Commentary For reprint orders, please contact [email protected]
The importance of partnership when outsourcing exploratory bioanalysis “Trust is the foundation of a solid partnership and for these relationships [client–CRO] to be successful one can never have too much trust (or information).” Keywords: contract research organization n CRO n discovery bioanalysis n nonregulatory outsourcing n partnership
Outsourcing of bioanalytical studies is not new. CROs have been offering these services for well over 30 years, and in drug development environments where regulatory standards are required [1,2] it has often been the preferred choice for smaller organizations who lack the internal capabilities and expertise. Mid to large pharmaceutical and healthcare companies also choose to partner with CROs to support their drug development activities as the need to meet portfolio demands through flexible resourcing limits the volume of work that can be supported in-house [3]. Outsourcing of drug discovery bioanalysis is not new either. The need for skilled individuals, ever reducing budgets and adaptable support has meant that companies have also relied on CROs to support the bioanalytical elements of their exploratory projects. What can be considered more recent is the evolution in drug discovery from in vivo PK screening approaches, through to more definitive PK, PK/PD and disposition studies. Ethical, fiscal and scientific pressures have meant that output from exploratory studies not only needs to generate standard PK parameters – such as clearance, AUC, bioavailability and volume of distribution – but also tissue distribution, metabolic fate and soluble biomarker measurements. This step change in early-stage project requirements has meant a shift in how CROs respond to Clients’ needs. High-throughput, generic bioanalytical methods still have a place in discovery bioanalysis; however, the need for skilled bioanalysts to determine if this approach is appropriate (and if not, how to best move forward), has meant CROs looking to align bioanalysts in support of Clients’ exploratory needs. While this has made the support of exploratory studies less prone to technical error (or misinterpretation due to inappropriate analytics),
nonregulatory bioanalysis is still fraught with complications that can easily misguide a project. In a lot of these instances, lack of information is the root cause of the problem, either through nonrelease of structure or nonsupply of relevant information to help develop appropriate analytics. Withholding compound structure is often driven by either Clients’ uneasiness with information being distributed in the absence of any protective intellectual property, or, if organisations are large enough, the companies’ legal department simply prohibiting it. Nonsupply of relevant information (i.e., metabolic fate) generally does not suffer from the same legal complication, but rather is usually an oversight due to supporting data being generated by a different department within the same CRO, a different CRO or even a different individual responsible for outsourcing that particular piece of work. However, in any of these situations, lack of this information can severely limit the CROs’ ability to develop an appropriate bioanalytical method. As reputable CROs provide their services within confidentiality disclosure agreements, nondisclosure agreements or material service agreements, supply of project-related material, which can aid in the bioanalytical support, is easily achievable. To exemplify the impact of withholding pertinent information from the supporting CRO, two case studies are provided here. In both cases, neither structure was supplied; however, the major impact to the projects were from nonsupply of study-related material and as such could have been avoided.
10.4155/BIO.14.10 © 2014 Future Science Ltd
Bioanalysis (2014) 6(6), 733–736
Graeme T Clark Cyprotex, 15 Beech Lane, Macclesfield, Cheshire, SK10 2DR, UK Tel.: +44 01625 505114 Fax: +44 01625 505199 [email protected]
Case study 1: absence of metabolic fate data In this example, Client A’s project had been through the hit-to-lead stage of discovery and ISSN 1757-6180
733
Commentary |
Clark was in lead optimization. The lead compound exhibited good PK characteristics and Client A had initiated a PK/PD study to validate their in vivo model. Unfortunately, a PK/PD disconnect was encountered where systemic exposure was deemed high enough to illicit a PD effect, but none was observed. As no information had been supplied (structure or supporting in vitro assays), the only option available to the CRO was to perform the bioanalysis using generic methodologies. While spiked samples performed adequately, the CRO observed a subtly different peak shape in the incurred samples (slight shouldering on the lead edge of the chromatographic peak). This observation was raised by the CRO to Client A; however, they deemed the results acceptable as they correlated well with previous PK studies. Why then was this disconnect observed?
