblotting Receptor binding
2 111 2
2 1 2
1/2 11 11
1/2 111 111
1/2 111 111
2 2 2
2 2 1/2
8–24 24 5
Gel electrophoresis Isoelectric focusing Nonreducing Reducing
2 2 111
1111 2 2
111 11 111
1 111 2
1 111 2
111 111 111
2 2 2
36 24 24
HPLC Hydrophobic interaction Ion exchange Reverse phase Size exclusion
1 2 1 111
1 11111 1 2
111 111 1111 2
11 2 11 11
11 1 2 111
111 111 1111 111
2 2 2 2
4 4 4 4
2 11111 1
2 1 2
2 11111 11
1111 1/2 11111
111 2 2
11 2 1111 2 11 2
2 1 1
Structural assays Circular dichroism Mass spectrometry Nuclear-magnetic resonance
Abbreviations: 11111, extremely informative; 1, only a little useful information is provided; 1/2, can provide minimal information in an indirect manner (e.g. if an immunoassay can detect a protein, it suggests that the structural conformation of the antibody-recognition sites may be correct); 2, no useful data provided. aTime for the procedure from start to finish is approximate and depends on whether the various systems are already set up and, in the case of gel electrophoresis, on the gel size.
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Standard curve Dilution of test sample Linear portion of response curve Relative activity
Concentration of sample (reciprocal dilution)
trends in Biotechnology
Assay response
or both? One can argue that, if a product has only one clinical activity, its bioassay should attempt to measure it. However, because potency measurements are a quality issue and should not to be used to relate to dosing in the clinic (clinical trials establish this), then any functional activity could, in theory, be used4. Although the different bioassay systems have disadvantages, most often lack of specificity or nonspecific interference of sample matrix with the cells, refinement of the assay technique and cell maintenance has resulted in bioassays that are as accurate (if not more so) than many physicochemical techniques; that is, a bioassay is only as good as its design and execution. The design of any bioassay must take into account factors that introduce variability, and the analysis of bioassays must test for variability if results are to be statistically valid. Although the design of in vitro bioassays can take many forms, a single point assay is definitely not valid. A titration of the test material has to be made and compared with a titration of a reference material, with particular attention paid to comparisons of the linear portion of the dose–response curve9 (Fig. 1). In order to reduce the effects of position within microtitre-plate assays, randomization of the position of sample titration curves within plates is recommended, as is the inclusion of a standard reference preparation on each plate10. Carefully controlled bioassays are technically demanding, relying heavily on the competence of the staff
Figure 1 A diagrammatic example of the design of a parallel-line bioassay. Samples and reference preparations are diluted in series to produce a dose–response curve. The displacement between the linear portions of the dose–response curves is representative of the relative activity of the two preparations and is calculated using parallel-line statistical analysis7.
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carrying out the assays to dilute and to pipette cytokine solutions accurately and reproducibly. However, attempts to automate biossays have been particularly successful when such tasks have been taken over by robots. The cost of bioassays varies, depending on the amount and sophistication of hardware used. The development of in vitro bioassay techniques has led to the increased use of microtitre-plate-based assays using continuous cell lines to bioassay the majority of cytokines. However, for some growth factors and cytokines (particularly the chemokines), there is no suitable cell-line-based bioassay that is appropriate for reproducible routine use. In this case, the development of receptor-based ‘biochemical bioassays’ are being developed: the binding of some cytokines to their receptor on cell surfaces induces the rapid phosphorylation of the receptor on tyrosine residues; the cells are then lysed and an immunoassay used to detect the level of phosphotyrosine being produced11. Although these assays are relatively rapid and reproducible, they are only of value for cytokines for which simple cell-line-based bioassays do not exist, because they still require the culture of cells or cell lines and involve an additional immunoassay step. How receptor phosphorylation relates to overall biological activity also requires further investigation. Current assays for chemokines involve the chemotaxis of fresh blood cells (usually neutrophils or T cells) through nitrocellulose membranes. These assays are very technically demanding and produce highly variable results. However, the binding of chemokines to their receptors induces Ca2+ fluxes, the magnitude of which correlates directly with the quantity of chemokine present12. These assays are currently being refined as alternatives to chemotaxis assays. As different manufacturers’ products, even the same product from the same cell source, can possess very different specific activities, mass cannot be used as a measure of the functional activity of a biological material13. In addition, using mass as an indicator of drug content is not helpful in itself, as one cannot possibly weigh a supplied drug, particularly if it contains excipients (as many biotherapeutics do). Therefore, a unit has to be defined for such activity, for without a single reference preparation, units with different definitions can occur. This causes confusion, leading to substantial interlaboratory variation in estimates of potency. The concept of a single internationally available potency standard has been developed under the auspices of the World Health Organization and shown to be extremely valuable in reducing the laboratory variability in potency estimates and enhancing the comparability of clinical and research studies14. International units and standards are vital if the biological potency of any preparation is to be assessed. Immunological assays Immunoassays are assays that use the binding of antibodies to a ligand to detect specific interactions, resulting in quantitation of that ligand. Immunoassays can be highly specific, quick and simple to carry out and require little expensive machinery. However, immunoassays do not measure a biological function15 and they cannot predict biological activity, because subtle changes in structure impacting greatly on biological
