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Feedback from a European Bioanalysis Forum survey on bioanalysis of drugs in tissues
Tissue analysis has always been a difficult discipline of bioanalysis. Laboratories that perform bioanalysis in tissue are facing a lot of challenges and questions before starting experiments, from a scientific/technical point of view regarding more regulated aspects. Actually, literature is poor regarding the more technical and scientific aspects but also beyond that no clear guidance is available on this topic and laboratories performing tissue analysis face real ambiguity regarding regulatory requirements, always with the risk of under- or over-validation of the assay. For all of these reasons bioanalysis in tissue became a frequently discussed topic within the European Bioanalytical Forum (EBF) organization. The EBF then decided to treat this as a specific topic, and carried out a survey that was done in two steps between 2012 and 2013. This paper represents an exhaustive summary of the result of this survey that includes themost important aspects of tissue bioanalysis. This survey provided the team a good starting point for their discussions and resulted in an EBF recommendation paper published separately.
Background Strategy regarding the bioanalysis of tissue samples, that is, quantification of endogenous and exogenous analytes in tissues in support of drug development is currently a hot topic in many bioanalytical laboratories. Laboratories performing these analyses are facing a lot of challenges and questions. The scope of these questions is very broad and ranges from “what are the best practices for tissue collection and homogenization?”, “how to perform tissue analysis in a correct, smart and efficient way?”, “what is requested/needed to ensure accurate performance of the assay?” to “what is the strategy from a regulatory perspective?”. Indeed, the community feels no clear guidance is available for tissue bioanalysis [1,2] . The resulting ambiguity on regulatory requirements may lead to applying inadequate processes or science and too stringent acceptance criteria, creating an inherent risk of under- or over-validating the assays [3–5] . For all of these reasons, bioanalysis of tissue samples has been frequently discussed within the European Bioanalysis Forum
10.4155/BIO.14.35 © 2014 Future Science Ltd
(EBF) since 2009. We decided then to invest time and effort into sharing current practices (technical and scientific) regarding the extent of method establishment (ME) performed by our member companies and on the level of regulatory compliance claimed at each stage of development. In continuation, an EBF topic team (TT) was formed in 2012 to gather more in depth information and ultimately issue an EBF recommendation. The team performed an extensive survey among the EBF members. The survey was launched in September 2012 and the first results were shared in a poster at the 5th EBF Open Symposium in Barcelona [6] . Following up on the first survey results, a follow-up survey was carried out in March 2013. The survey results formed the basis of our recommendation [7] . In this supporting manuscript, the TT is sharing and discussing the detailed results of both surveys. To include all aspects for tissue analysis – technical, scientific and regulatory aspects – the survey was organized into eight chapters:
Bioanalysis (2014) 6(12), 1617–1626
Nathalie Mokrzycki1, Pascal Delrat2, Marc De Meulder3, Eva Erbach4, Irene Lenthéric5, Morna McIntosh6, Pawel Dzygiel7 & Philip Timmerman*,3 1 Laboratoires Merck Sharp & DohmeChibret, Route de Marsat, Riom, 63963 Clermont-Ferrand Cedex 9, France 2 Technologie SERVIER, 25/27 rue Vignat, 45000 Orléans, France 3 Janssen Research & Development, a division of Janssen Pharmaceutica N.V. Turnhoutseweg 30, 2340 Beerse, Belgium 4 Bayer Pharma AG Global Drug Discovery, Global Early Development, Drug Metabolism & Pharmacokinetics Müllerstr. 178, Gebäude S109, Raum 220A 13353 Berlin, Germany 5 Harlan Laboratories S.A. Argenters, 6 08130 Santa Perpetua de Mogoda Barcelona, Spain 6 Charles River Laboratories, Bioanalysis & Immunology, Charles River Tranent, Edinburgh, EH33 1NE 7 Hoffman-La Roche AG, Grenzacherstrasse 124 CH-4070 Basel Switzerland *Author for correspondence: [email protected]
part of
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Tissue analysis: Quantification of endogenous and exogenous analytes in tissues in support of drug development.
understanding of the final objective. The results of the four first questions provide a good summary on the status of tissue analysis in the participating companies. The questions from the survey were:
EBF: European Bioanalytical Forum.
• Does your laboratory perform tissue analysis?
Method establishment: Strategy to define the appropriate method to analyze tissue samples.
• Does your laboratory analyze small molecules or large molecules?
Key terms
Survey: Investigation of the opinions or experience of a group of people by asking them questions. Bioanalytical method validation: All requirements as indicated in EMA/US FDA bioanalytical method validation guidances are assessed to validate the method.
• General questions; • Questions on quality and GLP status of analysis; • Questions on origin and types of tissue and reason for analysis; • Questions on technical and scientific points of consideration; • Questions on ME; • Questions on extent of qualification or validation and method parameters assessed; • Questions on impact of availability of matrices on bioanalytical study design;
• In which area do you mostly perform tissue analysis? A total of 22% of the responders performed tissue analysis weekly and 8.5% monthly, while 61% analyzed tissues sporadically (Table 1) . The question was refined in the second survey to understand at which stage of drug development tissue analysis was mostly performed. The answer was that 23% of the companies would assay tissues only in drug discovery, 12% in drug development and 65% in both stages of development. The majority of the participants were performing small molecule tissue bioanalysis using LC–MS/MS (21 out of 23 participants). Some of them were also involved in large biomolecules analysis either by LC–MS/MS or by ligand-binding assay (nine out of 23 participants).
• Question on protocol and reporting.
Question around quality & GLP status of analysis
In total, the surveys consisted of 68 questions. Some questions allowed the possibility for specific feedback on processes used in either drug discovery or drug development, as it was assumed that strategy for tissue bioanalysis could be different according to the stage of compound development. The surveys cover both small and large molecule bioanalysis. Twenty-three member companies, from Pharma and contract research organizations, all regularly involved in tissue analysis, participated in the survey. This gives our survey and the EBF recommendation on ME for tissue homogenates a solid basis and confirmed the great interest in this topic within the EBF community.
Many questions were around the level of quality applied to tissue bioanalysis and especially GLP compliance. In order to ensure a common understanding of the relationship between bioanalysis and GLP assumed in the survey, it was important to clarify how we defined GLP in the context of this survey to ensure accurate and unbiased answers. Member companies were asked whether they agree with the following definition: “Any analysis of study samples, which is performed using an a priori developed and validated method, with acceptance criteria predefined in a standard operating procedure (SOP; preferably in line with bioanalytical method validation [BMV] guidance, but can be different from acceptance criteria for plasma as mentioned in BMV guidance) and for which the process from sampling to analysis and reporting has been performed in compliance with Organization for Economic Cooperation and Development (OECD) -1, 8, 13 and 15 principles (including quality assurance oversight)”. Most of the companies (61%) agreed with this interpretation of the GLPs for the purpose of this survey,
Outcome of the survey General questions
The first section of the survey gauged the frequency and importance of tissue analysis within the community as well as whether small and/or large molecules were analyzed in tissue. The starting point was that companies frequently dealing with tissue bioanalysis would have a more structured bioanalytical strategy and a clear
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• How often does your laboratory perform tissue analysis?
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Feedback from a European Bioanalysis Forum survey on bioanalysis of drugs in tissues
while 22% did agree but applied GLP differently for tissue bioanalysis and the final 7% did not agree with it. In order to refine this, further questions were included in the survey: In our laboratory, we apply the following regulations when performing tissue analysis: • If you give GLP status, is it the same for all tissues in a particular study? • What is the driver to perform analysis under GLP conditions?