“Outsourcing discovery bioanalysis has suffered in the past from somewhat of a ‘throw it over the wall’ relationship between Client and CRO. ” Prior to the in vivo studies, Client A arranged for an in vitro rat hepatocyte metabolite identification assay to be performed. Again, the structure had not been disclosed as Client A was merely looking to identify the major route of clearance, any ‘unknown’ metabolites (i.e., due to dealkylation) could not have been identified, merely reported as atomic mass unit (amu) loss. The read out from the metabolite identification study, however, implied that the clearance was driven via Phase II conjugation; in particular two separate direct glucuronides were identified as the major metabolites (and confirmed by diagnostic loss of 176 amu [4]). As is standard with metabolite identification, much longer run times are applied in order to maximize the chances of chromatographically separating any metabolites (especially important if more than one isobaric metabolite is formed). These run times are typically 10–15 min in nature, compared with 1.0–2.0 min for bioanalysis, and tend to be developed such that the parent compound will elute at 70–80% organic modifier on the gradient profile. Under bespoke conditions the two identified glucuronides only eluted approximately 2.0 min before the parent compound, and as such were unlikely to chromatographically resolve from the parent under the much faster generic, bioanalytical method. 734
Bioanalysis (2014) 6(6)
Due to the observed PK/PD disconnect (and after the metabolite identification information had been received) the original generic HPLC conditions were modified from 5 to 95% organic modifier over a 2-min gradient to 5 to 60% organic modifier over a 3-min gradient, and the incurred samples were reanalyzed using this bespoke methodology. This improved methodology demonstrated that the chromatographic shoulder effect originally observed in the incurred samples was due to co-elution of the two glucuronides, which were appearing in the parent compound’s SRM channel. Phase II conjugates, glucuronides in particular, are known to break down in-source back to the parent compound [5,6], and as such can adversely affect quantification. Once a bespoke HPLC method had been developed they could be easily be resolved. Indeed the bespoke methodology demonstrated that parent compound was barely detected after 2 h; however, the major glucuronide was still present and quantifiable after 8 h. The coelution of the glucuronide(s) under the original generic HPLC conditions would have appeared to have been parent compound and quantified as such. This ‘mis-quantification’ explained the observed PK/PD disconnect as what the client thought was systemic exposure of compound was in fact the Phase II glucuronide conjugate, which did not illicit a pharmacological response. This root cause of this problem had been determined without the need for the client to release the structure (although it would have been extremely useful!); it was the supporting in vitro metabolite identification data that helped solve the issues and as a result of ineffective communication from Client A, the project’s lead compound had to be abandoned due to poor PK characteristics. Case study 2: absence of formulation data To reduce costs and animal utilization, Client B applied the technique of cassette dosing PK screening [7] during the hit-to-lead stage of their exploratory projects. Four compounds were cassetted per dose and each compound presented a different series of compounds. Client B did not release any structures; however, they did agree to up-front method development in order to: develop the optimum chromatography for a cassette of four compound and ensure the proposed formulation would not introduce any additional matrix effects. A recognised phenomenon in drug discovery when generic, future science group
The importance of partnership when outsourcing exploratory bioanalysis nonspecific extraction methods are applied, that is, protein precipitation [8]. In addition, Client B also provided data from an in vitro rat microsome assay that was performed to double check that there would no SRM crosstalk from common Phase I biotransformations. The study was completed successfully and the ‘best’ chemical series was determined and taken on to lead optimization and then into a PK/PD study. Upon analysis, although another PK/PD disconnect occurred, this time a PD effect was observed, but with limited systemic exposure. Client B asked for samples to be re-analyzed (for confirmation); however, due to low sample volume, certain incurred samples required diluting with control plasma (between one in ten and one in 50 depending on remaining sample volume) before they could be extracted. The reanalyzed samples returned much higher values than the initial analysis (after correction for dilution), which raised concern with Client