Editorial Special Focus Issue: Bioanalysis of Large Molecules by LC–MS
For reprint orders, please contact [email protected]
Can LC–MS/MS and ligand-binding assays live in harmony for large-molecule bioanalysis? “The biological drug is not a molecule of exactly one defined mass, but is rather a complex mixture of a multitude of molecules, varying in masses, due to micro-heterogeneity.” Keywords: assay development • bioanalytics • biologic • free drug • large molecule • ligand-binding assay • mass spectrometry
Remit of bioanalytics The evaluation of the pharmacokinetic (PK) properties and the establishment of exposure and the pharmacokinetic/pharmacodynamics (PK/PD) relationship of drug candidates plays a crucial role in drug development. Bioanalytics provides quantitative data for PK calculations. The bioanalytical strategy for large-molecule drug development includes quantification and assessment not only of the drug candidate, but also of potential biomarkers, soluble target and anti-drug antibodies. Assessment of entities other than drug candidates is important for large molecules as they can have an impact on the exposure or interfere with analysis of the drug. What is the analyte? Small-molecule analytes are well characterized molecules with distinct molecular weights, and are ideal candidates for LC–MS/ MS-based bioanalysis. LC–MS/MS-based methods have also been applied successfully for the quantification of small- to mid-size peptides with well-defined molecular weights. In contrast, therapeutic proteins are highly complex analytes. The biological drug is not a molecule of exactly one defined mass, but is rather a complex mixture of a multitude of molecules, varying in masses, due to microheterogeneity. This is a consequence of the biological production process, reflecting multiple post-translational modifications that can result in thousands of isoforms of a given biologic [1] . These individual isoforms may exhibit striking differences in potency, clearance rate and other key biological parameters.
10.4155/BIO.14.144 © 2014 Future Science Ltd
An in-depth understanding of the structure– function relationship of individual isoforms of a given biologic is often missing, despite intensive drug characterization work. Certain structure–function platform knowledge is available for monoclonal antibodies [2] , but is very limited for novel complex biologics, such as bi-specifics, targeted cytokines and antibody–drug conjugates (ADCs). So, the key question for biologics quantification is: what is the relevant analyte to measure? For an appropriate bioanalytical strategy, discrimination between target-binding competent/active isoforms and inactive isoforms due to post-translational modification, misfolding, degradation or aggregation, is of key importance. Focusing on the quantification of target-binding competent drug enables the generation of significant data to describe exposure without the need for full understanding of individual isoforms. Differentiation between active and inactive isoforms, sometimes isobaric forms, poses a huge analytical challenge for massbased analytical approaches. Because of the huge challenge of measuring whole protein molecules, the top-down approach could not provide any significant solution for the bioanalysis of large proteins. To reduce the complexity of the biological analyte, much interest has been generated in the last 3–5 years to employ a ‘bottom-up’ approach; which takes a complex biological sample in serum or plasma and converts the sample to a complex mixture via enzymatic digestion. The bottom-up approach then monitors a small piece of the original protein as a signa-
Bioanalysis (2014) 6(13), 1735–1737
Apollon Papadimitriou Author for correspondence: Roche Pharma Research & Early Development, Roche Innovation Center Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany Tel.: +49 8856 60 2693; Fax: +49 8856 60 792693; apollon.papadimitriou@ roche.com
Surendra Bansal Roche Pharma Research & Early Development, Roche Innovation Center New York, Roche TCRC Inc., 430 East 29th St., New York, NY 10016, USA
Julia Heinrich Roche Pharma Research & Early Development, Roche Innovation Center Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany
Roland F Staack Roche Pharma Research & Early Development, Roche Innovation Center Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany
part of
ISSN 1757-6180
1735
Editorial Papadimitriou, Bansal, Heinrich & Staack ture or surrogate peptide representative of the parent, native protein. Having digested the whole protein, the complexity of the protein isoforms is also removed and one is further away from knowing the identity of the active or inactive analyte with any certainty.
“
Appropriate immunoaffinity extraction of target-binding competent molecules combined with structural information from MS analysis should provide a clearer picture of the analyte.