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Table 1. How often does your laboratory perform tissue analysis?
Response (%)
Weekly
22.0
Monthly
8.5
Sporadically
61.0
Other (please specify)
8.5
Questions around origin & types of tissue & reason for analysis
• Does your company have experience with registration of studies including tissue analysis to authorities?
Next to GLP another important aspect driving the strategy of tissue bioanalysis was the origin and the type of tissues most frequently analyzed; hence further questions were included in the survey:
• In case of compound filing, are all tissue analyses carried out under GLP status?
• What are the three most abundant types of tissue analyzed?
• Did you get questions from the agency on the status of study sample analysis and the method validation (GLP or non-GLP or others)?
• From which species do you analyze tissues, indicate the three most frequent?
Approximately 17% of the participants answered that they performed analysis under GLP (Table 2) . For some of the participants, the extent of compliance to GLP would be different depending on the stage of drug development. The main reasons for claiming GLP compliance were either because the data were generated as part of a GLP study (73%) or because of customer request (60%, contract research organizations). Another reason was because data were used for pivotal decisions other than safety. Approximately 13% of the companies would follow the principles of GLP (i.e., same quality but not claiming GLP), and approximately 39% would use qualified assay (Table 2) [8] . The fact that the data were part of compound registration to authorities would not be a major driver for 77% of the companies to necessarily claim GLP. At this stage, 38% of the companies had experience with filing studies that included tissue analysis. No questions were asked by the health authorities on tissue analysis either regarding method qualification/validation or GLP or non-GLP status of the analysis.
The most frequently analyzed tissues were similar for both drug discovery and drug development: brain tissue for almost 95% and liver for almost 80% of the participants, followed by lung (47%), heart (26%) and kidney (21%). Tumor analysis was more the focus in drug discovery (26% of the participants). Several companies would also perform analysis in different other smaller tissues, including skin, uterus and small intestine (Table 3) . Not surprisingly, tissue analysis is mostly carried out in rodents. Rat is the most common species (77% of the participants) and equally frequent in drug discovery and drug development. For mouse, the frequency was similar to rat in drug discovery (73% of the participants) but less in drug development (54% of the participants). Nonrodents are less frequently included in tissue sampling in drug discovery and, if tissue analysis is performed for nonrodents, it is more common in drug development (Table 4) . Among nonrodent species the most common species analyzed in drug development is the dog (55%) and then nonhuman primates (NHP; 32%). Some
• What are the three main reasons for tissue analysis?
Table 2. The following regulations are applied when performing tissue analysis.
Response (%)
Fully GLP
17.4
According to GLP principles (i.e., same quality but non-GLP)
13.0
Using the principles of qualified assays
39.1
A mixture of above three depending on stage of development
17.4
Other
13.0
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Table 3. What are the three most abundant types of tissue analyzed?
Drug discovery (n)
Drug development (n)
Liver
11
12
Brain
16
14
Lung
6
6
Kidney
3
2
Heart
4
4
Muscle
1
0
Tumor
5
2
companies also analyze human tissue biopsy during drug development (Table 4) . The three main reasons for tissue analysis are almost the same in drug discovery and in drug development: pharmacokinetics (tissue distribution or tissue ratio), pharmacodynamics and toxicokinetics (Table 5) . During the team discussion, we translated the above three reasons into a four-tiered approach for tissue homogenate analysis as part of our recommendation [7] . Technical & scientific points of consideration Taking a representative sample
It is important to ensure that the tissue sample taken for analysis is representative for the whole tissue, taking into account the size of the whole organ and potential specific regional distribution differences. This may be an important factor with potential differences for small animals and large animals. For this reason the questions asked around the tissue sampling procedure were split-up for small (rats, mice) and large animals (dogs, NHP). Some of the questions of the survey tried to clarify these aspects:
• For large animals, at the time of homogenization, what is your preferred practice? In the case of small animals, generally the whole tissue is harvested (94% of the participants), and homogenization is also performed using the whole tissue. For large animals, only approximately 44% of the participants would take the whole tissue, approximately 31% would take a representative subsection and approximately 25% would take (a) subsection(s) without paying attention to potential regional distribution of the drug. The same trend was observed for homogenization (Tables 6–9) . Different process of harvesting/freezing samples & stability assessment
The process of harvesting/freezing samples is key in tissue analysis. Preserving drug from degradation to get reliable data represents an important point of consideration and different practices may be used for practical or scientific reasons. Is it more reliable to freeze the solid tissue directly or to homogenize before freezing? Investigation of stability in solid tissue may be challenging, if not impossible [9] . We need to acknowledge that we currently document the stability of the drug in tissue homogenate. In addition, even documenting stability in tissue homogenates is far from straightforward and subject to a lot of ambiguity and process options. Stability can be assessed at different stages in the analytical process. Some q uestions from the survey were related to this aspect: • Do you prefer to have the tissue homogenized immediately after harvesting?
• For small animals (mice, rat), how do you manage to take a homogenous sample from the tissue you need to analyze?
• In the case of fresh tissue being frozen prior to homogenization, what is your preferred process of harvesting/freezing?
• For larger animals (dog, NHP, other), how do you manage to take a homogenous sample from the tissue you need to analyze?
• When tissue is not homogenized just after harvesting, how long do you allow crude tissue to stay frozen before homogenization?
Table 4. From which species do you analyze tissues? Indicate the three most frequent.
Drug discovery (n)
Drug development (n)
Mouse
16
12
Rat
17
17
Dog
6
12
Nonhuman primates
2
7
Human
0
3
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• For small animals, at the time of homogenization, what is your preferred practice?
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• How do you store your tissue samples or tissue homogenates? To the first question, the answers went in all directions: 50% of the participants responded that they prefer to freeze the solid tissue immediately after harvesting ‘for convenience’. And interestingly, only 9% responded that they follow this process for scientific reason. Similarly, some of the companies would homogenize right after harvesting either for convenience (14%) or for scientific reasons (9%; Table 10).
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Feedback from a European Bioanalysis Forum survey on bioanalysis of drugs in tissues
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Table 5. Three main reasons for tissue analysis.
Drug discovery (n)
Drug development (n)
PK (distribution or tissue ratios)
15
13
PD (for PK/PD assessment)
14
9
Tox/TK (target tissue, TK/TD assessment)
11
16
PD: Pharmacodynamics; PK: Pharmacokinetics; TK: Toxicokinetics; Tox: Toxicology.
When solid tissue is frozen prior to homogenization, the preferred process of harvesting/freezing varies. After harvesting (or biopsy), tissues may be washed, dried and stored in the freezer (normal speed; 32% of the participants). Tissue may also be washed, dried, snap frozen (e.g., -80°C, liquid N2) and stored in the freezer (26% of the participants). Less commonly after harvesting (or biopsy), tissue is washed (e.g., phosphate buffered saline) and stored in the freezer (16% of the participants), or washed (e.g., phosphate buffered saline), snap frozen (e.g., -80°C, liquid N2) and stored in the freezer (16% of the participants). For question 3, which focuses on stability aspects, 59% of the participants kept solid tissue frozen until the homogenization and analysis would start. A total of 41% of the participants would take into account compound and stability data information (Table 11) . Storage of solid tissues or tissue homogenates follows similar processes. The vast majority stored them at -80°C. In both cases, approximately one-third considered knowledge of the compound important before choosing the appropriate temperature (Tables 12 & 13) .