B that the original concentration was correct and that an active Phase II conjugate had been formed and subsequently degraded back to parent upon storage at approximately -20°C. As a result Client B initiated an in vitro rat hepatocyte study with metabolite identification to determine what Phase II conjugates were being formed, however, no Phase II conjugates were observed. So why had the reanalyzed samples generated higher concentrations that the original analysis? After a great deal of discussion, Client B informed the bioanalytical CRO that the in-life portion of the PK/PD study had been performed at a different CRO than the original cassette PK study. Upon reviewing the in-life protocol it became apparent that a different formulation had been used in the PK/PD study compared with that which had been assessed in the original method development. To test the hypothesis that it was the different formulation affecting the quantification, the remaining samples were pooled and split into two aliquots. Aliquot 1 was analyzed neat, diluted with control plasma one in ten, one in 20 and one in 50 to provide a background response for the parent compound in the pooled sample. Aliquot 2 however, was spiked with an additional 500 ng/ml of parent compound and then analyzed neat and diluted with control plasma one in ten, one in 20 and one in 50. Comparison of the two sets of data indicated that the presence of the formulation introduced a significant ion suppression effect (80–90%) that was not removed until samples had been diluted at least one in 20. future science group
| Commentary
In this example, the lack of communication surrounding the change in formulation proved expensive to the project. Not only did the PK/PD study need to be repeated in order to obtain the correct systemic exposure, but an in vitro rat hepatocyte metabolite identification study had also been performed as a result of chasing a ‘red herring’. Should all the information been made available to the CRO performing the bioanalysis then a change in formation would have been flagged as a potential issue before any of the incurred samples had been consumed. Conclusion Outsourcing discovery bioanalysis has suffered in the past from somewhat of a ‘throw it over the wall’ relationship between Client and CRO. Things have improved greatly over the past few years, but presented here are two examples of how withholding information from the CRO can impact discovery projects not only in the progression an inappropriate candidate, but also in performing unnecessary assays. In these and other cases, projects not only suffer from delayed timelines, but poor communication also introduces additional costs to already tight discovery budgets. Although it is easy to try and lay the blame of this poor communication at the feet of the Clients, it is equally as important for CROs to ask for this information. Clients’ scientific expertise in bioanalytical sciences can range from highly experienced through to virtually none, and as such some Clients rely on CROs to act in more of a consultative capacity and ask the correct questions. Certain elements of a project, such as structure, will always be challenging to release for reasons mentioned previously; however, supporting data that can be aid in discovery bioanalysis support can be of benefit and explaining to the Client why this information is necessary builds trust between the two organizations. Trust is the foundation of a solid partnership and for these relationships to be successful one can never have too much trust (or information). Financial & competing interests disclosure The author has no 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. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. www.future-science.com
735
Commentary |
Clark
References European Medicines Agency, Committee for Medicinal Products for Human Use. Guideline on Bioanalytical Method Validation. European Medicines Agency, London, UK (2012).
5
2
US FDA. Guidance for Industry: Bioanalytical Method Validation. FDA, Silver Springs, MD, USA(2001).
6
3
Life Science Leader. www.lifescienceleader.com/doc/trends-instrategic-outsourcing-changes-in-partnerselection-0001
Yan Z, Caldwell GW, Jones WJ, Masucci JA. Cone voltage induced in-source dissociation of glucuronides in electrospray and implications in biological analyses. Rapid Commun. Mass Spectrom. 17(13), 1433–1442 (2003).
7
4
Levsen K, Schiebel HM, Behnke B et al. Structure elucidation of Phase II metabolites by tandem mass spectrometry: an overview. J. Chromatogr. A 1067(1–2), 55–72 (2005).
Nagilla R, Nord M, Mcatee JJ, Jolivette LJ. Cassette dosing for pharmacokinetic screening in drug discovery: comparison of clearance, volume of distribution, half-life, mean residence time, and oral bioavailability
1
736
Mei H, Morrison RA. Using Mass Spectrometry for Drug Metabolism Studies. CRC Press, Boca Raton, FL, USA, 59–98 (2009).