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activity may not affect antibody binding. For example, the gross change in structural integrity caused by proteolytic cleavage would not be detected in an immunoassay if the antibody recognition sites were unaffected. Immunoassays can be extremely reproducible and are particularly useful for detecting cytokines in samples that contain other substances that might affect bioassay results, such as those found in serum. However, immunoassays can be affected by the constituents of biological samples (matrix effects), particularly when dealing with biological fluids such as serum, cerebrospinal fluid and urine, and immunoassays must be tested very carefully for such effects and any found must be taken into account. For example, the reference material used to calibrate an immunoassay should ideally be made up in the same type of fluid as the samples, unless it has been shown that the formulation of the standard and samples is not relevant to their comparison in the assay. Although immunoassays are highly specific, this can be a problem for their use16. The antibodies that make up an immunoassay are usually raised against a cytokine from a particular source [such as Escherichia coli or Chinese hamster ovary (CHO) cells] and are rarely raised against natural material. Therefore, it is almost impossible to predict how well such antibodies recognize naturally derived cytokines in biological samples. Few cytokines have been purified from natural sources in sufficient quantity to carry out comparative experiments, but there is evidence to show that some antibodies can recognize differences between E. coli, CHO and yeast-derived material, suggesting that differential recognition of natural materials might well occur17. Recent work has also illustrated the problem of cytokines measured in the presence of soluble cytokine receptors and binding proteins18. It is extremely difficult to assess the true levels of unbound and bound cytokine using an immunoassay because of its dependency on temperature, time and the relative concentrations of the cytokine and binding protein. As the antibodies used in immunoassays are effectively in competition for binding the cytokine, it appears that environmental factors that affect the affinity of any of the cytokine-binding molecules have the potential radically to alter the results of the immunoassay. Immunoassays are also often employed to detect the presence of host-cell proteins in products by raising antibodies against a lysate of whole host cell and using these in an immunoassay format. The validity of this practice is questionable because there is no guarantee that the antibodies produced this way will result in an immunoassay that recognizes any of the host-cell proteins that evade purification processes19. There have been several recent developments in immunoassay technology for the detection of cytokines, one example of which is an immunoassay format with improved sensitivity – dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)20. The DELFIA system is based on the theory of time-resolved fluoroimmunoassay using europium (Eu)-labelled antibodies. Eu emits fluoresecent light that is detected to a higher degree than the colour changes used in standard enzyme-linked immunosorbant assays (ELISAs). This results in an immunoassay that is 10–100 times more sensitive than current ELISAs. TIBTECH AUGUST 1999 (VOL 17)
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The use of electrochemiluminescence has allowed the sensitivity of immunoassays to be increased and has resulted in assays that appear to be less affected by matrix effects. Typically, these assays take the form of a cytokine bound to an antibody on a bead, which is then sandwiched to an antibody labelled with ruthenium (Ru). An electrical potential applied to this complex produces light, which can then be detected and quantified21. Recent biosensor developments allow the interaction of cytokines and cytokine-binding proteins (such as antibodies and soluble receptors) to be measured accurately, a process termed ‘biospecific interaction analysis’. A biosensor can be defined as an instrument that combines a biological recognition mechanism with a sensing device or transducer. One example of biosensor technology employs surface-plasmon resonance (SPR) to monitor biomolecular interactions. SPR is an optical technique that uses changes in the reflectance of light off a thin metal film to detect changes in the concentration of macromolecules in a surface layer of solution in contact with the metal film. The use of biosensors enables real time on and off rates to be calculated, in addition to much-more-detailed information on cytokine binding than can easily be produced using conventional techniques22. Physicochemical analysis There are many physicochemical techniques available for the analysis of cytokines (Table 2). The widespread use of mass spectrometry (MS) has had the most recent impact on protein characterization and produces extremely accurate data on the molecular mass (and hence amino acid content) of