Almost all participants would prefer mechanical homogenization (such as grinding, blending, sonication and bead beating) above chemical (saponification) or biochemical (enzymatic treatment) homogenization. Mechanical techniques are used irrespective of the nature of the tissue, soft tissue such as liver or lung or hard tissue such as bone, eye or skin. Automated tissue homogenization equipment is not commonly used and individual manual homogenization is performed in 60% of the companies. For large molecules, 50% of the companies performing such analysis would introduce protease inhibitors. To the question on preference of homogenization at the in-life facility, 63% of the companies answered they preferred to do it in the bioanalytical laboratory rather than in-life laboratory due to the broader experience of bioanalytical laboratory scientists (89%) and/or more for practical reasons because analysis is done right after homogenization (22%). From the survey it is clear that the currently used process of homogenization and storage of tissue is driven more by practical considerations than scientific reasons.
Process for homogenization
Questions on ME
Multiple tools are available to homogenize tissue and there is ample literature available. Some of the questions from the survey that we want to focus on in this manuscript were:
As mentioned previously, ME for tissue homogenates is a challenge in many bioanalytical laboratories. The effort and the resources spent will typically depend on the objectives and the stage of drug development. The survey tried to explore whether generic methods or more focused methods were used and whether plasma methods were adapted systematically to tissue analysis:
• What is your preferred technique for tissue homogenization? • Do you prefer to have the tissues homogenized at the in-life facility? • In your laboratory (or the laboratory you work with), do you use automated tissue homogenization equipment?
• In drug discovery (pre-GLP), do you use a generic approach to analyze tissue samples? • In drug discovery (pre-GLP), what does your generic approach look like?
Table 6. For small animals, at the time of sacrifice, how do you prefer the tissue to be sampled?
Response (%)
We prefer to take the whole tissue
94.1
We combine subsections from the tissue
0.0
We take one representative subsection from the tissue
0.0
We take (a) subsection(s) and do not consider potential regional concentration differences
5.9
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Table 7. For small animals, at the time of homogenization, what is your preferred practice?
Response (%)
We prefer to homogenize all of the complete tissue sample (the whole tissue or all of the available subsections)
94.1
We take a representative subsection from the available tissue material
0.0
We take (a) subsection(s) from the available tissue material and do not consider potential regional concentration differences
5.9
Table 8. For large animals, at the time of sacrifice, how do you prefer the tissue to be sampled?
Response (%)
We prefer to take the whole tissue
43.8
We combine subsections from the tissue
0.0
We take one representative subsection from the tissue
31.3
We take (a) subsection(s) and do not consider potential regional concentration differences
25.0
• In drug discovery (pre-GLP), if you do not use a generic approach, how do you establish your assays? • In drug development, do you use a generic approach to analyze tissue samples? • In drug development, what does your generic approach look like? • In drug development, if you don’t use a generic approach, how do you establish your assays? In drug discovery and in drug development, respectively, 72 and 92% of the participants would use the method that is in place for plasma and apply this to tissue homogenates by making only small changes to
the sample preparation. In drug discovery, 17% of the participants choose a generic process. In drug discovery as well as in drug development, respectively, 75 and 54% of the participants establish a different assay per compound. However, the tissue type is not considered when developing the assay and the tissue assay is used for all tissues for that particular compound. In drug discovery as well as in drug development, respectively, 25 and 31% of the participants establish a different assay per compound where tissue type is not considered when developing the assay. The extent of time spent to define the appropriate method to analyze tissue samples would depend on the purpose of the tissue sample analysis for the specific studies.
Table 9. For large animals, at the time of homogenization, what is your preferred practice?
Response (%)
We prefer to homogenize all of the complete tissue sample (the whole tissue or all of the available subsections)
62.5
We take a representative subsection from the available tissue material
12.5
We take (a) subsection(s) from the available tissue material and do not consider potential regional concentration differences
25.0
Table 10. Do you prefer to have the tissue homogenized immediately after harvesting?
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Response (%)
Yes, for convenience
14.0
Yes, we feel this is a scientific requirement
9.0
Yes, but we have difficulties in managing the logistics of this preferred process
14.0
Yes, but only for soft tissue. Less a requirement for hard tissues
0.0
No, for convenience, we prefer to freeze the tissue prior to homogenization
50.0
No, for scientific reasons, we prefer to freeze the tissue prior to homogenization
9.1
Other (please specify)
4.5
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Feedback from a European Bioanalysis Forum survey on bioanalysis of drugs in tissues
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Table 11. When tissue is not homogenized just after harvesting, how long do you allow crude tissue to stay frozen before homogenization?
Response (%)
Less than 1 week
5.9
Less than 1 month
5.9
As long as needed until we can start the homogenization/analysis
58.8
It depends on study design, some samples may be older than others
11.8
It will depend on compound and stability data information
41.2
Questions on extent of qualification or validation: method parameters assessed
• Do you assess parameters mentioned in US FDA/EMA guidance principles for ME?
guidance, as soon as tissue analysis is performed within a GLP study in drug development (Table 15) . For 45% of the companies all FDA/EMA parameters are assessed during qualification or validation of the method. Among the companies that responded ‘no’ to the above question, approximately 50% would not assess one or more validation parameter: for example, calibration curve and/or selectivity and/or accuracy and precision and/or dilution integrity and/or matrix effect and/or carry over. A total of 70% would not assess stability and 64% would skip ISR. For ligand binding studies, hook effect would not be assessed in 21% of the companies. Usually, acceptance criteria of FDA/EMA guidance would be applied for 55% of the participants. When changing matrix or species some companies would reassess some parameters and the main parameters assessed in both cases are calibration curve, accuracy and precision, selectivity and stability. Carry over, matrix effect, dilution integrity and ISR would come in second position.
• Do you apply acceptance criteria according to FDA/EMA guidance principles?
Questions on impact of availability of matrices on bioanalytical study design
• Do you accept acceptance criteria 4–6-15/20 for calibration curve?
With respect to the use of control matrix for calibration standards (CALs) or quality controls (QCs), several options can be considered. Either the same control
No specific guidance is available to guide the performance characteristics of tissue analysis methods as is for plasma. To what extent does a method for the analysis of tissue samples needs to be validated? What makes scientific sense? What is doable? Do we need to fully validate or is qualification a better approach? Should we allow for less stringent performance parameters? According to what was discussed within the EBF, scientists attempt at least to refer to BMV guidance [1,2] to demonstrate performance of the method. But it becomes obvious that some performance qualification parameters may be difficult to establish and that some BMV guidance criteria may be hard to meet. Hence, we asked the following questions: • Across all drug development phases, do you use method validation or qualification?
• Do you assess the following parameters: assessment of lower limit of quantification, accuracy and precision, selectivity–specificity, matrix effect, carry over, dilution integrity, incurred sample reproducibility (ISR)? • Do you perform stability on tissue homogenate? (Table 14) . A screening method is mainly used for tissue analysis in drug discovery. A qualified method with widened acceptance criteria compared with the BMV guidance is used in drug discovery as well as in nonGLP studies in drug development. Companies tend to start using validated methods, either with widened or the same acceptance criteria than in the BMV
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Table 12. How do you store your solid tissue samples (nonhomogenized)?
Response (%)
At -20°C
5.9
At -80°C
64.7
One of the above depending on compound or tissue
29.4
Table 13. How do you store your tissue homogenates?
