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European Biopharmaceutical Review

Flow Cytometry Outsourcing

As drug development of biological medicines increasingly demands custom complex assays to address compound-specific questions and biomarker requirements, flow cytometry technology is quickly advancing into an advantageous solution for meeting these needs.

The landscape of medicine is changing. Small molecule compounds flooded the market in the 20th century, whereas the 21st century has seen a rise in large molecule drugs. It is estimated that by 2015 half of all marketed drugs will be biologics and the pipelines of both large pharma and biotech companies are already filling with potential new biologic therapies (1). It is not surprising that biologic therapies are attractive prospects for commercial drug development. Many have demonstrated superior efficacy with less side effects due to the targeted nature of their mode of action, and the difficulty and time required for their development offers some protection from generic competition. Understandably, the clinical development of biologics does require significant scientific expertise and considerable expense. In addition to safety, efficacy and dosage concerns, clinical trials involving biologic drug candidates must consider immunogenicity and the inclusion of biomarkers to optimally assess in vivo effects.

Critical Path

Initiative In 2004 the US Food and Drug Administration introduced the Critical Path Initiative with the intent of modernising drug development by incorporating recent scientific advances, such as genomics and advanced cell analysis technologies, into the process (2). The outcome should be better science during clinical trials, accelerated and improved drug development, and healthier patients. As a result, clinical trial laboratory testing is becoming more complex and sophisticated testing is required to evaluate new drug candidates. For biologic drug candidates, laboratory testing during clinical trials now typically includes technologies not historically used in this arena, such as microarray and flow cytometry.

Flow Cytometry

Flow cytometry is capable of highly advanced, complex cellular analyses. A flow cytometer is an instrument equipped with lasers, detectors and a fluidics system capable of multiple measurements from a single cell (3). As cells flow past one or more laser beams, scattered and fluorescence light signals are emitted and captured, allowing sophisticated analysis software to identify specific cellular subpopulations and perform phenotypic and/or functional measurements of the cells. Flow cytometry originated as a cellular analysis tool in basic research laboratories (4). Decades later, the requirement to gather cell-based information from patients with oncological or immunological disease, as well as the availability of affordable bench-top cytometers, fuelled the migration of this technology into the clinical diagnostic laboratory (5). Now, decades later, the transition within the drug development industry from small to large molecule compounds has driven the implementation of flow cytometric testing within clinical trials.

Biomarkers

Biomarkers are becoming increasingly more important in trial design, with the trend in clinical development expected to shift toward biomarker-driven trials instead of ‘endpoint’ type outcomes. An informative biomarker included in a clinical trial can alert investigators early on as to whether the drug effect is as expected. Early indication that a drug is working or not can significantly affect the total cost of bringing a new drug to market. Because of the nature of biological drugs, cell-based analyses will be necessary during the drug development process. Between 2003 and 2010, the FDA approved 32 new drugs for oncology; more than half of these were granted accelerated approval, which allows the FDA to approve a new drug using surrogate endpoints expected to predict clinical benefits (6). Cellular assays are being used to fill biomarker needs, and implementation within biological drug development is expected to grow over the next decade. In fact, biomarkers and genomics top the list of services that biopharmaceutical sponsors anticipate to grow the fastest for central laboratory outsourcing needs (7).

 Customisation for Outsourcing

Burdened with expiring patents, shrinking pipelines and escalating financial pressures, the pharmaceutical industry is outsourcing more of its research to contract research organisations (CROs) (8). However, contrary to small molecule safety testing (for example, chemistry, haematology and urinalysis), development of biological medicines requires custom complex assays to address compoundspecific needs. Test menus no longer meet clinical trial outsourcing needs; protocol-specific questions differ for biologic and non-biologic compounds. It is likely that a custom assay will be required for every new biologic in the market; for example a receptor occupancy assay must be specific for the compound and its target and cannot be applied to another biologic. In many instances the assay costs will be higher for the custom work, since the same assay cannot be utilised for other compounds. Furthermore, outsourcing requirements for each biologic will be unique, unlike conventional small molecule drug trials. Outsourced work should be entrusted to a laboratory with both scientific expertise in immunological assays and balanced global infrastructure to conduct Phase 2 and 3 studies. A trend in the drug development industry has been, and is predicted for the foreseeable future, ever increasing outsourcing to CROs (9).