Bioanalysis (2014) 6(6)
obtained by cassette and discrete dosing in rats. J. Pharm. Sci. 100(9), 3862–3874 (2011). 8
Larger PJ, Breda M, Fraier D, Hughes H, James CA. Ion-suppression effects in liquid chromatography–tandem mass spectrometry due to a formulation agent, a case study in drug discovery bioanalysis. J. Pharm. Biomed. Anal. 39(1–2), 206–216 (2005).
future science group
The importance of partnership when outsourcing exploratory bioanalysis “Trust is the foundation of a solid partnership and for these relationships [client–CRO] to be successful one can never have too much trust (or information).” Keywords: contract research organization n CRO n discovery bioanalysis n nonregulatory outsourcing n partnership
Outsourcing of bioanalytical studies is not new. CROs have been offering these services for well over 30 years, and in drug development environments where regulatory standards are required [1,2] it has often been the preferred choice for smaller organizations who lack the internal capabilities and expertise. Mid to large pharmaceutical and healthcare companies also choose to partner with CROs to support their drug development activities as the need to meet portfolio demands through flexible resourcing limits the volume of work that can be supported in-house [3]. Outsourcing of drug discovery bioanalysis is not new either. The need for skilled individuals, ever reducing budgets and adaptable support has meant that companies have also relied on CROs to support the bioanalytical elements of their exploratory projects. What can be considered more recent is the evolution in drug discovery from in vivo PK screening approaches, through to more definitive PK, PK/PD and disposition studies. Ethical, fiscal and scientific pressures have meant that output from exploratory studies not only needs to generate standard PK parameters – such as clearance, AUC, bioavailability and volume of distribution – but also tissue distribution, metabolic fate and soluble biomarker measurements. This step change in early-stage project requirements has meant a shift in how CROs respond to Clients’ needs. High-throughput, generic bioanalytical methods still have a place in discovery bioanalysis; however, the need for skilled bioanalysts to determine if this approach is appropriate (and if not, how to best move forward), has meant CROs looking to align bioanalysts in support of Clients’ exploratory needs. While this has made the support of exploratory studies less prone to technical error (or misinterpretation due to inappropriate analytics),
nonregulatory bioanalysis is still fraught with complications that can easily misguide a project. In a lot of these instances, lack of information is the root cause of the problem, either through nonrelease of structure or nonsupply of relevant information to help develop appropriate analytics. Withholding compound structure is often driven by either Clients’ uneasiness with information being distributed in the absence of any protective intellectual property, or, if organisations are large enough, the companies’ legal department simply prohibiting it. Nonsupply of relevant information (i.e., metabolic fate) generally does not suffer from the same legal complication, but rather is usually an oversight due to supporting data being generated by a different department within the same CRO, a different CRO or even a different individual responsible for outsourcing that particular piece of work. However, in any of these situations, lack of this information can severely limit the CROs’ ability to develop an appropriate bioanalytical method. As reputable CROs provide their services within confidentiality disclosure agreements, nondisclosure agreements or material service agreements, supply of project-related material, which can aid in the bioanalytical support, is easily achievable. To exemplify the impact of withholding pertinent information from the supporting CRO, two case studies are provided here. In both cases, neither structure was supplied; however, the major impact to the projects were from nonsupply of study-related material and as such could have been avoided.
10.4155/BIO.14.10 © 2014 Future Science Ltd
Bioanalysis (2014) 6(6), 733–736
Graeme T Clark Cyprotex, 15 Beech Lane, Macclesfield, Cheshire, SK10 2DR, UK Tel.: +44 01625 505114 Fax: +44 01625 505199 [email protected]
Case study 1: absence of metabolic fate data In this example, Client A’s project had been through the hit-to-lead stage of discovery and ISSN 1757-6180
733
Commentary |
Clark was in lead optimization. The lead compound exhibited good PK characteristics and Client A had initiated a PK/PD study to validate their in vivo model. Unfortunately, a PK/PD disconnect was encountered where systemic exposure was deemed high enough to illicit a PD effect, but none was observed. As no information had been supplied (structure or supporting in vitro assays), the only option available to the CRO was to perform the bioanalysis using generic methodologies. While spiked samples performed adequately, the CRO observed a subtly different peak shape in the incurred samples (slight shouldering on the lead edge of the chromatographic peak). This observation was raised by the CRO to Client A; however, they deemed the results acceptable as they correlated well with previous PK studies. Why then was this disconnect observed?