Response (%)
At -20°C
11.8
At -80°C
52.9
One of the above depending on compound or tissue
35.3
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Table 14. Stability assessment: do you perform stability assessment on tissue homogenate? Time scale/processing technique
Yes, a priori
No
No, we rely on plasma data
As in the US FDA/EMA criteria for assays
Short term
8
6
3
4
1 week
3
6
3
1
F/T
9
5
3
4
Processed sample
9
4
3
4
F/T: Freeze/thaw.
matrix as the matrix of the study samples is used to prepare CALs and QCs or a surrogate control matrix is used due to difficult supply or for ethical reasons [10] . Plasma can also be used since the tissue method is often derived from the plasma method. The following question did explore those various options: • What is your preferred bioanalytical design to analyze tissue samples in a study?
in early stage of development (drug discovery) other approaches can be considered. Questions on protocol & reporting
Finally the EBF TT wanted to extend the survey to more practical considerations and more specifically to management of procedures, to protocol and report documentation and to archiving data from tissue bioanalysis:
The preferred analytical design for tissue analysis is to prepare CALs and QCs in homogenate from target tissue (53%). A total of 20% of the participants prepare CALs and QCs in plasma. Alternative designs are also used, for example:
• Does your laboratory have a specific SOP for tissue analysis?
• CALs prepared in buffer or extraction solvent and QCs prepared in homogenate from target tissue;
• If tissue analysis is part of a study, does your laboratory have a specific section on tissue analysis within the protocol of the main study (Tox/TK study)?
• Standards in plasma and QCs in homogenate. Finally the survey seems to suggest that the strategy will depend on the stage of development with the preference to try and use study samples matrix (depending on supply) for CALs and QCs in drug development while
• Does your laboratory include tissue analysis in the protocol of the main study (Tox/TK study, etc)?
• Does your laboratory specify the status (GLP/non-GLP) of the tissue analysis in the protocol? • Does your laboratory provide description/guidance of tissue collection in the protocol?
Table 15. Across all drug development phases, do you use method validation or qualification?
Drug discovery (n)
Drug development non-GLP studies (n)
Drug development GLP studies (n)
We use screening methods, i.e., look at ratios between tissue levels or plasma/tissue levels
6
0
0
We use qualified methods (i.e., absolute concentrations) using widened acceptance criteria
12
11
4
We use qualified methods (i.e., absolute concentrations) using acceptance criteria in line with BMV
3
9
3
We use validated (i.e., absolute concentrations) using widened acceptance criteria
0
2
8
We use validated (i.e., absolute concentrations) using acceptance criteria in line with BMV
1
2
8
BMV: Bioanalytical method validation.
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Feedback from a European Bioanalysis Forum survey on bioanalysis of drugs in tissues
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• Do you ‘archive/store’ tissue homogenates after analysis?
Table 16. Does your laboratory provide description/guidance of tissue collection in the protocol?
• How does your laboratory report data of tissue analysis?
Yes (n)
No (n)
Size of tissue sample to be collected
13
7
The majority of the participants (62%) have no specific SOP in place, while 20% of the companies do have a SOP for tissue analysis. Approximately 67% of the companies would include tissue analysis in the main study protocol either in GLP or nonGLP studies, and 33% would manage this as a separate study. Among the companies that include tissue analysis in the main study, 50% of the companies use a specific section of the protocol. The other would have this in the general statement of the bioanalysis section. Approximately 70% of the companies would specify the status (GLP or non-GLP) in the protocol. The content of the protocol regarding tissue collection or preprocessing varied. Some would specify the size of the tissue sample to be collected (65% of the participants), the (sub)section of tissue sample to be collected (45% of the participants), and/or the description of tissue preprocessing/handling (83% of the participants; Table 16 ). Almost 50% of the companies would archive tissue homogenate as they do for plasma samples. Some (~20%) would archive but with a duration independent from plasma samples. Approximately 24% would archive at sponsor request. The remaining companies would discard the samples.
(Sub)section of tissue sample to be collected
9
9
Description of tissue preprocessing/handling
15
3
Conclusion As mentioned at the beginning of this manuscript, the EBF is committed to investing time and effort to share current practices (technical and scientific) on the extent of ME performed by the member companies and on the level of regulatory compliance claimed at each stage of development. More specifically, the focus of this survey was to understand the frequency of tissue analysis, the level of experience, the level of quality (scientific/regulatory) and/or of compliance to GLP, the rationale for tissue analysis, the nature of the samples collected and the samples measured and finally the strategy of bioanalysis applied to sample measurement. The diversity of answers in some aspects highlights the fact that tissue analysis is very complex and dependent on scientific purpose. The extent of ME will depend on the future use of the data. But whether regulatory or exploratory, it appears that what matters most for all participants is the fit for purpose of ME to ensure adequate quality of the data to allow valid decision-making. This survey provided the team a good starting point for their discussions and resulted in an EBF recommendation.
Executive summary • Within the bioanalytical community, 61% of laboratories perform tissue analysis sporadically and only 22% on weekly basis. • Only around 17% of the participants of the survey perform analysis under GLP, others would follow GLP principles. • Brain and liver are the most frequently analyzed tissues in drug discovery and drug development. Tissue analysis is mostly carried out in rodents. • Taking a representative sample, the process of harvesting/freezing samples and consequently stability assessment, as well as the homogenization process are key scientific and technical points that needs to be taken into consideration. • Method establishment: the survey showed that in drug discovery and drug development the method used is mainly the method that is in place for plasma analysis with small changes in sample preparation. Methods are mainly established per compound. • A qualified method with widened acceptance criteria compared to the bioanalytical method validation guidance is used in drug discovery as well as in non-GLP studies in drug development. Companies tend to start using validated methods, either with widened or the same acceptance criteria as the bioanalytical method validation guidance, as soon as tissue analysis is performed within a GLP study in drug development. • The preferred analytical design for tissue analysis is to prepare calibration standards or quality controls in homogenates from target tissue (53%). A total of 20% of the participants prepare calibration standards and quality controls in plasma. • The majority of participants (62%) have no specific standard operating procedure in place while 20% of the companies do have an standard operating procedure for tissue analysis.
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Special Report Mokrzycki, Delrat, Meulder et al. Future perspective This survey on bioanalysis of drug in tissue among the EBF community provided the team a good starting point for discussions and resulted in an EBF recommendation published in this issue of the journal. Acknowledgments The EBF subteam on tissue analysis thanks all EBF participants in this survey without whom getting this extensive overview would not have been possible.
References Papers of special note have been highlighted as: • of interest 1
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US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM). Guidance for Industry. Bioanalytical Method Validation. CDER, Rockville, MD, USA (2001). www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf
Financial & competing interests disclosure The authors have 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.
•
Exhaustive review of tissue sample analysis for small molecules using LC–MS/MS.
6
Towards a recommendation for tissue analysis. http://bcn2012.europeanbioanalysisforum.eu/slides/ day%203/ix%20tiered%20approach/4_timmerman.pdf
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Timmerman P, Mokrzycki N, Delrat P et al. Recommendations from the European Bioanalysis Forum on method establishment for tissue homogenates. Bioanalysis 6(12), 1647–1656 (2014).
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EMA. Guidelines on bioanalytical method validation. EMA, London, UK (2011). www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2011/08/WC500109686.pdf
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Timmerman P, Herling C, Stoellner D et al. European Bioanalysis Forum recommendation on method establishment and bioanalysis of biomarkers in support of drug development. Bioanalysis 4(15), 1883–1894 (2012).
