Method validation considerations for LC–MS/MS bioanalysis of protein drugs Perspective
Table 1. Comparison of method validation parameters of protein ligand-binding assays and those recommended for protein LC–MS/MS methods.
Parameter Calibration curve regression function
Selectivity/specificity using individual matrix lots (include hemolyzed, lipemic or disease state as appropriate), analyze blanks and LLOQ samples (include standard zero for PrD-LCMS)
Protein LBA
Nonlinear with four or five parameter logistic fit anchor points used
10 lots, 80% of lots fortified at the LLOQ quantify within 25% of the nominal
Fortified with available ‘related molecules’ (ADA, soluble target, circulating ligand) or concomitant drugs accuracy within 25%
Matrix effects individual lots; hemolyzed, lipemic or disease state matrix as appropriate MS signal using individual matrix lots
Recovery (including digestion efficiency) Matrix stability Stock and working solution stability Change in critical assay reagents Critical assay reagent stability NA NA
Within 20% of nominal determine at each storage temperature
May not be required if covered by CofA
May need revalidation
Appropriate testing/stability programs for LBA reagents may be required
Protein LC–MS/MS, using a surrogate peptide (recommended)
Linear fit recommended when possible; nonlinear model may be acceptable with some affinity-capture methods
6–10 lots blanks: <20% of LLOQ <5% of IS LLOQ: 80% of lots fortified at the LLOQ quantify within 25% of the nominal
With affinity capture methods, fortified with available ‘related molecules’ (ADA, soluble target, circulating ligand) or concomitant drugs, as appropriate accuracy within 25%
MF in six lots compare surrogate and SIL-IS peptides in processed matrix and reagent blanks IS-normalized CV within 15% across lots
Overall recovery including digestion should be reproducible. Recoveries for individual steps may be evaluated for troubleshooting
Within 20% of nominal determine at each storage temperature
Compare old versus freshly prepared solutions Protein must be digested; mean values within 10% is justified
Primarily a concern for protein reagents; confirm by consistent and acceptable accuracy and precision in at least one run
Stability is demonstrated by prevalidation testing and acceptability of analytical runs
ADA: Anti-drug antibodies; CofA: Certificate of analysis; LBA: Ligand-binding assays; MF: Matrix factor; NA: Not applicable; SIL-IS: Stable isotope-labeled IS. Adapted with permission from [11] © AAPS (2015).
Matrix effect Matrix effect is a well-known phenomenon in LC– MS/MS assays. It refers
to the matrix-related sup-
pression and/or enhancement of analyte ionization (usually in electrospray mode) when other ions or substances derived from the sample co-elute with the analyte, resulting in potential sample-to-sample inconsistency in the analyte response versus concen- tration. Matrix effects may be quantified by measuring matrix factor (MF) which has been defined as the ratio of the analyte response in the presence of matrix (mea- sured by analyzing blank matrix spiked with analyte postextraction) to the response in absence of matrix (pure solution of the analyte spiked at the nominal concentration). This is an EMA requirement for chro- matographic assays [8]. For PrD-LCMS methods, MF should be determined for the surrogate peptide
future science group
and determination of IS normalized MF is recom- mended. One of the experimental challenges in deter- mining MF is to ensure the results are not affected by nonspecific binding losses of the peptides, a common phenomenon for the nonmatrix comparator samples. To determine MF, six individual lots (fewer may be required for rare matrices and inbred animals) may be evaluated by comparing the signals for the surrogate and the IS peptides in presence and absence of matrix. The IS normalized MF coefficient of variation should be within 20%.
Recovery & digestion efficiency Recovery calculations for a PrD-LCMS method are similar to those used for a small molecule, but the over- all process is more complex because of the proteolytic digestion itself along with any affinity purification steps
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