”
In contrast to the LC–MS approach, an appropriate ligand-binding assay (LBA), using a target-capture format, enables detection of all target-binding competent species, independent of the respective micro-heterogeneity of a given analyte. This requires the availability of adequate reagents and appropriate method development. Such an approach reduces the complexity and focuses quantification on target-binding competent isoforms, but in parallel limits the unique quantification of specific selected isoforms. Quantification of free or total drug? The complexity further increases by taking into consideration the potential impact of drug binding partners like soluble/shedded target, natural binding proteins, or anti-drug antibodies. Soluble binding partners might bind and consequently inactivate the drug. Therefore, differentiation between free and total drug concentrations is of special importance for PK/PD relationship for large molecules [3] . Whether a differentiation between free and total drug really needs to be implemented into the bioanalytical strategy is certainly product specific, and depends amongst other parameters on concentration of the soluble binding partners and the administered dose of the drug candidate [4] . The development of an immune response against the drug might also potentially result in neutralization of the administered drug and poses the bioanalytical challenge to prove ‘active exposure’ in samples where anti-drug antibodies are present. Total drug quantification in the presence of a prominent immune response and the absence of a suitable PD or safety biomarker makes it difficult to determine active drug exposure. This limitation is relevant for LC–MS/MS signature peptide approaches (especially without immuno-affinity extraction), as well as for generic LBAs that do not discriminate between active and total drug [5,6] . Even LC–MS/MS methods, using signature peptides derived from the binding regions (e.g., complementarity determining regions for antibody drugs), cannot deduce on binding the capacity/activity because information on 3D structure and complex formation is lost [7] . In this
1736
Bioanalysis (2014) 6(13)
case, additional complementary ex vivo potency assays may be required to characterize exposure. Hybrid immunoaffinity MS approaches – true synergies? As mentioned above, LBAs enable a selective quantification of target-binding competent drug, but provide limited information on isoforms. In contrast, MS methods without immunoaffinity extraction are limited to total drug quantification only. Top-down MS-based methods, when realized, could provide high-resolution data on individual isoforms. In the meantime, combination of LBAs and LC–MS technologies (hybrid methods) could synergistically provide better quantitative information. Appropriate immunoaffinity extraction of target-binding competent molecules combined with structural information from MS analysis should provide a clearer picture of the analyte. Current hybrid protocols, combine target-specific immune-extraction with signature-peptide-based LC–MS/MS quantification. Currently a full potential of hybrid protocols is not exploited. Due to the enzymatic digestion and subsequent peptide quantification of the immuno-purified analyte fraction, structural information is missed out. Since both approaches equally depend on appropriate reagents, the huge advantage of LC–MS/MS-based methods of short development timelines is lost without providing additional value compared with the LBA. In addition, the immunoaffinity extraction step requires careful evaluation, as it may bias results by disturbing the equilibrium between drug and potential binding partners during the extraction procedure. The free analyte QC concept, as applied for LBAs, may be helpful for appropriate method development [8] . Definition of a bioanalytical strategy The definition of the relevant analyte (e.g., free or total drug) during establishment of the BA strategy is crucial and requires a joint effort of multiple stakeholders from different disciplines [9] . The definition of the ‘right’ drug analyte is certainly highly dependent on the biological question that needs to be answered. Technical aspects such as availability of reagents or technologies are important parameters, as well as the project stage. The bioanalytical strategy may differ between discovery and development phases and might co-evolve together with the understanding of the structure–function relation of a given product. At early stages, specific reagents might be lacking and the development of a specific target capture assay might not be possible. In such cases, and if the information of total drug concentration is sufficient, the use of ‘generic’ LBAs [5,6] or LC–MS/MS signature peptide approaches without a specific extraction procedure,
future science group
Can LC–MS/MS & ligand-binding assays live in harmony for large-molecule bioanalysis?
