Customizing MLR’s

Fit-for-purpose MLR’s:

If a mixed lymphocyte reaction (MLR) is to be conducted in such a way that relevant and actionable data is obtained, then the MLR experimental design must be adapted to take into account the different material properties and mechanisms of action of each test article or therapeutic when designing a study. Customizing the MLR can be as simple as adjusting culture media for solubility (e.g. therapeutic in DMSO) or it can be as complex as optimizing cell concentrations and timepoints to obtain accurate cytokine data.

For example, testing an antibody therapeutic for checkpoint inhibition will require a different approach than using the MLR to assess potential immunosuppressive properties of a demineralized bone matrix. The former can easily be spiked into the culture as a liquid, whereas the latter is often a dense slurry or paste that requires titrating by volume or mass. Biomaterials pose unique challenges as they are unable to diffuse like aqueous therapeutic formulations, meaning that careful consideration is needed to ensure that the responder test cells of the MLR are evenly spread across the material at high enough density for a measurable response.

MLR Optimization:

Below is a list of the most common factors to consider when designing an MLR. Each of these factors should be either optimized or verified as appropriate for the intended use (e.g. fit for purpose).

• • Cell source and type: allo, auto, xeno, isolated T-cells, PBMCs, etc.
  • Assay format: one-way MLR, two-way MLR, DC/T-cell
  • Concentration: titrated therapeutic and test cells
    • Can often depend on plate type: flat bottom, v-bottom, u-bottom, 96-well, 24-well, etc.
  • Culture conditions: media, incubation time, gas mixture, agitation
  • Readouts: acute cytokines, intracellular cytokines, proliferation, phenotypic changes, etc.
  • Controls: negative and positive controls, comparative controls to commercial competitor, autologous controls, allogeneic controls, longitudinal controls, readout specific controls, etc.

Regulatory Requirements:

The degree to which an MLR will need to be optimized or customized depends heavily on the phase for which the study is being performed, as well as its intended purpose. As researchers work with regulatory agencies to bring their product to market, these agencies may provide additional recommendations on desired assay performance requirements.

Three basic regulatory classifications in which an MLR assay may be performed under are Research Use Only (RUO), Good Lab Practices (GLP), and Clinical Laboratory Improvement Amendments (CLIA). There are also other regulatory and hybrid systems (GMP and GCLP) but those are beyond the scope of this page. Each of these have different requirements that must be met.

Research Use Only (RUO)-

RUO studies are essentially unregulated (neither GLP nor CLIA). As such, they do not need to adhere to strict guidelines for documentation or assay verification. They cannot be used for patient diagnostics or treatment decisions.

RUO assays are often appropriate for pre-clinical research and development, where the assay may only be performed a limited number of times. RUO studies do not have to undergo validation, qualification, or even extensive development. The degree to which the assay should be optimized and characterized will depend entirely on the individual project and how confident the researchers want to be in the results. For projects requiring the testing of large quantities of samples, or repeated testing over longer periods of time, it is often advised that optimization and qualification be performed.

Though an assay may only be run under RUO, that does not mean that the testing facility should not follow GLP or CLIA standards. Reagents and equipment should always be routinely maintained, performance verified, and monitored. Xeno Diagnostics adheres to a strict GCLP standard and RUO assays are all performed as “GLP-like”. Ideally, the primary difference between RUO and GLP are the documentation requirements. An RUO assay may not include as much documentation for pre-planning (study plan), as extensive characterization (validation), nor as detailed quality assurance monitoring as a GLP study. In all other respects, GLP guidelines are followed to ensure that good science is being performed.

Good Laboratory Practice (GLP)-

GLP is a management system whose framework was written into law as the Code of Federal Regulations (CFR) Title 21: Food and Drugs, Part 58: Good laboratory practice for nonclinical laboratory studies (21 CFR Part 58). GLP guidelines are intended to assure quality and integrity of data, facilitate study reconstruction, and provide overall accountability of nonclinical studies that are intended to support the production of food additives, cosmetics, therapeutics, or medical devices.

GLP regulations cover many areas beyond the performance of an assay. Briefly, some of those areas are material storage, facility management, quality assurance, personnel and director qualification and responsibilities, archival and documentation, equipment, standard operating procedures (SOPs), and electronic systems. An MLR performed under GLP must be thoroughly optimized (developed) and characterized (validated). Both the culture method (MLR) and its chosen readout should be validated, and depending on the analytical method used, there may be recommended industry guidance that should be followed.

Clinical Laboratory Improvement Amendments (CLIA)-

The Clinical Laboratory Improvement Amendments (CLIA) of 1988 was intended to regulate all non-research laboratory testing performed on humans, with the objective to ensure patient results are accurate, reliable, and timely. Much like GLP, CLIA is a management system that covers similar areas from testing personnel, facility management, documentation, testing, quality assurance and more. The specifics and differences between GLP and CLIA are beyond the scope of this page; however, it is important to know that like GLP, CLIA has its own guidelines that determine how an assay should be developed, and what criteria an assay must meet to be considered validated and ready for use.   

Xeno Diagnostic’s MLR Services:

From evaluating mechanism of action to potency, and from immunogenicity to transplant tolerance, the MLR is a versatile platform that can be utilized for many therapeutics in the development pipeline. With the available variations in format and readouts, Xeno Dx experts are adept at providing custom MLRs tailored to fit the exact needs of the therapeutic. Every therapeutic is unique, so the MLR should be as well. Contact us to learn more about how we can leverage the MLR to your benefit.