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Neubert H, Fountain S, King L et al. Tissue bioanalysis of biotherapeutics and drug targets to support PK/PD. Bioanalysis 4(21), 2589–2604 (2012).
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Overview of contemporary applications of quantitative bioanalysis in tissue compartments.
Zhang J, Reimer MT, Ji QC et al. Accurate determination of an immunosuppressant in stented swine tissues with LC–MS/MS. Anal. Bioanal. Chem. 387(8), 2745–2756 (2012).
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Chen S, Wu J-T, Huang R et al. Evaluation of surrogate matrices for standard curve preparation in tissue. Bioanalysis 4(21), 2579–2587 (2012).
4
Ho S. Tissue analysis – a powerful tool for drug discovery and development. Bioanalysis 4(21), 2545–2547 (2012).
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Xue Y-J, Gao H, Ji Q et al. Bioanalysis of drug in tissue: current status and challenges. Bioanalysis 4(21), 2637–2653 (2012).
Bioanalysis (2014) 6(12)
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Feedback from a European Bioanalysis Forum survey on bioanalysis of drugs in tissues
Tissue analysis has always been a difficult discipline of bioanalysis. Laboratories that perform bioanalysis in tissue are facing a lot of challenges and questions before starting experiments, from a scientific/technical point of view regarding more regulated aspects. Actually, literature is poor regarding the more technical and scientific aspects but also beyond that no clear guidance is available on this topic and laboratories performing tissue analysis face real ambiguity regarding regulatory requirements, always with the risk of under- or over-validation of the assay. For all of these reasons bioanalysis in tissue became a frequently discussed topic within the European Bioanalytical Forum (EBF) organization. The EBF then decided to treat this as a specific topic, and carried out a survey that was done in two steps between 2012 and 2013. This paper represents an exhaustive summary of the result of this survey that includes themost important aspects of tissue bioanalysis. This survey provided the team a good starting point for their discussions and resulted in an EBF recommendation paper published separately.
Background Strategy regarding the bioanalysis of tissue samples, that is, quantification of endogenous and exogenous analytes in tissues in support of drug development is currently a hot topic in many bioanalytical laboratories. Laboratories performing these analyses are facing a lot of challenges and questions. The scope of these questions is very broad and ranges from “what are the best practices for tissue collection and homogenization?”, “how to perform tissue analysis in a correct, smart and efficient way?”, “what is requested/needed to ensure accurate performance of the assay?” to “what is the strategy from a regulatory perspective?”. Indeed, the community feels no clear guidance is available for tissue bioanalysis [1,2] . The resulting ambiguity on regulatory requirements may lead to applying inadequate processes or science and too stringent acceptance criteria, creating an inherent risk of under- or over-validating the assays [3–5] . For all of these reasons, bioanalysis of tissue samples has been frequently discussed within the European Bioanalysis Forum
10.4155/BIO.14.35 © 2014 Future Science Ltd
(EBF) since 2009. We decided then to invest time and effort into sharing current practices (technical and scientific) regarding the extent of method establishment (ME) performed by our member companies and on the level of regulatory compliance claimed at each stage of development. In continuation, an EBF topic team (TT) was formed in 2012 to gather more in depth information and ultimately issue an EBF recommendation. The team performed an extensive survey among the EBF members. The survey was launched in September 2012 and the first results were shared in a poster at the 5th EBF Open Symposium in Barcelona [6] . Following up on the first survey results, a follow-up survey was carried out in March 2013. The survey results formed the basis of our recommendation [7] . In this supporting manuscript, the TT is sharing and discussing the detailed results of both surveys. To include all aspects for tissue analysis – technical, scientific and regulatory aspects – the survey was organized into eight chapters:
Bioanalysis (2014) 6(12), 1617–1626
Nathalie Mokrzycki1, Pascal Delrat2, Marc De Meulder3, Eva Erbach4, Irene Lenthéric5, Morna McIntosh6, Pawel Dzygiel7 & Philip Timmerman*,3 1 Laboratoires Merck Sharp & DohmeChibret, Route de Marsat, Riom, 63963 Clermont-Ferrand Cedex 9, France 2 Technologie SERVIER, 25/27 rue Vignat, 45000 Orléans, France 3 Janssen Research & Development, a division of Janssen Pharmaceutica N.V. Turnhoutseweg 30, 2340 Beerse, Belgium 4 Bayer Pharma AG Global Drug Discovery, Global Early Development, Drug Metabolism & Pharmacokinetics Müllerstr. 178, Gebäude S109, Raum 220A 13353 Berlin, Germany 5 Harlan Laboratories S.A. Argenters, 6 08130 Santa Perpetua de Mogoda Barcelona, Spain 6 Charles River Laboratories, Bioanalysis & Immunology, Charles River Tranent, Edinburgh, EH33 1NE 7 Hoffman-La Roche AG, Grenzacherstrasse 124 CH-4070 Basel Switzerland *Author for correspondence: [email protected]
part of
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Tissue analysis: Quantification of endogenous and exogenous analytes in tissues in support of drug development.
understanding of the final objective. The results of the four first questions provide a good summary on the status of tissue analysis in the participating companies. The questions from the survey were:
EBF: European Bioanalytical Forum.
• Does your laboratory perform tissue analysis?
Method establishment: Strategy to define the appropriate method to analyze tissue samples.
• Does your laboratory analyze small molecules or large molecules?
Key terms
Survey: Investigation of the opinions or experience of a group of people by asking them questions. Bioanalytical method validation: All requirements as indicated in EMA/US FDA bioanalytical method validation guidances are assessed to validate the method.
• General questions; • Questions on quality and GLP status of analysis; • Questions on origin and types of tissue and reason for analysis; • Questions on technical and scientific points of consideration; • Questions on ME; • Questions on extent of qualification or validation and method parameters assessed; • Questions on impact of availability of matrices on bioanalytical study design;
• In which area do you mostly perform tissue analysis? A total of 22% of the responders performed tissue analysis weekly and 8.5% monthly, while 61% analyzed tissues sporadically (Table 1) . The question was refined in the second survey to understand at which stage of drug development tissue analysis was mostly performed. The answer was that 23% of the companies would assay tissues only in drug discovery, 12% in drug development and 65% in both stages of development. The majority of the participants were performing small molecule tissue bioanalysis using LC–MS/MS (21 out of 23 participants). Some of them were also involved in large biomolecules analysis either by LC–MS/MS or by ligand-binding assay (nine out of 23 participants).
• Question on protocol and reporting.
Question around quality & GLP status of analysis
In total, the surveys consisted of 68 questions. Some questions allowed the possibility for specific feedback on processes used in either drug discovery or drug development, as it was assumed that strategy for tissue bioanalysis could be different according to the stage of compound development. The surveys cover both small and large molecule bioanalysis. Twenty-three member companies, from Pharma and contract research organizations, all regularly involved in tissue analysis, participated in the survey. This gives our survey and the EBF recommendation on ME for tissue homogenates a solid basis and confirmed the great interest in this topic within the EBF community.
