Webinar Q&A follow up: biomarker assays – bioanalytical meets CLIA

Thank you everyone who attended our webinar ‘Biomarker assays – bioanalytical meets CLIA’ in association with ICON. Below are responses to the questions posed by our audience during the live event. We hope this is a useful resource and thank those who submitted these thoughtful questions.

(Q) How do you classify a CLIA validation outside the USA? New EU IVDR regulations will come into play during 2022, which will provide more stringent regulations for labs based in the EU. Global laboratory networks such as ICON Central Labs will operate off the same global procedure and will maintain CAP accreditation across all labs therefore ensuring the CAP/CLIA standard is applied globally.

(Q) How can you determine if the data from exploratory endpoints, which may be included in a submission, will not be used in a regulatory decision? It should be understood that any data collected as part of a clinical protocol may be submitted to a regulator. The use of biomarker data can range from internal decisions supporting (or not) further clinical development decisions for a therapeutic, provide supporting evidence for safety or efficacy, to supporting a label claim. FDA issued a Guidance on Multiple Endpoints in Clinical Trials in January 2017 that discussed the hierarchy of families of endpoints. Using a biomarker as an exploratory endpoint may still include clinically important events but those are expected to occur too infrequently to show a treatment effect sufficient for a regulatory decision. It would not be unreasonable that subsequent clinical protocols might list a particular biomarker as secondary or primary endpoints once an effect could be correlated to its measurement.

(Q) FDA BMV requires QC to be within 15%. This is for acceptance of the method, but when using the method why do labs still use 15% when actually the %2B/-2SD of the method indicates and accept/reject should be much tighter, meaning “failed” runs are accepted. Do you think acceptance of a run should be 15% or a “real” tighter limit? The FDA BMV is a guidance document and outlines current regulatory thinking on bioanalysis. One should never forgo sound scientific judgement and if the measurement of a biomarker requires tighter tolerances for decision making then those specifications should be called out in a sample analysis plan. Conversely, if an internal decision can be made with looser tolerances of method accuracy, then this should also be taken into consideration when defining run acceptance criteria during sample analysis.

(Q) Is minimum dilution required for pk assays? (following the procedure as per FDA) The minimum required dilution was defined in a 2003 Whitepaper (DeSilva, et al., Pharmaceutical Research, 20, 1885, 2003) as the minimum magnitude of dilution to which a sample must be subjected to optimize accuracy and precision in an assay run with a specified standard and sample diluent. If a particular method is not impacted by matrix effects, then a sample may not require dilution (unless the instrument response is above the dynamic range of the method).

(Q) With increasingly global studies, what are the equivalent regulations to CLIA? I am used to saying GLP or GCP and having everyone know what I am talking about, but CLIA is US-only? CLIA is a US centric standard that is and can be held by labs both inside and outside of the US. Global laboratory networks such as ICON Central Labs will operate off the same global procedure and will maintain CAP accreditation across all labs therefore ensuring the CAP/CLIA standard is applied globally. New EU IVDR regulations will come into play during 2022, which will provide more stringent regulations for labs based in the EU.

(Q) Do ASRs always need to be produced according to GMP standard? FDA issued a guidance document answering frequently asked questions on Commercially Distributed Analyte Specific Reagents (ASR) in 2007. ASRs are medical devices that are regulated by the FDA. They are subject to general controls, including current Good Manufacturing Practices to adequately control the risks associated with using these devices given that they are building blocks of LDTs.

(Q) An LDT can also be used as an IVD class III in a single site PMA, right? The exact intent of the question and use of the LDT is not clear. However, given the high risk associated with class III devices it is advisable to discuss specifics with a regulatory professional prior to providing results from the potential class III device to clinical sites.

(Q) Is testing for pre-existing antibodies in patients prior to say AAV administration considered an LDT? Also, does this become a CDx as the program proceeds through the development cycle (if we are using the test to exclude patients with pre-existing antibody)? Developing, validating and performing a screening assay to measure pre-existing antibodies to a therapeutic can be challenging because commercial reagents (e.g., positive controls, detection antibodies) often do not exist. As such the work is often done by an individual laboratory and is therefore a laboratory developed test. FDA have deferred a decision to issue a final guidance regulating LDTs and the laboratory must therefore rely on a 2014 FDA draft guidance. For further insights, please see the FDA Discussion Paper on Laboratory Developed Tests (LDTSs) issued January 13, 2017.

An LDT can continue to be used throughout the clinical development of the specific therapeutic. The laboratory performing the LDT may seek regulatory approval for the assay (i.e., premarket approval (PMA) to then evolve the LDT into a companion diagnostic). More often, the laboratory will collaborate with an in vitro diagnostic (IVD) company and the drug developer to produce a diagnostic kit after the IVD manufacturer obtains an investigational device exemption (IDE) to utilize the assay in clinical trials.

(Q) CDRH does not agree with dilution linearity when clinical cut off is higher than assay ULOQ. Any comment on this? An optimally designed clinical assay would ensure the clinical cut off relevant to the intended use statement sat within the AMR of the assay rather than the CRR. Have you considered consulting CLSI EP34?