could be applied. At later phases in development for the establishment of a PK/PD correlation, a more detailed characterization of active exposure may require more specific bioanalytical solutions. The selected strategies may vary between companies due to differing infrastructural background, such as, for example, availability of in-house facilities to quickly and cost-effectively provide appropriate reagents or adequate MS support. The selection of the bioanalytical strategy should be driven by the question that needs to be answered: total or active drug? In some cases even both entities may be needed to interpret PK in the context of safety and efficacy. Conclusion & outlook: parallel worlds, complementing methods, or hybrid? LC–MS/MS-based methods have evolved into alternative tools for the quantification of therapeutic proteins in biological matrices. Despite the fast development times they suffer from the lack of information on drug activity as they provide only total drug concentration, independent of its physico-chemical state and interaction with other binding partners [7,10] . Similar capabilities and limitations also apply to generic platform LBAs [5,6] , which do not require specific tool development but are equally limited on total drug information. So in cases where information on total drug is sufficient and no reagents for specific immunoassay are available, both methods may be equally suitable. In contrast, specific target-capture LBAs can provide information/quantification of target-binding competent/active drug concentrations, but require time-consuming development of specific reagents. The biological question should drive appropriate technology selection with the aim References
to provide relevant quantitative data with a reasonable effort under defined circumstances. Conducting LBA and MS-based assays in separated worlds, independent from each other, should be avoided. Rather, the strengths of both technologies should complement each other and lead to synergy. ‘Hybrid’ approaches, that is the use of immunoaffinity extraction procedures to enable sensitive and selective MS detection of specific isoforms, offers the power of combined forces. The full potential of hybrid protocols is not yet exploited. Since the current hybrid methods do not provide a clear benefit compared with LBAs, LBAs remain the gold standard for bioanalysis of large molecules. With the expected further development of MS methods in the future, hybrid approaches may uncover their full potential by providing a detailed structural picture of selected, immunopurified functional isoforms on the intact protein level, thus enabling quantification and structural characterization in parallel. Multifunctional biologics will pose a special challenge in this context. Ideally, we would be able to provide a complete quantitative and qualitative bioanalytical picture in a complex biologic mixture of analytes using appropriate combinations of technologies, including LBAs, LC–MS and also cell-based functional assays. Financial & competing interests disclosure All authors are employed by F Hoffmann-La Roche, Ltd. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. 6
Stubenrauch K, Wessels U, Essig U, Kowalewsky F, Vogel R, Heinrich J. Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic quantification of human Fab fragments in non-human serum samples including cynomolgus monkey samples. J. Pharm. Biomed. Anal. 72, 208–215 (2013).
1
Kozlowski S, Swann P. Current and future issues in the manufacturing and development of monoclonal antibodies. Adv. Drug Deliv. Rev. 58(5–6), 707–722 (2006).
2
Harris RJ. Heterogeneity of recombinant antibodies: linking structure to function. Dev. Biol. (Basel) 122, 117–127 (2005).
7
Roskos LK, Schneider A, Vainshtein I et al. PK–PD modeling of protein drugs: implications in assay development. Bioanalysis 3(6), 659–675 (2011).
van de Merbel NC, Bronsema KJ, Nemansky M. Protein quantification using LC–MS: can it make a difference? Bioanalysis 4(17), 2113–2116 (2012).
8
Staack RF, Jordan G, Dahl U, Heinrich J. Free analyte QC concept: a novel approach to prove correct quantification of free therapeutic protein drug/biomarker concentrations. Bioanalysis 6(4), 485–496 (2014).
9
Dudal S, Staack RF, Stoellner D et al. How the bioanalyst plays a key role in interdisciplinary project teams in the development of biotherapeutics – a reflection of the European Bioanalysis Forum. Bioanalysis 6(10), 1339–1348 (2014).
10
Kay RG, Roberts A. Bioanalysis of biotherapeutic proteins and peptides: immunological or MS approach? Bioanalysis 4(8), 857–860 (2012).