Many questions were around the level of quality applied to tissue bioanalysis and especially GLP compliance. In order to ensure a common understanding of the relationship between bioanalysis and GLP assumed in the survey, it was important to clarify how we defined GLP in the context of this survey to ensure accurate and unbiased answers. Member companies were asked whether they agree with the following definition: “Any analysis of study samples, which is performed using an a priori developed and validated method, with acceptance criteria predefined in a standard operating procedure (SOP; preferably in line with bioanalytical method validation [BMV] guidance, but can be different from acceptance criteria for plasma as mentioned in BMV guidance) and for which the process from sampling to analysis and reporting has been performed in compliance with Organization for Economic Cooperation and Development (OECD) -1, 8, 13 and 15 principles (including quality assurance oversight)”. Most of the companies (61%) agreed with this interpretation of the GLPs for the purpose of this survey,
Outcome of the survey General questions
The first section of the survey gauged the frequency and importance of tissue analysis within the community as well as whether small and/or large molecules were analyzed in tissue. The starting point was that companies frequently dealing with tissue bioanalysis would have a more structured bioanalytical strategy and a clear
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• How often does your laboratory perform tissue analysis?
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Feedback from a European Bioanalysis Forum survey on bioanalysis of drugs in tissues
while 22% did agree but applied GLP differently for tissue bioanalysis and the final 7% did not agree with it. In order to refine this, further questions were included in the survey: In our laboratory, we apply the following regulations when performing tissue analysis: • If you give GLP status, is it the same for all tissues in a particular study? • What is the driver to perform analysis under GLP conditions?
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Table 1. How often does your laboratory perform tissue analysis?
Response (%)
Weekly
22.0
Monthly
8.5
Sporadically
61.0
Other (please specify)
8.5
Questions around origin & types of tissue & reason for analysis
• Does your company have experience with registration of studies including tissue analysis to authorities?
Next to GLP another important aspect driving the strategy of tissue bioanalysis was the origin and the type of tissues most frequently analyzed; hence further questions were included in the survey:
• In case of compound filing, are all tissue analyses carried out under GLP status?
• What are the three most abundant types of tissue analyzed?
• Did you get questions from the agency on the status of study sample analysis and the method validation (GLP or non-GLP or others)?
• From which species do you analyze tissues, indicate the three most frequent?
Approximately 17% of the participants answered that they performed analysis under GLP (Table 2) . For some of the participants, the extent of compliance to GLP would be different depending on the stage of drug development. The main reasons for claiming GLP compliance were either because the data were generated as part of a GLP study (73%) or because of customer request (60%, contract research organizations). Another reason was because data were used for pivotal decisions other than safety. Approximately 13% of the companies would follow the principles of GLP (i.e., same quality but not claiming GLP), and approximately 39% would use qualified assay (Table 2) [8] . The fact that the data were part of compound registration to authorities would not be a major driver for 77% of the companies to necessarily claim GLP. At this stage, 38% of the companies had experience with filing studies that included tissue analysis. No questions were asked by the health authorities on tissue analysis either regarding method qualification/validation or GLP or non-GLP status of the analysis.
The most frequently analyzed tissues were similar for both drug discovery and drug development: brain tissue for almost 95% and liver for almost 80% of the participants, followed by lung (47%), heart (26%) and kidney (21%). Tumor analysis was more the focus in drug discovery (26% of the participants). Several companies would also perform analysis in different other smaller tissues, including skin, uterus and small intestine (Table 3) . Not surprisingly, tissue analysis is mostly carried out in rodents. Rat is the most common species (77% of the participants) and equally frequent in drug discovery and drug development. For mouse, the frequency was similar to rat in drug discovery (73% of the participants) but less in drug development (54% of the participants). Nonrodents are less frequently included in tissue sampling in drug discovery and, if tissue analysis is performed for nonrodents, it is more common in drug development (Table 4) . Among nonrodent species the most common species analyzed in drug development is the dog (55%) and then nonhuman primates (NHP; 32%). Some
• What are the three main reasons for tissue analysis?
Table 2. The following regulations are applied when performing tissue analysis.
Response (%)
Fully GLP
17.4
According to GLP principles (i.e., same quality but non-GLP)
13.0
Using the principles of qualified assays
39.1
A mixture of above three depending on stage of development
17.4
Other
13.0
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Table 3. What are the three most abundant types of tissue analyzed?
Drug discovery (n)
Drug development (n)
Liver
11
12
Brain
16
14
Lung
6
6
Kidney
3
2
Heart
4
4
Muscle
1
0
Tumor
5
2
companies also analyze human tissue biopsy during drug development (Table 4) . The three main reasons for tissue analysis are almost the same in drug discovery and in drug development: pharmacokinetics (tissue distribution or tissue ratio), pharmacodynamics and toxicokinetics (Table 5) . During the team discussion, we translated the above three reasons into a four-tiered approach for tissue homogenate analysis as part of our recommendation [7] . Technical & scientific points of consideration Taking a representative sample
It is important to ensure that the tissue sample taken for analysis is representative for the whole tissue, taking into account the size of the whole organ and potential specific regional distribution differences. This may be an important factor with potential differences for small animals and large animals. For this reason the questions asked around the tissue sampling procedure were split-up for small (rats, mice) and large animals (dogs, NHP). Some of the questions of the survey tried to clarify these aspects:
• For large animals, at the time of homogenization, what is your preferred practice? In the case of small animals, generally the whole tissue is harvested (94% of the participants), and homogenization is also performed using the whole tissue. For large animals, only approximately 44% of the participants would take the whole tissue, approximately 31% would take a representative subsection and approximately 25% would take (a) subsection(s) without paying attention to potential regional distribution of the drug. The same trend was observed for homogenization (Tables 6–9) . Different process of harvesting/freezing samples & stability assessment
The process of harvesting/freezing samples is key in tissue analysis. Preserving drug from degradation to get reliable data represents an important point of consideration and different practices may be used for practical or scientific reasons. Is it more reliable to freeze the solid tissue directly or to homogenize before freezing? Investigation of stability in solid tissue may be challenging, if not impossible [9] . We need to acknowledge that we currently document the stability of the drug in tissue homogenate. In addition, even documenting stability in tissue homogenates is far from straightforward and subject to a lot of ambiguity and process options. Stability can be assessed at different stages in the analytical process. Some q uestions from the survey were related to this aspect: • Do you prefer to have the tissue homogenized immediately after harvesting?
• For small animals (mice, rat), how do you manage to take a homogenous sample from the tissue you need to analyze?
• In the case of fresh tissue being frozen prior to homogenization, what is your preferred process of harvesting/freezing?
• For larger animals (dog, NHP, other), how do you manage to take a homogenous sample from the tissue you need to analyze?
• When tissue is not homogenized just after harvesting, how long do you allow crude tissue to stay frozen before homogenization?
Table 4. From which species do you analyze tissues? Indicate the three most frequent.
Drug discovery (n)
Drug development (n)
Mouse
16
12
Rat
17
17
Dog
6
12
Nonhuman primates
2
7
Human
0
3
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• For small animals, at the time of homogenization, what is your preferred practice?
Bioanalysis (2014) 6(12)
• How do you store your tissue samples or tissue homogenates? To the first question, the answers went in all directions: 50% of the participants responded that they prefer to freeze the solid tissue immediately after harvesting ‘for convenience’. And interestingly, only 9% responded that they follow this process for scientific reason. Similarly, some of the companies would homogenize right after harvesting either for convenience (14%) or for scientific reasons (9%; Table 10).
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Feedback from a European Bioanalysis Forum survey on bioanalysis of drugs in tissues
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Table 5. Three main reasons for tissue analysis.
Drug discovery (n)
Drug development (n)
PK (distribution or tissue ratios)
15
13
PD (for PK/PD assessment)
14
9
Tox/TK (target tissue, TK/TD assessment)
11
16
PD: Pharmacodynamics; PK: Pharmacokinetics; TK: Toxicokinetics; Tox: Toxicology.