a protein. The technique involves ionizing a protein and measuring various parameters (e.g. time of flight and mass:charge ratios of the resulting ionized protein species) to estimate the mass of the original material. Two of the most popular methods to achieve ionization are matrix-assisted laser-desorption ionization (MALDI) and electrospray ionization (ESI)23. MS is claimed to have a mass resolution of 0.1% and an accuracy of 0.01%, and to be able to detect contaminants at 1%. However, ESI, in particular, requires sample desalting, which can affect the nature of the material being investigated, and both MALDI and ESI are not as yet particularly good at resolving complex mixtures of protein species24. In addition, not all protein species ionize equally and therefore might not be correctly represented. It must also be noted that, although MS provides valuable information about the amino acid content of proteins as a whole, it does not provide information about the actual sequence of the protein unless peptide mapping is used in addition. Other techniques, such as nuclear magnetic resonance (NMR) or circular dichroism (CD) can provide information about the higher-order structure of proteins25. NMR involves applying strong magnetic fields to proteins in solution and analysing the absorption or emission of electromagnetic radiation by the nuclei of the constituent atoms. NMR is an extremely powerful technique and can provide a highly complex ‘fingerprint’ of the secondary and tertiary structure of a protein but it requires milligram quantities of protein and is affected by the presence of sugars, organic solvents and detergents. TIBTECH AUGUST 1999 (VOL 17)
CD is an adsorption technique that detects changes in left and right circularly polarized ultraviolet (UV) light passing through a protein solution. Near-UV CD detects absorption by aromatic residues (chromophores) and disulphide linkages, as well as changes in tertiary structure. Far-UV CD shows changes in secondary structures (a helices and b-pleated sheets). CD provides a less complex profile of protein structure than NMR and is essentially a comparative technique, able to illustrate differences in protein structures, although the exact nature of those changes at a molecular level are more difficult to establish. Chromatographic techniques [e.g. high-performance liquid chromatography (HPLC)] are, taken together, probably the most informative of the various physicochemical techniques, as they are often able to resolve the majority of protein species in the sample, allowing further detailed characterization in association with other techniques26. Reverse-phase HPLC can detect 0.1% impurities under optimal conditions and works in the microgram range, but is denaturing to the protein. Subtle changes might not necessarily be detected within large proteins and unequal denaturation can cause problems in resolving power. Size-exclusion HPLC is not as resolving as reverse-phase HPLC, being able to detect only 0.5% aggregates, but is nondenaturing. Ion-exchange HPLC is able to detect the most subtle of changes, even single amino acid changes if that change alters the charge of the protein. HPLC techniques are most useful when used in a panel of tests, particularly in association with peptide mapping, when they allow most known differences between protein species to be detected27. In general, it appears that the more informative the physicochemical technique, the more complex the hardware (or, possibly, the exclusivity of use to a few laboratories) and therefore the greater the costs involved. The latest developments in physicochemical analysis involve the use of existing techniques in an automated sequence. For example, tandem MS involves the use of two mass spectrometers in sequence and gives a much greater analytical potential, with products produced by the first being selected and reanalysed by the second. HPLC systems can also be linked directly to mass spectrometers so that peptides separated by chromatography can be further characterized by MS in one automated process28. One of the most advanced developments in MS is the combination of ionization technology with Fouriertransform ion-cyclotron resonance (FTICR) mass spectrometry29. ESI has been combined with FTICR to produce a technique so sensitive that it can detect mass changes of 1 Da (the equivalent of a single hydrogen atom). Although this technology is far beyond that available for the majority of laboratories, it indicates the future trends and possibilities within this field. Which test to use and why? The primary aim of characterizing a protein product during its development as a therapeutic agent is to gain as much insight into its composition and activity as possible, as this information is essential to ensure the quality of the product. The quality of a biological product involves batch-to-batch consistency, appropriate potency and stability, and other factors4. As described in the previous sections, no single analytical procedure
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a
b
CytokineÐHSA aggregates
HSA
CytokineÐHSA adducts
Cytokine 10 mg
1.0 mg 10 mg 0.5 mg 5 mg 25 mg
Figure 2 Examples of how the choice of technique and sample handling affect the results of physicochemical analysis. (a) A silver stain of an SDS-PAGE gel of a cytokine product formulated in the presence of albumin, illustrating a wide range of protein bands that does not discriminate between species related to the cytokine, albumin, adducts or other contaminants present. (b) An immunoblot of a SDS-PAGE gel of the same product as in (a), illustrating only those protein species containing the cytokine, including cytokine–albumin adducts. The quantity of the product loaded is shown at the bottom of the blot and shows the dose-related detection of various bands.