(Q) What options are there for bioanalytical labs to conduct inter laboratory platform comparison? If two (or more) laboratories generate study data for the same analyte in the same study or across multiple studies wherein the data are being submitted for regulatory decision, the laboratories must perform a method cross-validation. That does not necessarily imply that the same analytical platform need be used, only that the concentration results obtained from each method are comparable. Most commonly, the laboratories would share sets of Quality Controls (low, medium and high) that are analyzed in at least triplicate and when feasible, study samples (n ≥ 30) that span the ample concentration range. Bias might be assessed by Bland-Altman plots or Deming regression.

(Q) What general TEa do you use? Are the TEa specifications set during assay development/validation and do they need to be lowered according to patient risk associated with the effectiveness of a device? Analyte specific TEa’s, which define the total amount of error that can be tolerated without impacting an accurate clinical assessment, are assigned according to numerous rules including:

    1. Regulatory requirements imposed by bodies such as CLIA
    2. Industry guiding bodies such as NGSP
    3. In house studies, which assess the acceptable analytical imprecision and bias of a method
    4. Occasionally via understanding the biological variation of a method.

Where possible it is optimal to set the TEa during the development/validation process and where the practical capabilities of an assay allow – it is optimal to lower the TEa to better improve assay performance and thereby the quality of results created for clinical assessments.

(Q) Is the biomarker in your presentation mainly a large molecule? Will the strategy presented for context-of-use apply to other types of biomarkers? While it is true that a good majority of biomarkers are large molecules, the concept of context-of-use applies equally to small molecules. A pharmacodynamic biomarker could be endogenous metabolites such as shown in the Crigler-Najjar genetic disorder case study (i.e., the clearance of bilirubin and the correlated appearance of bilirubin glucuronide). The bioanalytical methods to measure bilirubin and bilirubin glucuronide would be validated in accordance with FDA BMV Guidance.

Cellular biomarkers would similarly follow best scientific practices for their measurement and a flow cytometry method would be assessed in accordance with its intended use.

(Q) Could you give an outline as to what determines bioanalytical validation vs CLIA, you have mentioned both, but it is not clear what the major differences between the two are? Bioanalytical validations follow FDA BMV Guidance to ensure that the data are reliable. The validation will determine if the method is specific or selective for the analyte, accurate and precise, range of quantitation, and the stability of the analyte during sample collection, handling and storage.

A CLIA validation will perform similar assessments to determine the reliability of the data and assure that the test performs as intended. The analytical validation will assess accuracy, precision, sensitivity, reportable range and a reference range for the analyte (i.e., clinical relevance).

(Q) Can you address FDA requirements for clinically validating a biomarker assay?

Please see:


(Q) You mentioned CLIA is applicable for labs based in the US. What is required for labs outside the US testing US patient samples? CLIA is a US centric standard that is and can be held by labs both inside and outside of the US. The intent of the regulation is to stipulate the minimum standards for laboratories reporting clinical results for US subjects. Global laboratory networks such as ICON Central Labs will operate off the same global procedure and will maintain CAP accreditation across all labs therefore ensuring the CAP/CLIA standard is applied globally.

(Q) Why is an IVD assay qualified as ‘FDA exempt’? And why? Most class I and some class II IVD devices are exempt from pre-market notification 510(k) requirements, subject to certain limitations. A device may be exempt from 510(k) requirements if the FDA determines that a 510(k) is not required to provide reasonable assurance of safety and effectiveness for the device.

(Q) Would you recommend that the quality control material used for a biomarker validation, matches where possible the study samples? While the FDA BMV Guidance does indicate that a reference standard should be identical to the analyte it is acknowledged that endogenous protein biomarkers are not easily obtained and most commonly recombinant versions are used. In either case, the identity, purity and stability of the biomarker should be known.

(Q) What platforms are most commonly used at ICON? Within ICON’s Bioanalytical Laboratory, we utilize SCIEX LC—MS platforms for the measurement of small molecule biomarkers and various immunoassay platforms (MSD, Biotek, Tecan, Gyros) to measure protein biomarkers. Within our CLIA laboratory we utilize a wide array of platforms depending on the biomarker. For cellular biomarkers we utilize BD FACSymphony A3 flow cytometers. For cytokine biomarkers we would commonly use Luminex, Ella or Quanterix Simoa. For other protein biomarkers we might employ COBAS 4800 or Roche e601 systems.

(Q) In your sickle cell case study – what would the approach be if both situations are encountered in a protocol – i.e., if you need to screen patients into the study and also follow the effect of therapy – two assays validated under CLIA or BMV, or a single assay that addresses both regulations? The intended use and how results are being reported defined our approach. Because confirmation of sickle cell disease for patient screening did not require measurement of sickle cell haemoglobin the need to have a CLIA method validated was not required. The disappearance of abnormal haemoglobin was considered to be a secondary pharmacodynamic endpoint and therefore we validated the biomarker assay in accordance with FDA BMV Guidance.

If we were to encounter a requirement to use a biomarker assay for patient stratification or dose decisions as well as a pharmacodynamic endpoint, ICON would choose to validate two methods; viz., a CLIA method for clinical reporting and a bioanalytical method for the PD endpoint.

(Q) To transition a bioanalytical validated assay to a CLIA assay, do you need to revalidate or only validate additional parameters required for the CLIA assay? The transition is not so much a revalidation as it is in assessing clinical relevance of the lab developed test or commercial kit. The process of establishing a lab developed test or an IVD is in most aspects identical to what you would perform for a bioanalytical method validation. The CLIA validation is therefore the assessment of additional parameters, e.g., reference range, related to its use in clinical development.

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