Director of Scientific Services AIT Bioscience Indianapolis, Indiana
As the applications of novel formulations and drug approaches including gene therapy, make traction in the drug development world. Bioanalysts are challenged with addressing the cutting edge of analysis. Sometimes this requires novel technologies, unique troubleshooting approaches, reanalysis of validation requirements and introductions to analysis of new tissues. Sometimes it involves all of these. The analysis of small molecule drug candidate X must take place in bone as the unique formulation of this drug is applied directly to bone during a surgical procedure with an intent to reduce systemic exposure. A validation of drug X was conducted taking into account the traditional measures of valid quantitation but also addressing ensuing gaps and challenges discovered along the way. This presentation will discuss the need to develop and qualify a cleaning protocol for homogenization equipment between samples; the suitability of samples for analysis and potential impact to quantitation, in this case whether the presence of residual flesh could bias results; as well as the establishment of the correct matrix for stability establishment and additional requirements upon processing to that suitable matrix. The analysis of biotherapeutic Y required analysis in multiple selected matrices, including bone collagen scaffold and bone marrow, based on an understanding of the matrices targeted for protein expression after administration of a dosed gene therapy. An existing ligand-binding (LBA) kit for analysis of the expressed protein was commercially available and was used to assess protein expression across a series of tissues. Unexpected quantitation of the expressed protein was encountered in the bone tissue. The utilization of immunocapture-LC-HRMS as a perpendicular tool to the LBA assessment allowed for investigation of the ligand binding results. IP-LC-HRMS utilized a surrogate peptide approach to quantitate the expressed protein. This approach demonstrated that the protein levels observed using the LBA kit did not reflect unwanted protein expression in the bone tissue, but rather was a problem of assay specificity for the LBA kit assay in this matrix, a problem that was resolved utilizing the orthogonal IP-LC-HRMS technique. Both of these examples showcase the evolving nature of bioanalysis and the versatility required of bioanalysts as new and unusual matrices move from secondary qualification to primary validations.
Describe the challenges of bone as a bioanalytical matrix including implications and tactics of flesh removal, bone processing, and recovery quantitation.
Observe both small and large molecule analysis from bone matrix utilizing LBA, LC/MS/MS and hybrid IP/LC/HRMS.
Understand the additional bioanalytical experiments that may be required to confirm valid quantitation and specificity from bone matrices.