“Outsourcing discovery bioanalysis has suffered in the past from somewhat of a ‘throw it over the wall’ relationship between Client and CRO. ” Prior to the in vivo studies, Client A arranged for an in vitro rat hepatocyte metabolite identification assay to be performed. Again, the structure had not been disclosed as Client A was merely looking to identify the major route of clearance, any ‘unknown’ metabolites (i.e., due to dealkylation) could not have been identified, merely reported as atomic mass unit (amu) loss. The read out from the metabolite identification study, however, implied that the clearance was driven via Phase II conjugation; in particular two separate direct glucuronides were identified as the major metabolites (and confirmed by diagnostic loss of 176 amu [4]). As is standard with metabolite identification, much longer run times are applied in order to maximize the chances of chromatographically separating any metabolites (especially important if more than one isobaric metabolite is formed). These run times are typically 10–15 min in nature, compared with 1.0–2.0 min for bioanalysis, and tend to be developed such that the parent compound will elute at 70–80% organic modifier on the gradient profile. Under bespoke conditions the two identified glucuronides only eluted approximately 2.0 min before the parent compound, and as such were unlikely to chromatographically resolve from the parent under the much faster generic, bioanalytical method. 734
Bioanalysis (2014) 6(6)
Due to the observed PK/PD disconnect (and after the metabolite identification information had been received) the original generic HPLC conditions were modified from 5 to 95% organic modifier over a 2-min gradient to 5 to 60% organic modifier over a 3-min gradient, and the incurred samples were reanalyzed using this bespoke methodology. This improved methodology demonstrated that the chromatographic shoulder effect originally observed in the incurred samples was due to co-elution of the two glucuronides, which were appearing in the parent compound’s SRM channel. Phase II conjugates, glucuronides in particular, are known to break down in-source back to the parent compound [5,6], and as such can adversely affect quantification. Once a bespoke HPLC method had been developed they could be easily be resolved. Indeed the bespoke methodology demonstrated that parent compound was barely detected after 2 h; however, the major glucuronide was still present and quantifiable after 8 h. The coelution of the glucuronide(s) under the original generic HPLC conditions would have appeared to have been parent compound and quantified as such. This ‘mis-quantification’ explained the observed PK/PD disconnect as what the client thought was systemic exposure of compound was in fact the Phase II glucuronide conjugate, which did not illicit a pharmacological response. This root cause of this problem had been determined without the need for the client to release the structure (although it would have been extremely useful!); it was the supporting in vitro metabolite identification data that helped solve the issues and as a result of ineffective communication from Client A, the project’s lead compound had to be abandoned due to poor PK characteristics. Case study 2: absence of formulation data To reduce costs and animal utilization, Client B applied the technique of cassette dosing PK screening [7] during the hit-to-lead stage of their exploratory projects. Four compounds were cassetted per dose and each compound presented a different series of compounds. Client B did not release any structures; however, they did agree to up-front method development in order to: develop the optimum chromatography for a cassette of four compound and ensure the proposed formulation would not introduce any additional matrix effects. A recognised phenomenon in drug discovery when generic, future science group
The importance of partnership when outsourcing exploratory bioanalysis nonspecific extraction methods are applied, that is, protein precipitation [8]. In addition, Client B also provided data from an in vitro rat microsome assay that was performed to double check that there would no SRM crosstalk from common Phase I biotransformations. The study was completed successfully and the ‘best’ chemical series was determined and taken on to lead optimization and then into a PK/PD study. Upon analysis, although another PK/PD disconnect occurred, this time a PD effect was observed, but with limited systemic exposure. Client B asked for samples to be re-analyzed (for confirmation); however, due to low sample volume, certain incurred samples required diluting with control plasma (between one in ten and one in 50 depending on remaining sample volume) before they could be extracted. The reanalyzed samples returned much higher values than the initial analysis (after correction for dilution), which raised concern with Client B that the original concentration was correct and that an active Phase II conjugate had been formed and subsequently degraded back to parent upon storage at approximately -20°C. As a result