3
4
5
Staack RF, Jordan G, Heinrich J. Mathematical simulations for bioanalytical assay development: the (un-)necessity and (im-)possibility of free drug quantification. Bioanalysis 4(4), 381–395 (2012). Stubenrauch K, Wessels U, Lenz H. Evaluation of an immunoassay for human-specific quantitation of therapeutic antibodies in serum samples from non-human primates. J. Pharm. Biomed. Anal. 49(4), 1003–1008 (2009).
future science group
Editorial
www.future-science.com
1737
For reprint orders, please contact [email protected]
Can LC–MS/MS and ligand-binding assays live in harmony for large-molecule bioanalysis? “The biological drug is not a molecule of exactly one defined mass, but is rather a complex mixture of a multitude of molecules, varying in masses, due to micro-heterogeneity.” Keywords: assay development • bioanalytics • biologic • free drug • large molecule • ligand-binding assay • mass spectrometry
Remit of bioanalytics The evaluation of the pharmacokinetic (PK) properties and the establishment of exposure and the pharmacokinetic/pharmacodynamics (PK/PD) relationship of drug candidates plays a crucial role in drug development. Bioanalytics provides quantitative data for PK calculations. The bioanalytical strategy for large-molecule drug development includes quantification and assessment not only of the drug candidate, but also of potential biomarkers, soluble target and anti-drug antibodies. Assessment of entities other than drug candidates is important for large molecules as they can have an impact on the exposure or interfere with analysis of the drug. What is the analyte? Small-molecule analytes are well characterized molecules with distinct molecular weights, and are ideal candidates for LC–MS/ MS-based bioanalysis. LC–MS/MS-based methods have also been applied successfully for the quantification of small- to mid-size peptides with well-defined molecular weights. In contrast, therapeutic proteins are highly complex analytes. The biological drug is not a molecule of exactly one defined mass, but is rather a complex mixture of a multitude of molecules, varying in masses, due to microheterogeneity. This is a consequence of the biological production process, reflecting multiple post-translational modifications that can result in thousands of isoforms of a given biologic [1] . These individual isoforms may exhibit striking differences in potency, clearance rate and other key biological parameters.
10.4155/BIO.14.144 © 2014 Future Science Ltd
An in-depth understanding of the structure– function relationship of individual isoforms of a given biologic is often missing, despite intensive drug characterization work. Certain structure–function platform knowledge is available for monoclonal antibodies [2] , but is very limited for novel complex biologics, such as bi-specifics, targeted cytokines and antibody–drug conjugates (ADCs). So, the key question for biologics quantification is: what is the relevant analyte to measure? For an appropriate bioanalytical strategy, discrimination between target-binding competent/active isoforms and inactive isoforms due to post-translational modification, misfolding, degradation or aggregation, is of key importance. Focusing on the quantification of target-binding competent drug enables the generation of significant data to describe exposure without the need for full understanding of individual isoforms. Differentiation between active and inactive isoforms, sometimes isobaric forms, poses a huge analytical challenge for massbased analytical approaches. Because of the huge challenge of measuring whole protein molecules, the top-down approach could not provide any significant solution for the bioanalysis of large proteins. To reduce the complexity of the biological analyte, much interest has been generated in the last 3–5 years to employ a ‘bottom-up’ approach; which takes a complex biological sample in serum or plasma and converts the sample to a complex mixture via enzymatic digestion. The bottom-up approach then monitors a small piece of the original protein as a signa-
Bioanalysis (2014) 6(13), 1735–1737
Apollon Papadimitriou Author for correspondence: Roche Pharma Research & Early Development, Roche Innovation Center Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany Tel.: +49 8856 60 2693; Fax: +49 8856 60 792693; apollon.papadimitriou@ roche.com
Surendra Bansal Roche Pharma Research & Early Development, Roche Innovation Center New York, Roche TCRC Inc., 430 East 29th St., New York, NY 10016, USA
Julia Heinrich Roche Pharma Research & Early Development, Roche Innovation Center Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany
Roland F Staack Roche Pharma Research & Early Development, Roche Innovation Center Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany
part of
ISSN 1757-6180
1735
Editorial Papadimitriou, Bansal, Heinrich & Staack ture or surrogate peptide representative of the parent, native protein. Having digested the whole protein, the complexity of the protein isoforms is also removed and one is further away from knowing the identity of the active or inactive analyte with any certainty.
“
Appropriate immunoaffinity extraction of target-binding competent molecules combined with structural information from MS analysis should provide a clearer picture of the analyte.
