Bioanalytical Support in the Development of BiologicsThe Need for Sensitivity Combined with Broad Assay Range – Case Studies

Dr. Christian Pieper

Summary

Biologics are highly complex molecules which havetheir origin in biological processes and are used to ob-tain high efficient drugs. The most common class of bi-ologics are monoclonal and bi-specific antibodies whichcan be difficult in finding of optimal dosing. As a con-sequence of safety considerations, initial dosing in clin-ical trials is often very low and may require ultra sensi-tive ligand-binding assay (LBA) support. In addition,the therapeutic dose of antibody drugs can vary sig-nificantly, depending on binding targets, potency andphysiological effects. For dose range finding, not onlysensitivity but equally important broad assay range isa key feature. Most ultra sensitive LBA technologieshowever, lack continuous dynamic range. Exponentialsignal amplification on Immuno-PCR (IPCR) basedImperacer® platform combines broad assay range ofbetter than 4 logs with excellent sensitivities for op-timal PK sample testing support.

Background

Macro molecular drugs differ from small molecule drugsin many aspects including size, structural complexityand production. Utilizing biotechnological and bio-chemical methods, biologics are either purified fromnatural sources and modified or are re-engineered andoptimized from the gene of interest. Both cases leadto extreme specialized and highly effective therapeuticmolecules. One example for the unexpected high ef-ficacy with significant consequences for future clinicaltrials was the TGN1412 tragedy in 2006. The appli-cation of an anti CD28 antibody to test persons in afirst-in-man-study led to severe immune reaction withmulti organ dysfunction syndrome. Although the ini-tial dose was low, regulatory authorities as a conse-quence adapted the initial dose calculation to reducethe overall risk.In fact, in the beginning of a trial the final dosing isunknown and many unpredictable factors like ADME(adsorption, distribution, metabolism and elimination)or other physiological effects can influence the dosing.Thus, dosing will be tested in a broad range startingwith low concentrations. A broad dynamic range ofthe analytical method is therefore indispensable for op-timal evaluation of dosing and ultimately to generatefull PK profiles. The formation of anti-drug antibodies(ADA) is another fact to keep in mind, which posi-tively correlates with the duration of application andthe re-introduction of the drug. Immunogenicity leadsto faster proteolytic elimination and increased clear-ance, leading to reduced detectable amounts in pa-

Fig. 1: Imperacer® Technology. Exponential signal amplifi-cation of antibody binding events by qPCR boostsimmuno-assay signal read out.

tient’s samples, again asking for sensitive LBA meth-ods.In summary, when it comes to biologics testing, ultrasensitive assay platforms are highly recommended toaccount for low dosing or fast clearance. However, theoptimal biologic assay should, in addition to sensitiv-ity, provide a broad assay range to avoid cumbersomeand time-consuming re-testing of “above limit of quan-tification” (ALQ) samples wherever possible.Immuno-PCR based Imperacer® technology (see fig-ure 1) combines ultra sensitivity with broad assayrange, by exponential qPCR signal amplification ofimmunoassay binding events. As a CRO special-ized on ultra sensitive assay development with qual-ity bioanalysis, Chimera Biotec will identify the bestsuited technology for the requirements of each trial.The case studies listed below represent ultra sensitiveImperacer® assays to quantify typical antibody biolog-ics.

Case Study 1: Monoclonal Antibody Drug

As part of a technology evaluation study, anImperacer® assay to quantify a therapeutic antibodyfor cancer treatment, was developed with preliminaryassay range of 2 pg/ml – 32 ng/ml and good accuracyand precision (see figure 2). Using two anti-idiotypicantibodies, a bridging assay was established with abroad detection range of greater 4 orders of magnitude.Optimal assay performance was found at a sample di-

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lution ratio of 1:5 in a specific Any Source® (ChimeraBiotec) sample dilution buffer, corresponding to a totalneat sample requirement of 12 µl per run in duplicate.Selectivity testing at 6 pg/ml was tested in 10 humanserum samples from healthy volunteers to define LLOQlevel. Prozone (hook) effect was found starting around1 µg/ml, however dilution linearity confirmed that con-centrations up to 10 µg/ml can be diluted into assayrange. Higher concentrations are accessible, howeverwere not tested at this point. This assay, as developedfor a feasibility evaluation, has the potential to signifi-cantly reduce time and effort in LBA PK sample test-ing support for this biologic, as the sponsor uses twodifferent platforms so far. The assay is not yet fully op-timized and can be further adapted towards additionalstudy requirements prior to method validation.

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Fig. 2: Recovery plot for calibration curve and matching QCsfor quantification of an antibody (drug). Assay rangeis 2 pg/ml – 32 ng/ml.

Case Study 2: Bi-specific Antibody Drug

An Imperacer® assays to support the development ofa bi-specific antibody therapy in cancer treatment wasestablished with an assay range of 1 pg/ml – 6 ng/mland good accuracy and precision (see figure 3). Aimingfor phase III support in human plasma samples, assayfeasibility was carried out with high dosed cynomolgusmonkey plasma samples from a previous TOX study.Sample parallelism was confirmed up to 1:10,000,000-fold dilution. With 30 µl sample volume per analysis induplicate, the protocol is suitable for both preclinicaland clinical testing support.

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Fig. 3: Calibration curve for quantification of a bi-specific an-tibody with an assay range of 1 pg/ml – 6 ng/ml.

Case Study 3: Diabody Drug

Diabodies are dimers of single-chain variable fragments(scFv) which are fusion proteins of the heavy- and light-chain variable regions of immunoglobulins, intercon-nected by a short peptide linker. As the linker is toshort to allow pairing of the two domains, they areforced to dimerize with their complementary domainfrom another chain and form a diabody.Towards clinical PK sample testing support of an on-cology therapeutic bi-specific diabody, an Imperacer®

assay ranging from 100 fg/ml – 800 pg/ml was devel-oped (see figure 4). In the assay, the diabody drugis bridging the diabody target and an anti-idiotypicdetection antibody. With sub-pg/ml sensitivity, theassay range of better than 4 orders of magnitude wasdesigned towards the expected dosing regime. Yet, asthe upper plateau is not reached, the assay range canbe extended to even higher, upper diabody concentra-tion if needed. In addition, the assay was evaluatedfor microsampling support, with a massive reductionin required neat sample volume to less than 5 µl for adouble determination.

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Fig. 4: Calibration curve for quantification of a bi-specificdiabody (drug) with an assay range of 100 fg/ml –800 pg/ml.

Summary

The vast range for drug concentrations in PK sampletesting during the development of biologics poses prac-tical issues in bioanalytic support. While sensitivity isneeded to capture low dosing or late time points despitefast clearance, high drug concentrations need to be tol-erable to reduce re-testing effort and time. Broad assayranges combined with sub pg/ml sensitivities make theImperacer® platform the LBA technique of choice forbiologics support.

Contact

Dr. Christian Pieper, Senior Project ManagerChimera Biotec GmbHT EU: +49-231-9742840 | US: +1-617-8616018B pieper@chimera-biotec.comm www.chimera-biotec.com

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