Client B initiated an in vitro rat hepatocyte study with metabolite identification to determine what Phase II conjugates were being formed, however, no Phase II conjugates were observed. So why had the reanalyzed samples generated higher concentrations that the original analysis? After a great deal of discussion, Client B informed the bioanalytical CRO that the in-life portion of the PK/PD study had been performed at a different CRO than the original cassette PK study. Upon reviewing the in-life protocol it became apparent that a different formulation had been used in the PK/PD study compared with that which had been assessed in the original method development. To test the hypothesis that it was the different formulation affecting the quantification, the remaining samples were pooled and split into two aliquots. Aliquot 1 was analyzed neat, diluted with control plasma one in ten, one in 20 and one in 50 to provide a background response for the parent compound in the pooled sample. Aliquot 2 however, was spiked with an additional 500 ng/ml of parent compound and then analyzed neat and diluted with control plasma one in ten, one in 20 and one in 50. Comparison of the two sets of data indicated that the presence of the formulation introduced a significant ion suppression effect (80–90%) that was not removed until samples had been diluted at least one in 20. future science group
| Commentary
In this example, the lack of communication surrounding the change in formulation proved expensive to the project. Not only did the PK/PD study need to be repeated in order to obtain the correct systemic exposure, but an in vitro rat hepatocyte metabolite identification study had also been performed as a result of chasing a ‘red herring’. Should all the information been made available to the CRO performing the bioanalysis then a change in formation would have been flagged as a potential issue before any of the incurred samples had been consumed. Conclusion Outsourcing discovery bioanalysis has suffered in the past from somewhat of a ‘throw it over the wall’ relationship between Client and CRO. Things have improved greatly over the past few years, but presented here are two examples of how withholding information from the CRO can impact discovery projects not only in the progression an inappropriate candidate, but also in performing unnecessary assays. In these and other cases, projects not only suffer from delayed timelines, but poor communication also introduces additional costs to already tight discovery budgets. Although it is easy to try and lay the blame of this poor communication at the feet of the Clients, it is equally as important for CROs to ask for this information. Clients’ scientific expertise in bioanalytical sciences can range from highly experienced through to virtually none, and as such some Clients rely on CROs to act in more of a consultative capacity and ask the correct questions. Certain elements of a project, such as structure, will always be challenging to release for reasons mentioned previously; however, supporting data that can be aid in discovery bioanalysis support can be of benefit and explaining to the Client why this information is necessary builds trust between the two organizations. Trust is the foundation of a solid partnership and for these relationships to be successful one can never have too much trust (or information). Financial & competing interests disclosure The author has no 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. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. www.future-science.com
735
Commentary |
Clark
References European Medicines Agency, Committee for Medicinal Products for Human Use. Guideline on Bioanalytical Method Validation. European Medicines Agency, London, UK (2012).
5
2
US FDA. Guidance for Industry: Bioanalytical Method Validation. FDA, Silver Springs, MD, USA(2001).
6
3
Life Science Leader. www.lifescienceleader.com/doc/trends-instrategic-outsourcing-changes-in-partnerselection-0001
Yan Z, Caldwell GW, Jones WJ, Masucci JA. Cone voltage induced in-source dissociation of glucuronides in electrospray and implications in biological analyses. Rapid Commun. Mass Spectrom. 17(13), 1433–1442 (2003).
7
4
Levsen K, Schiebel HM, Behnke B et al. Structure elucidation of Phase II metabolites by tandem mass spectrometry: an overview. J. Chromatogr. A 1067(1–2), 55–72 (2005).
Nagilla R, Nord M, Mcatee JJ, Jolivette LJ. Cassette dosing for pharmacokinetic screening in drug discovery: comparison of clearance, volume of distribution, half-life, mean residence time, and oral bioavailability
1
736
Mei H, Morrison RA. Using Mass Spectrometry for Drug Metabolism Studies. CRC Press, Boca Raton, FL, USA, 59–98 (2009).
Bioanalysis (2014) 6(6)
obtained by cassette and discrete dosing in rats. J. Pharm. Sci. 100(9), 3862–3874 (2011). 8
Larger PJ, Breda M, Fraier D, Hughes H, James CA. Ion-suppression effects in liquid chromatography–tandem mass spectrometry due to a formulation agent, a case study in drug discovery bioanalysis. J. Pharm. Biomed. Anal. 39(1–2), 206–216 (2005).
future science group