Model System Using RO Assay

Since monoclonal antibody-based drug therapeutics bind to specific cell surface receptors, it is often important and useful to quantify the receptor occupancy of a specific cell subpopulation. Receptor occupancy assays use fluorescence-based techniques and flow cytometry to measure the receptors occupied by a drug. Early approaches using this concept were conducted using neutrophils and platelets, but any cell subset present in peripheral blood is amenable to this technique (10,11). Receptor occupancy assays require substantial scientific expertise for their development as these are complex, custom methods (12). A typical approach will involve the use of competitive (binding is blocked by the investigational drug) and non-competitive (binds to a different epitope of the antigen targeted by the investigational drug) fluorochrome-conjugated monoclonal antibodies (see Figure 1). In addition, specific cell subpopulations may need to be labelled and differentiated; for example, T or B lymphocytes, to clarify effects. The outcome is a complex flow cytometry panel, generated with multiple controls for use in clinical trials. The end result is a potential biomarker, customised for a particular biological drug, which may yield useful information relevant to dosage, safety and efficacy (13-15).

Custom Complex Assay Challenges

Custom work is usually required for the development, characterisation, standardisation and implementation of laboratory analyses each time a new biological drug is brought to trial. Whereas a standard test-menu chemistry panel may be applicable for several small molecule compounds, receptor occupancy assays for large molecule drugs are unique to the particular biological compound. Thus, each cellbased assay requires development, optimisation and validation prior to implementation for patient samples. The validation process often utilises in vitro incubation with the drug under investigation in order to devise a model system where plasma membrane proteins are occupied by drugs to mimic the in vivo state. Within the clinical trial itself, the drug-receptor binding step will occur upon infusion of the compound.

Cell-based assays commonly incorporate leucocytes in the test model and therefore whole blood from healthy volunteers is usually collected for validation experiments. If the receptor to which the drug binds is not expressed on the cell surface of leucocytes from healthy volunteers, innovative approaches must be undertaken to identify an appropriate sample type for validation that will adequately mimic the clinical scenario. Some examples include: leucocytes isolated from healthy volunteers, which are activated in vitro to express the desired cellular antigen; cell lines which express the marker of interest, used as a homogeneous suspension or spiked into healthy whole blood samples; or cellular blood products which may be acquired from commercial vendors from donors of an appropriate disease state.

Although much of the routine clinical safety testing is automated, flow cytometry methods in contrast are mostly manual procedures. Typically, custom flow cytometry assays include manual pipetting procedures to label cells with fluorochrome conjugated monoclonal antibodies and lyse undesired red blood cells. Manual sample handling is then required for centrifugation, decanting of excess wash fluid and transfer of samples for acquisition on the flow cytometer. Resultant listmode data files must then be analysed on a custom basis by experienced scientists to obtain valid results. With cell-based assays being compound- and trial-specific, it is often not cost-effective or feasible to automate the method procedures. Batching of samples is not always possible due to stability concerns or worthwhile in trials where infrequent sampling and low enrolment rate results in a low production pace.

Sample stability is a major concern in global clinical trials, especially for cellular assays using whole blood. Typically, blood is collected in vacuum tubes through venipuncture, before being packaged and transported via a courier to the testing laboratory. The challenge is to ensure delivery within the determined timeframe where the testing results will not be skewed due to changes that may have occurred during transport. Cells start to die as soon as the blood sample is removed from the body. In general one to two days post-collection is required for sample transport, although it is often more challenging in remote geographical areas. Logistically, unexpected weather, volcanic eruptions, earthquakes and other ‘acts of God’ can challenge sample transportation from the clinical site to the testing laboratory, with international shipping regulations and customs causing additional delay. A well-characterised assay is essential in determining how to differentiate the effects of shipment, post-collection storage effects, disease state and drug effects on cell death in clinical samples.

Advantages of Outsourcing

Central laboratories with the capability for customised assay development and validation, who are proficient in transitioning and implementing a standardised assay into global clinical trials, can offer both large pharma and small biotech companies fit-for-purpose biomarker outsourcing solutions. Collaboration between the sponsor and outsourcing laboratory scientists is essential to define appropriate testing (see Table 1). Initial assay development, or characterisation of a sponsor developed method for precision, stability and accuracy may be required in outsourcing. The sponsor may request a qualification run(s) with split samples to ensure the results obtained at the outsourced laboratory are comparable to results obtained by the assay developers. Alternatively, a technology transfer may be appropriate where several samples are tested that exhibit varied expression levels of the desired results, such as healthy donors, diseased state, spiked samples or cell lines. Well-established communication between the sponsor and the outsourcing laboratory is vital for streamlined assay development and successful implementation.

The central laboratory offers a significant degree of standardisation to the extent possible for custom cellbased assays implemented globally.

Identical flow cytometry platforms, trained personnel, validated SOP driven methods, and centralised result peer review together serve to minimise variation in the final data set. In fact, validation experimental designs often incorporate assessment of inter-instrument and inter-analyst differences observed within a method. This ensures seamless delivery of standardised results when there is an unexpected instrument failure or changes in personnel – this is especially important in trials that endure for several years.