can provide all the data required to ensure the quality of a biological product and so a range of tests is required to form the specifications for the product on its release. Although a panel of physicochemical tests can provide a great deal of information about the structure of protein components, impurities and so on, the biological assay is the only assay that can provide a measure of biological activity30. A bioassay is defined as a ‘functional’ assay, and no physicochemical test can measure function. However, for some peptide hormones that are less complex in structure than the majority of cytokines, physicochemical tests characterized over many years can now be used as surrogate correlates for biological activity. Examples of the type of information that can be produced by the various analytical techniques are provided in Table 2, but the following points must be considered if any technique used to characterize a protein is to provide useful data: assay selection; assay design; sample handling; validity testing; interpreting results; and presenting the findings. For example, sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) can provide data about the proteins within a product, separated nominally by mass. It must be noted, however, that not only do different proteins bind SDS to different extents but they do not all pass through a gel matrix similarly (with or without reducing agents), as differential retention of higher structure occurs. The use of stains to detect proteins within the gel is equally important. Silver stains are highly sensitive and are the most-often used in the characterization of protein products. However, not only is the technique used for silver staining highly laboratory specific but it also tends to vary in sensitivity from day to day31. The development of such staining patterns is dependent on factors including time, temperature, the nature of the gel and the age of materials used. The effects of these must be explored and controlled if such a technique is to provide valid information.
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As discussed earlier, not only must we be cautious when using any assay to make sure that the data are relevant but the assay must also be designed in light of the material it is testing. Some cytokine therapeutics are formulated with albumin and this impacts heavily on the types of test required to analyse the product – assays must be altered to accommodate the presence of albumin, and the problem of the existence of drug–albumin adducts also occurs. Silver staining of SDS-PAGE gels of albumin-containing products is virtually useless as it produces a wealth of bands that require immunoblotting to distinguish between the drug and albumin, and to pick up any adduct formation (Fig. 2). In fact, special tests have to be designed to measure adduct formation, so that suitable limits can be applied. The quantity of sample loaded plays a vital role in the results: one can load enough sample to see tens of protein bands or amounts so small as to see only one or two (Fig. 2b). This problem relates to almost any physicochemical test and seriously compromises their validity if one considers that any physicochemical test can be designed to provide as little or as much information as is required. Therefore, the decision of which test to use when analysing a product depends heavily on the nature of the product itself, and can involve the adaptation of standard tests to provide information specific to that product. For example, one must consider the analysis of heterodimeric protein products differently from monomeric products, in addition to other issues such as N-terminal heterogeneity, oxidation, methylation and aggregation. Thus, the current regulatory approach of case-by-case consideration of biologicals must also apply to the analysis of cytokine products, where no single set of rules would be appropriate for all products. When analysing cytokines, one must also consider the stage of manufacture at which a material is being tested, and for what purpose. For example, during pharmacokinetic analysis, the range of useful techniques is limited in many cases by the low amount of material to be measured and by the problems of detecting cytokines in serum. Physicochemical analysis is not appropriate for serum samples because of the amount of interfering substances, and one is limited to the immunological and biological detection methods already discussed. Such issues are also involved in the choice of test during the manufacturing process, where the product is becoming purer and contaminating materials are being removed, allowing for more sophisticated analysis as the process continues. The storage and handling of the material influences physicochemical analysis and has to be carefully considered (Fig. 3). Conclusion There are now many methods for the characterization of cytokines and growth factors that together can provide a great deal of information about the structure and composition of cytokine therapeutic products. No single test is able wholly to characterize a protein and so a range of tests is required. The selection of which tests to use depends heavily on the type of product and the information required. Once a test has been selected, it must be designed and validated for the test material. Even though increasingly sophisticated analytical techniques are continually being developed, it is important TIBTECH AUGUST 1999 (VOL 17)
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Figure 3 A size-exclusion HPLC separation of a cytokine drug substance that is stored frozen. Thawing the material at time zero and testing it after storage at 48C for the times indicated illustrates the formation of dimer on freezing and its gradual dissociation on thawing. The storage and handling of this material can be seen to influence the physicochemical analysis and so this has to be considered carefully. Abbreviation: A214 , absorbance at 214 nm.