”
In contrast to the LC–MS approach, an appropriate ligand-binding assay (LBA), using a target-capture format, enables detection of all target-binding competent species, independent of the respective micro-heterogeneity of a given analyte. This requires the availability of adequate reagents and appropriate method development. Such an approach reduces the complexity and focuses quantification on target-binding competent isoforms, but in parallel limits the unique quantification of specific selected isoforms. Quantification of free or total drug? The complexity further increases by taking into consideration the potential impact of drug binding partners like soluble/shedded target, natural binding proteins, or anti-drug antibodies. Soluble binding partners might bind and consequently inactivate the drug. Therefore, differentiation between free and total drug concentrations is of special importance for PK/PD relationship for large molecules [3] . Whether a differentiation between free and total drug really needs to be implemented into the bioanalytical strategy is certainly product specific, and depends amongst other parameters on concentration of the soluble binding partners and the administered dose of the drug candidate [4] . The development of an immune response against the drug might also potentially result in neutralization of the administered drug and poses the bioanalytical challenge to prove ‘active exposure’ in samples where anti-drug antibodies are present. Total drug quantification in the presence of a prominent immune response and the absence of a suitable PD or safety biomarker makes it difficult to determine active drug exposure. This limitation is relevant for LC–MS/MS signature peptide approaches (especially without immuno-affinity extraction), as well as for generic LBAs that do not discriminate between active and total drug [5,6] . Even LC–MS/MS methods, using signature peptides derived from the binding regions (e.g., complementarity determining regions for antibody drugs), cannot deduce on binding the capacity/activity because information on 3D structure and complex formation is lost [7] . In this
1736
Bioanalysis (2014) 6(13)
case, additional complementary ex vivo potency assays may be required to characterize exposure. Hybrid immunoaffinity MS approaches – true synergies? As mentioned above, LBAs enable a selective quantification of target-binding competent drug, but provide limited information on isoforms. In contrast, MS methods without immunoaffinity extraction are limited to total drug quantification only. Top-down MS-based methods, when realized, could provide high-resolution data on individual isoforms. In the meantime, combination of LBAs and LC–MS technologies (hybrid methods) could synergistically provide better quantitative information. Appropriate immunoaffinity extraction of target-binding competent molecules combined with structural information from MS analysis should provide a clearer picture of the analyte. Current hybrid protocols, combine target-specific immune-extraction with signature-peptide-based LC–MS/MS quantification. Currently a full potential of hybrid protocols is not exploited. Due to the enzymatic digestion and subsequent peptide quantification of the immuno-purified analyte fraction, structural information is missed out. Since both approaches equally depend on appropriate reagents, the huge advantage of LC–MS/MS-based methods of short development timelines is lost without providing additional value compared with the LBA. In addition, the immunoaffinity extraction step requires careful evaluation, as it may bias results by disturbing the equilibrium between drug and potential binding partners during the extraction procedure. The free analyte QC concept, as applied for LBAs, may be helpful for appropriate method development [8] . Definition of a bioanalytical strategy The definition of the relevant analyte (e.g., free or total drug) during establishment of the BA strategy is crucial and requires a joint effort of multiple stakeholders from different disciplines [9] . The definition of the ‘right’ drug analyte is certainly highly dependent on the biological question that needs to be answered. Technical aspects such as availability of reagents or technologies are important parameters, as well as the project stage. The bioanalytical strategy may differ between discovery and development phases and might co-evolve together with the understanding of the structure–function relation of a given product. At early stages, specific reagents might be lacking and the development of a specific target capture assay might not be possible. In such cases, and if the information of total drug concentration is sufficient, the use of ‘generic’ LBAs [5,6] or LC–MS/MS signature peptide approaches without a specific extraction procedure,
future science group
Can LC–MS/MS & ligand-binding assays live in harmony for large-molecule bioanalysis?
