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Perspective Duggan, Vazvaei & Jenkins Executive summary


• The use of LC–MS/MS to quantify proteins in biological matrices using surrogate peptides derived from proteolytic digestion (PrD- LCMS) is being increasingly applied to regulated protein bioanalysis.


• No current regulatory guidances specifically apply to PrD-LCMS methods. • This paper discusses recommendations published in a consortium white paper on validation criteria for PrD-LCMS bioanalytical methods.


General consensus • The consortium white paper recommends using the applicable ligand-binding assay (LBA) validation criteria for PrD-LCMS methods. • Recommended QC accuracy and precision would use the 4–6–20 rule. Validation parameters & acceptance criteria for LC–MS protein methods • For standards and QCs, the 20% criteria apply, but they are expanded to 25% accuracy and precision for QCs and standards at the LLOQ.


• For each run two-thirds of the QCs must meet acceptance criteria of within 20% CV (for multiple QCs) and within +/- 20% of the nominal concentration, and 50% must meet criteria at each QC level.


• Calibration curve requirements generally agree with those for small molecule LC–MS methods. Selectivity/specificity • Hybrid PrD-LCMS methods apply both LBA and chromatographic procedures, necessitating re-examination of selectivity and specificity.


• The validation procedure for selectivity/specificity in PrD-LCMS is similar to that used for small molecule LC–MS methods. • Using a minimum of six lots of blank matrix spiked at the LLOQ, 80% of the LLOQ samples should quantify within 25% of the nominal with < 5% interference due to the IS.


Matrix effect • Matrix effect measurements for PrD-LCMS methods are similar to small molecule LC–MS/MS methods, using normally six individual lots.


• Matrix factor is defined as the ratio of the analyte response in the presence of matrix after extraction to that in absence of matrix. Recovery & digestion efficiency • A typical digestion efficiency procedure where a surrogate peptide is used without predigestion affinity capture set is outlined below (each set is run in triplicate): – Set A: the analyte protein therapeutic spiked in to matrix predigestion at L/M/H nominal levels, then digested and analyzed. – Set B: the analyte surrogate peptide spiked in to matrix predigestion at concentrations equimolar to the analyte protein (L/M/H), then digested and analyzed.


– Set C: extracted/digested/quenched blank matrix is spiked with the analyte peptide postdigestion at concentrations equimolar to the analyte protein (L/M/H), then analyzed.


• Parameters are calculated form the peak area from each sample set as follows: – Digestion efficiency: peak area Set A /peak area Set B. – Recovery: peak area Set B/peak area Set C.


Protein assay stability considerations • Protein therapeutic stability should be monitored under employed usage and storage conditions as directed in the current guidances.


• Since PrD-LCMS utilizes a surrogate peptide, the use of monitoring peptides from other portions of the analyte protein molecule can help to provide confidence in stability measurements.


• PrD-LCMS methods must be tested for any factor that could cause biases due to precipitation, aggregation or absorption . • The peptide (or intact protein) stable labeled IS must be periodically tested to determine that it sufficiently corrects for extraction and mass spectrometric variation.


Critical assay reagents • Critical assay reagents that are very specific for an LBA and are difficult to make and maintain may also be an integral part of a PrD-LCMS assay, primarily those that utilize immunoaffinity capture.


• Stability and maintenance procedures must be clearly established for these immunocapture reagents as they are key elements of the assay.


• Assay monitoring procedures with accompanying assessment/maintenance must be developed and maintained for critical assay reagents.


Discussion & conclusion • The application of PrD-LCMS to protein bioanalysis in regulated studies is a very important and growing field. • Here we expand upon some of the concepts developed in the industry consortium white paper published in AAPSJ, January 2015. • These discussions clarify some differences between the criteria for small molecules, LBA assays and those proposed for PrD-LCMS assays.


• Proposed PrD-LCMS validation criteria are only recommendations that will continue to evolve as industry experience grows.


1394


Bioanalysis (2015) 7(11)


future science group


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