Selection of a superior outsourcing laboratory requires consideration of scientific capability, flexibility to customise, and infrastructure to manage and perform the trial requirements. If all trends continue, biopharmaceuticals will continue to increase in number as physicians and patients strive for better therapeutic modalities. Likewise, the use of flow cytometry for clinical trial testing will continue to increase, as evaluation of cell-based biomarker assays become a necessity in the assessment of biologic drug candidates (16-19).

References

  1. Sood N, Rise of biologics, www.ngpharma.com, 2010
  2. Food and Drug Administration, Challenge and opportunity on the critical path to new medical products, United States Department of Health and Human Services, March 2004
  3. Stewart CC, Clinical applications of flow cytometry: immunologic methods for measuring cell membrane and cytoplasmic antigens, Cancer 69: pp1,543-1,552, 1992
  4. Robinson JP, Mack Fulwyler in his own words, Cytometry 67A: pp61-67, 2005
  5. Preffer FI, Flow cytometry, Diagnostic Immunopathology, Raven Press, New York: pp453-473, 1988
  6. Wadman M, FDA speedier than European agency on cancer drug approvals, blogs.nature.com, 16 June 2011
  7. Industry Standard Research, The state of the central labs market: p9, 2010
  8. Ledford H, Contract investigators, Nature 466: pp280-281, 2010
  9. Wadman M, The quiet rise of the clinical contractor, Nature 441: pp22-23, 2006
  10. Brunkhorst BA, Lazzari KG, Strohmeier G, Weil G, and Simons ER, Calcium changes in immune complex stimulated human neutrophils: simultaneous measurement of receptor occupancy and activation reveals full population stimulus binding but subpopulation activation, J Biol Chem 266:13,035-13,043, 1991
  11. Tsao PW, Bogarth JM, Jackson SA, Forsythe MS, Flint SK and Mousa SA, Platelet GPIIb/IIIa receptor occupancy studies using a novel fluoresceinated cyclic Arg-Gly-Asp peptide, Thromb Res 77: pp543-556, 1995
  12. Woska JR et al, Small molecule LFA-1 antagonists compete with an anti- LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow cytometry based receptor occupancy assay, J Immunol Methods 277: pp101-115, 2003
  13. Reinhart K et al, CD14 receptor occupancy in severe sepsis: results of a phase I clinical trial with a recombinant chimeric CD14 monoclonal antibody, Crit Care Med 32: pp1,100-1,108, 2004
  14. Waibler Z, Sender LY, Kamp C, Muller Berghaus J, Liedert B, Schneider CK, Lower J, and Kalinke U, Toward experimental assessment of receptor occupancy: TGN1412 revisited, J Allergy Clin Immunol 122: pp890-892, 2008
  15. Muller PY and Brennan FR, Safety assessment and dose selection for first in human clinical trials with immunomodulatory monoclonal antibodies, Clin Pharmacol Ther 85: pp247-258, 2009
  16. Hill C, Wu D, Ferbas J, Litwin V and Reddy M, Regulatory compliance and method validation, In: Litwin V and Marder P, eds, Flow Cytometry in Drug Discovery and Development, Wiley-Liss, 2010
  17. Wu D, Patti-Diaz L and Hill C, Development and validation of flow cytometry methods for pharmacodynamic clinical biomarkers, Bioanalysis 2: pp1,617-1,626, 2010
  18. O’Hara DM, Xu Y, Liang Z, Reddy MP, Wu DY and Litwin V, Recommendations for the validation of flow cytometric testing during drug development: assays, J Immunol Meths 363: pp120-134, 2011
  19. Green CL, Brown L, Stewart JJ, Xu Y, Litwin V and McCloskey TW, Recommendations for the validation of flow cytometric testing during drug development: instrumentation, J Immunol Meths 363: pp104-119, 2011

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Carla G Hill specialises in transitioning flow cytometry biomarker assays into clinical trials, as evidenced by her published articles. She serves on the MetroFlow NY/NJ Flow Cytometry Steering Committee and the Medical Laboratory Technology Advisory Commission at Mercer County Community College, New Jersey. Her credentials include an MS Biology from Georgian Court University in New Jersey, a BS in Microbiology from Pennsylvania State University, Pennsylvania, and Medical Technology ASCP certification. Email: carla.hill@iconplc.com

Thomas W McCloskey specialises in the validation of complex novel cell-based assays for use in clinical trials. He obtained his BS in Biology from Hofstra University, New York, and his PhD in Immunology from Rutgers University, New Jersey. Thomas has authored more than 40 manuscripts. In 1998, Thomas won the Presidential Award of Excellence, which recognised him as the ‘Top Young Investigator in Flow Cytometry’ in a worldwide competition. Email: thomas.mccloskey@iconplc.com

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