could be applied. At later phases in development for the establishment of a PK/PD correlation, a more detailed characterization of active exposure may require more specific bioanalytical solutions. The selected strategies may vary between companies due to differing infrastructural background, such as, for example, availability of in-house facilities to quickly and cost-effectively provide appropriate reagents or adequate MS support. The selection of the bioanalytical strategy should be driven by the question that needs to be answered: total or active drug? In some cases even both entities may be needed to interpret PK in the context of safety and efficacy. Conclusion & outlook: parallel worlds, complementing methods, or hybrid? LC–MS/MS-based methods have evolved into alternative tools for the quantification of therapeutic proteins in biological matrices. Despite the fast development times they suffer from the lack of information on drug activity as they provide only total drug concentration, independent of its physico-chemical state and interaction with other binding partners [7,10] . Similar capabilities and limitations also apply to generic platform LBAs [5,6] , which do not require specific tool development but are equally limited on total drug information. So in cases where information on total drug is sufficient and no reagents for specific immunoassay are available, both methods may be equally suitable. In contrast, specific target-capture LBAs can provide information/quantification of target-binding competent/active drug concentrations, but require time-consuming development of specific reagents. The biological question should drive appropriate technology selection with the aim References
to provide relevant quantitative data with a reasonable effort under defined circumstances. Conducting LBA and MS-based assays in separated worlds, independent from each other, should be avoided. Rather, the strengths of both technologies should complement each other and lead to synergy. ‘Hybrid’ approaches, that is the use of immunoaffinity extraction procedures to enable sensitive and selective MS detection of specific isoforms, offers the power of combined forces. The full potential of hybrid protocols is not yet exploited. Since the current hybrid methods do not provide a clear benefit compared with LBAs, LBAs remain the gold standard for bioanalysis of large molecules. With the expected further development of MS methods in the future, hybrid approaches may uncover their full potential by providing a detailed structural picture of selected, immunopurified functional isoforms on the intact protein level, thus enabling quantification and structural characterization in parallel. Multifunctional biologics will pose a special challenge in this context. Ideally, we would be able to provide a complete quantitative and qualitative bioanalytical picture in a complex biologic mixture of analytes using appropriate combinations of technologies, including LBAs, LC–MS and also cell-based functional assays. Financial & competing interests disclosure All authors are employed by F Hoffmann-La Roche, Ltd. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. 6
Stubenrauch K, Wessels U, Essig U, Kowalewsky F, Vogel R, Heinrich J. Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic quantification of human Fab fragments in non-human serum samples including cynomolgus monkey samples. J. Pharm. Biomed. Anal. 72, 208–215 (2013).
1
Kozlowski S, Swann P. Current and future issues in the manufacturing and development of monoclonal antibodies. Adv. Drug Deliv. Rev. 58(5–6), 707–722 (2006).
2
Harris RJ. Heterogeneity of recombinant antibodies: linking structure to function. Dev. Biol. (Basel) 122, 117–127 (2005).
7
Roskos LK, Schneider A, Vainshtein I et al. PK–PD modeling of protein drugs: implications in assay development. Bioanalysis 3(6), 659–675 (2011).
van de Merbel NC, Bronsema KJ, Nemansky M. Protein quantification using LC–MS: can it make a difference? Bioanalysis 4(17), 2113–2116 (2012).
8
Staack RF, Jordan G, Dahl U, Heinrich J. Free analyte QC concept: a novel approach to prove correct quantification of free therapeutic protein drug/biomarker concentrations. Bioanalysis 6(4), 485–496 (2014).
9
Dudal S, Staack RF, Stoellner D et al. How the bioanalyst plays a key role in interdisciplinary project teams in the development of biotherapeutics – a reflection of the European Bioanalysis Forum. Bioanalysis 6(10), 1339–1348 (2014).
10
Kay RG, Roberts A. Bioanalysis of biotherapeutic proteins and peptides: immunological or MS approach? Bioanalysis 4(8), 857–860 (2012).
3
4
5
Staack RF, Jordan G, Heinrich J. Mathematical simulations for bioanalytical assay development: the (un-)necessity and (im-)possibility of free drug quantification. Bioanalysis 4(4), 381–395 (2012). Stubenrauch K, Wessels U, Lenz H. Evaluation of an immunoassay for human-specific quantitation of therapeutic antibodies in serum samples from non-human primates. J. Pharm. Biomed. Anal. 49(4), 1003–1008 (2009).
future science group
Editorial
www.future-science.com
1737