for those concerned with the manufacture of clinical products to consider the true importance of any characterization in terms of clinical use and necessity. Requirements for highly extensive analysis could significantly slow the development of products and yet have no real impact on the value of a material as a therapeutic agent. It is thus extremely important that both industry and regulators have experience of handling biological products in a variety of tests using different conditions, not only to explore how a technique can characterize a protein but also to show how that protein can be used to validate the technique. Acknowledgments I wish to thank the participants in the The Biological Characterization and Assay of Cytokines and Growth Factors meeting, held at National Institute for Biological Standards and Control, 10–12 September 1997, for discussions that led to this article. Proceedings of this
meeting will appear in the Developments in Biological Standardisation series, Vol. 97. In addition, I am grateful to C. Dolman in the Immunobiology division at NIBSC for the HPLC data included in Fig. 3. References 1 Mire-Sluis, A. R. and Thorpe, R. (1998) Cytokines (1st edn), Academic Press 2 Oppenheim, J. J., Rossio, J. L. and Gearing, A. J. H. (1993) Clinical Applications of Cytokines: Role in Pathogenesis, Diagnosis and Therapy (1st edn), Oxford University Press 3 Thomson, A. (1998) The Cytokine Handbook (3rd edn), Academic Press 4 Mire-Sluis, A. R., Gaines-Das, R., Gerrard, T., Padilla, A. and Thorpe, R. (1996) Biologicals 24, 351–361 5 Townsend, R. R. and Hotchkiss, A. T. (1997) Techniques in Glycobiology (1st edn), Marcel Dekker 6 Wadhwa, M., Bird, C., Page, L., Mire-Sluis, A. R. and Thorpe, R. (1995) in Cytokines: A Practical Approach (Balkwill, F. R., ed.), pp. 357–391, IRL Press 7 Mire-Sluis, A. R. and Thorpe, R. (1998) J. Immunol. Methods 211, 199–210 8 Bartley, T. D. et al. (1994) Cell 77, 1117–1124 9 Thorpe, R., Wadhwa, M. and Mire-Sluis, A. R. (1997) Dev. Biol. Stand. 91, 79–88 10 Gaines Das, R. E. and Meager, A. (1995) Biologicals 23, 285–297 11 Sadick, M. D. et al. (1996) Anal. Biochem. 235, 207–214 12 Monteclaro, F. S., Hidenor, A. and Charo, I. F. (1997) Methods Enzymol. 288, 70–83 13 Mire-Sluis, A. R. (1997) Pharm. Sci. 3, 15–18 14 Mire-Sluis A. R., Gaines Das, R. and Padilla, A. (1998) J. Immunol. Methods 216, 103–116 15 Mire-Sluis, A. R., Gaines-Das, R. and Thorpe, R. (1995) J. Immunol. Methods 186, 161–164 16 Tsang, M. L. and Weatherbee, J. A. (1996) Pharmacol. Ther. 10, 55–62 17 Bird, C., Wadhwa, M. and Thorpe, R. (1991) Cytokine 3, 562–567 18 Jung, T. et al. (1998) J. Immunol. Methods 217, 41–50 19 Mire-Sluis, A. R. (1999) Biodrugs 11, 367–376 20 Ogata, A. et al. (1992) J. Immunol. Methods 148, 15–22 21 Grimshaw, C., Gleason, C., Chojnicki, E. and Young, J. (1997) J. Pharm. Biomed. Anal. 16, 605–612 22 Wong, R. L., Mytych, D., Jacobs, S., Bordens, R. and Swanson, S. J. (1997) J. Immunol. Methods 209, 1–15 23 Chapman, J. R. (1997) Protein and Peptide Analysis by Mass Spectrometry (1st edn), Humana Press 24 Jones, C., Mulloy, B. and Thomas, A. H. (1993) Spectroscopic Methods and Analyses (1st edn), Humana Press 25 Creighton, T. E. (1997) Protein Structure: A Practical Approach (2nd edn), IRL Press 26 Swadesh, J. (1997) HPLC: Practical and Industrial Applications (1st edn), CRC Press 27 Hancock, W. S. (1996) New Methods in Peptide Mapping for the Characterization of Proteins (1st edn), CRC Press 28 Burlinghame, A. L. and Carr, S. A. (1996) Mass Spectrometry in the Biological Sciences (1st edn), Humana Press 29 Green, M. K., Vestling, M. M., Johnston, M. V. and Larsen, B. S. (1998) Anal. Biochem. 260, 204–211 30 Mire-Sluis, A. R. (1993) Biologicals 21, 131–144 31 Gersten, D. M. (1996) Gel Electrophoresis: Proteins (1st edn), Wiley
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