When solid tissue is frozen prior to homogenization, the preferred process of harvesting/freezing varies. After harvesting (or biopsy), tissues may be washed, dried and stored in the freezer (normal speed; 32% of the participants). Tissue may also be washed, dried, snap frozen (e.g., -80°C, liquid N2) and stored in the freezer (26% of the participants). Less commonly after harvesting (or biopsy), tissue is washed (e.g., phosphate buffered saline) and stored in the freezer (16% of the participants), or washed (e.g., phosphate buffered saline), snap frozen (e.g., -80°C, liquid N2) and stored in the freezer (16% of the participants). For question 3, which focuses on stability aspects, 59% of the participants kept solid tissue frozen until the homogenization and analysis would start. A total of 41% of the participants would take into account compound and stability data information (Table 11) . Storage of solid tissues or tissue homogenates follows similar processes. The vast majority stored them at -80°C. In both cases, approximately one-third considered knowledge of the compound important before choosing the appropriate temperature (Tables 12 & 13) .
Almost all participants would prefer mechanical homogenization (such as grinding, blending, sonication and bead beating) above chemical (saponification) or biochemical (enzymatic treatment) homogenization. Mechanical techniques are used irrespective of the nature of the tissue, soft tissue such as liver or lung or hard tissue such as bone, eye or skin. Automated tissue homogenization equipment is not commonly used and individual manual homogenization is performed in 60% of the companies. For large molecules, 50% of the companies performing such analysis would introduce protease inhibitors. To the question on preference of homogenization at the in-life facility, 63% of the companies answered they preferred to do it in the bioanalytical laboratory rather than in-life laboratory due to the broader experience of bioanalytical laboratory scientists (89%) and/or more for practical reasons because analysis is done right after homogenization (22%). From the survey it is clear that the currently used process of homogenization and storage of tissue is driven more by practical considerations than scientific reasons.
Process for homogenization
Questions on ME
Multiple tools are available to homogenize tissue and there is ample literature available. Some of the questions from the survey that we want to focus on in this manuscript were:
As mentioned previously, ME for tissue homogenates is a challenge in many bioanalytical laboratories. The effort and the resources spent will typically depend on the objectives and the stage of drug development. The survey tried to explore whether generic methods or more focused methods were used and whether plasma methods were adapted systematically to tissue analysis:
• What is your preferred technique for tissue homogenization? • Do you prefer to have the tissues homogenized at the in-life facility? • In your laboratory (or the laboratory you work with), do you use automated tissue homogenization equipment?
• In drug discovery (pre-GLP), do you use a generic approach to analyze tissue samples? • In drug discovery (pre-GLP), what does your generic approach look like?
Table 6. For small animals, at the time of sacrifice, how do you prefer the tissue to be sampled?
Response (%)
We prefer to take the whole tissue
94.1
We combine subsections from the tissue
0.0
We take one representative subsection from the tissue
0.0
We take (a) subsection(s) and do not consider potential regional concentration differences
5.9
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Table 7. For small animals, at the time of homogenization, what is your preferred practice?
Response (%)
We prefer to homogenize all of the complete tissue sample (the whole tissue or all of the available subsections)
94.1
We take a representative subsection from the available tissue material
0.0
We take (a) subsection(s) from the available tissue material and do not consider potential regional concentration differences
5.9
Table 8. For large animals, at the time of sacrifice, how do you prefer the tissue to be sampled?
Response (%)
We prefer to take the whole tissue
43.8
We combine subsections from the tissue
0.0
We take one representative subsection from the tissue
31.3
We take (a) subsection(s) and do not consider potential regional concentration differences
25.0
• In drug discovery (pre-GLP), if you do not use a generic approach, how do you establish your assays? • In drug development, do you use a generic approach to analyze tissue samples? • In drug development, what does your generic approach look like? • In drug development, if you don’t use a generic approach, how do you establish your assays? In drug discovery and in drug development, respectively, 72 and 92% of the participants would use the method that is in place for plasma and apply this to tissue homogenates by making only small changes to
the sample preparation. In drug discovery, 17% of the participants choose a generic process. In drug discovery as well as in drug development, respectively, 75 and 54% of the participants establish a different assay per compound. However, the tissue type is not considered when developing the assay and the tissue assay is used for all tissues for that particular compound. In drug discovery as well as in drug development, respectively, 25 and 31% of the participants establish a different assay per compound where tissue type is not considered when developing the assay. The extent of time spent to define the appropriate method to analyze tissue samples would depend on the purpose of the tissue sample analysis for the specific studies.
Table 9. For large animals, at the time of homogenization, what is your preferred practice?
Response (%)
We prefer to homogenize all of the complete tissue sample (the whole tissue or all of the available subsections)
62.5
We take a representative subsection from the available tissue material
12.5
We take (a) subsection(s) from the available tissue material and do not consider potential regional concentration differences
25.0
Table 10. Do you prefer to have the tissue homogenized immediately after harvesting?
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Response (%)
Yes, for convenience
14.0
Yes, we feel this is a scientific requirement
9.0
Yes, but we have difficulties in managing the logistics of this preferred process
14.0
Yes, but only for soft tissue. Less a requirement for hard tissues
0.0
No, for convenience, we prefer to freeze the tissue prior to homogenization
50.0
No, for scientific reasons, we prefer to freeze the tissue prior to homogenization
9.1
Other (please specify)
4.5
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Feedback from a European Bioanalysis Forum survey on bioanalysis of drugs in tissues
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Table 11. When tissue is not homogenized just after harvesting, how long do you allow crude tissue to stay frozen before homogenization?
Response (%)
Less than 1 week
5.9
Less than 1 month
5.9
As long as needed until we can start the homogenization/analysis
58.8
It depends on study design, some samples may be older than others
11.8
It will depend on compound and stability data information
41.2
Questions on extent of qualification or validation: method parameters assessed
• Do you assess parameters mentioned in US FDA/EMA guidance principles for ME?
guidance, as soon as tissue analysis is performed within a GLP study in drug development (Table 15) . For 45% of the companies all FDA/EMA parameters are assessed during qualification or validation of the method. Among the companies that responded ‘no’ to the above question, approximately 50% would not assess one or more validation parameter: for example, calibration curve and/or selectivity and/or accuracy and precision and/or dilution integrity and/or matrix effect and/or carry over. A total of 70% would not assess stability and 64% would skip ISR. For ligand binding studies, hook effect would not be assessed in 21% of the companies. Usually, acceptance criteria of FDA/EMA guidance would be applied for 55% of the participants. When changing matrix or species some companies would reassess some parameters and the main parameters assessed in both cases are calibration curve, accuracy and precision, selectivity and stability. Carry over, matrix effect, dilution integrity and ISR would come in second position.
• Do you apply acceptance criteria according to FDA/EMA guidance principles?
Questions on impact of availability of matrices on bioanalytical study design
• Do you accept acceptance criteria 4–6-15/20 for calibration curve?
With respect to the use of control matrix for calibration standards (CALs) or quality controls (QCs), several options can be considered. Either the same control
No specific guidance is available to guide the performance characteristics of tissue analysis methods as is for plasma. To what extent does a method for the analysis of tissue samples needs to be validated? What makes scientific sense? What is doable? Do we need to fully validate or is qualification a better approach? Should we allow for less stringent performance parameters? According to what was discussed within the EBF, scientists attempt at least to refer to BMV guidance [1,2] to demonstrate performance of the method. But it becomes obvious that some performance qualification parameters may be difficult to establish and that some BMV guidance criteria may be hard to meet. Hence, we asked the following questions: • Across all drug development phases, do you use method validation or qualification?
• Do you assess the following parameters: assessment of lower limit of quantification, accuracy and precision, selectivity–specificity, matrix effect, carry over, dilution integrity, incurred sample reproducibility (ISR)? • Do you perform stability on tissue homogenate? (Table 14) . A screening method is mainly used for tissue analysis in drug discovery. A qualified method with widened acceptance criteria compared with the BMV guidance is used in drug discovery as well as in nonGLP studies in drug development. Companies tend to start using validated methods, either with widened or the same acceptance criteria than in the BMV
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Table 12. How do you store your solid tissue samples (nonhomogenized)?
Response (%)
At -20°C
5.9
At -80°C
64.7
One of the above depending on compound or tissue
29.4
Table 13. How do you store your tissue homogenates?
Response (%)
At -20°C
11.8
At -80°C
52.9
One of the above depending on compound or tissue
35.3
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Table 14. Stability assessment: do you perform stability assessment on tissue homogenate? Time scale/processing technique
Yes, a priori
No
No, we rely on plasma data
As in the US FDA/EMA criteria for assays
Short term
8
6
3
4
1 week
3
6
3
1
F/T
9
5
3
4
Processed sample
9
4
3
4
F/T: Freeze/thaw.
matrix as the matrix of the study samples is used to prepare CALs and QCs or a surrogate control matrix is used due to difficult supply or for ethical reasons [10] . Plasma can also be used since the tissue method is often derived from the plasma method. The following question did explore those various options: • What is your preferred bioanalytical design to analyze tissue samples in a study?
in early stage of development (drug discovery) other approaches can be considered. Questions on protocol & reporting
Finally the EBF TT wanted to extend the survey to more practical considerations and more specifically to management of procedures, to protocol and report documentation and to archiving data from tissue bioanalysis:
The preferred analytical design for tissue analysis is to prepare CALs and QCs in homogenate from target tissue (53%). A total of 20% of the participants prepare CALs and QCs in plasma. Alternative designs are also used, for example:
• Does your laboratory have a specific SOP for tissue analysis?
• CALs prepared in buffer or extraction solvent and QCs prepared in homogenate from target tissue;
• If tissue analysis is part of a study, does your laboratory have a specific section on tissue analysis within the protocol of the main study (Tox/TK study)?
• Standards in plasma and QCs in homogenate. Finally the survey seems to suggest that the strategy will depend on the stage of development with the preference to try and use study samples matrix (depending on supply) for CALs and QCs in drug development while
• Does your laboratory include tissue analysis in the protocol of the main study (Tox/TK study, etc)?
• Does your laboratory specify the status (GLP/non-GLP) of the tissue analysis in the protocol? • Does your laboratory provide description/guidance of tissue collection in the protocol?
Table 15. Across all drug development phases, do you use method validation or qualification?
Drug discovery (n)
Drug development non-GLP studies (n)
Drug development GLP studies (n)
We use screening methods, i.e., look at ratios between tissue levels or plasma/tissue levels
6
0
0
We use qualified methods (i.e., absolute concentrations) using widened acceptance criteria
12
11
4
We use qualified methods (i.e., absolute concentrations) using acceptance criteria in line with BMV
3
9
3
We use validated (i.e., absolute concentrations) using widened acceptance criteria
0
2
8
We use validated (i.e., absolute concentrations) using acceptance criteria in line with BMV
1
2
8
BMV: Bioanalytical method validation.
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• Do you ‘archive/store’ tissue homogenates after analysis?
Table 16. Does your laboratory provide description/guidance of tissue collection in the protocol?
• How does your laboratory report data of tissue analysis?
Yes (n)
No (n)
Size of tissue sample to be collected
13
7
The majority of the participants (62%) have no specific SOP in place, while 20% of the companies do have a SOP for tissue analysis. Approximately 67% of the companies would include tissue analysis in the main study protocol either in GLP or nonGLP studies, and 33% would manage this as a separate study. Among the companies that include tissue analysis in the main study, 50% of the companies use a specific section of the protocol. The other would have this in the general statement of the bioanalysis section. Approximately 70% of the companies would specify the status (GLP or non-GLP) in the protocol. The content of the protocol regarding tissue collection or preprocessing varied. Some would specify the size of the tissue sample to be collected (65% of the participants), the (sub)section of tissue sample to be collected (45% of the participants), and/or the description of tissue preprocessing/handling (83% of the participants; Table 16 ). Almost 50% of the companies would archive tissue homogenate as they do for plasma samples. Some (~20%) would archive but with a duration independent from plasma samples. Approximately 24% would archive at sponsor request. The remaining companies would discard the samples.
(Sub)section of tissue sample to be collected
9
9
Description of tissue preprocessing/handling
15
3
Conclusion As mentioned at the beginning of this manuscript, the EBF is committed to investing time and effort to share current practices (technical and scientific) on the extent of ME performed by the member companies and on the level of regulatory compliance claimed at each stage of development. More specifically, the focus of this survey was to understand the frequency of tissue analysis, the level of experience, the level of quality (scientific/regulatory) and/or of compliance to GLP, the rationale for tissue analysis, the nature of the samples collected and the samples measured and finally the strategy of bioanalysis applied to sample measurement. The diversity of answers in some aspects highlights the fact that tissue analysis is very complex and dependent on scientific purpose. The extent of ME will depend on the future use of the data. But whether regulatory or exploratory, it appears that what matters most for all participants is the fit for purpose of ME to ensure adequate quality of the data to allow valid decision-making. This survey provided the team a good starting point for their discussions and resulted in an EBF recommendation.
Executive summary • Within the bioanalytical community, 61% of laboratories perform tissue analysis sporadically and only 22% on weekly basis. • Only around 17% of the participants of the survey perform analysis under GLP, others would follow GLP principles. • Brain and liver are the most frequently analyzed tissues in drug discovery and drug development. Tissue analysis is mostly carried out in rodents. • Taking a representative sample, the process of harvesting/freezing samples and consequently stability assessment, as well as the homogenization process are key scientific and technical points that needs to be taken into consideration. • Method establishment: the survey showed that in drug discovery and drug development the method used is mainly the method that is in place for plasma analysis with small changes in sample preparation. Methods are mainly established per compound. • A qualified method with widened acceptance criteria compared to the bioanalytical method validation guidance is used in drug discovery as well as in non-GLP studies in drug development. Companies tend to start using validated methods, either with widened or the same acceptance criteria as the bioanalytical method validation guidance, as soon as tissue analysis is performed within a GLP study in drug development. • The preferred analytical design for tissue analysis is to prepare calibration standards or quality controls in homogenates from target tissue (53%). A total of 20% of the participants prepare calibration standards and quality controls in plasma. • The majority of participants (62%) have no specific standard operating procedure in place while 20% of the companies do have an standard operating procedure for tissue analysis.
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Special Report Mokrzycki, Delrat, Meulder et al. Future perspective This survey on bioanalysis of drug in tissue among the EBF community provided the team a good starting point for discussions and resulted in an EBF recommendation published in this issue of the journal. Acknowledgments The EBF subteam on tissue analysis thanks all EBF participants in this survey without whom getting this extensive overview would not have been possible.
References Papers of special note have been highlighted as: • of interest 1
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Financial & competing interests disclosure The authors have 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.
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