Method validation considerations for LC–MS/MS bioanalysis of protein drugs Perspective
IS stability When using a stable isotope-labeled peptide IS (SIL-IS peptide) or an intact stable isotope-labeled protein IS (SIL-IS protein) where the isotopic label itself is known to be stable; then the determination of IS stability is generally not necessary, provided that stability is being determined independently for the unlabeled version of this molecule (analyte peptide or protein) under the same storage conditions. One important consideration for the IS is that it be periodi- cally tested to be suitable for its purpose in the assay. This means that the specific chromatographic signal of the IS remains sufficient for the assay; and there should be no significant chromatographic interfer- ence from the IS on the analyte channel, such that any measured interference from the IS should be less than 20% of the LLOQ peak area response of the analyte (per current guidance recommendations for chromato- graphic assays [8,9]). The reproducibility of the IS signal in terms of chromatographic peak area and retention time should also be monitored continually as is done for small molecule assays, and it should be consistent throughout validation and sample assay.
Matrix stability In some cases therapeutic protein stability might be affected by the age of the biological matrix due to vary- ing enzyme activity, fluctuations in the matrix pH or by storage and handling of the matrix prior to use. An example of this, which has been discussed in various bioanalytical meetings, is one where analyte recovery from a once frozen and thawed sample is different from that of a freshly spiked and immediately processed com- parator. Therefore, sample processing and handling conditions as well as matrix age and storage conditions should be thoroughly investigated for their influence on apparent analyte stability, and these conditions should be optimized during method development.
Processed sample stability Stability of processed samples should be evaluated by comparing aged QC extracts, stored under the tempera- ture and conditions to be used in the production assay, with a freshly processed calibration curve. This stability determination is the same for PrD-LCMS as is currently specified in guidances for small molecules [8,9], and the specific definition of the processed sample is the final protein digest (after any additional purification steps) in the state that it will be used for final injection.
Critical assay reagents The L4 Global Harmonization Consortium white- paper [13] defines critical reagents as reagents that are critical in LBAs. These are reagents that are difficult
future science group
to make, acquire or substitute. They suggest that this definition could be extended to the capture antibody used in PrD-LCMS assays utilizing immunoaffinity capture. While many reagents such as the reference standard, IS and other solutions used for PrD-LCMS assays may be considered as critical reagents, they are reagents and standards for which stability and main- tenance methods are normally clearly established and routinely validated. In this article, we discuss criticality of protease enzymes and the immunocapture reagents used for isolation or enrichment at the protein and/or peptide level. It is important to note that these are usu- ally added in excess in PrD-LCMS assays. In addition, due to the high selectivity of LC–MS/MS, the specific- ity of the immunocapture reagents is not as critical as it is for LBAs. In cases where new lots of such reagents must be qualified, a fit-for-purpose approach may be applied such that the reagent can be accepted based upon its functionality in the assay. It may be useful in some cases to examine the specific assay parameters impacted by the reagent, for example, to verify the digestion efficiency of the assay when a new lot of the proteolytic enzyme is first being used. The demonstra- tion of one acceptable precision and accuracy run or an acceptable analytical run may be sufficient to indicate reagent suitability; but, in cases where the reagent is questionable or requires rigorous testing, a more thor- ough test of precision and accuracy or a partial valida- tion may be required. In any case, the level of criticality of reagents, their production procedure or procurement options and any other considerations important to the assay should be documented and described in the ana- lytical method. Since many assays are utilized over several years, the long-term availability and stability of critical reagents should be carefully undertaken.
Discussion & conclusion The application of PrD-LCMS is rapidly growing as a complementary and/or alternative technique to conven- tional LBAs, and there is the clear need for a regulatory guidance for application of this technique to regulated studies. In the absence of such a guidance (at the time of this writing), a group of industry practitioners have drafted a whitepaper on recommendations for the valida- tion of PrD-LCMS methods. The White Paper, ‘Recom- mendations for Validation of LC–MS/MS Bioanalytical Methods for Protein Biotherapeutics,’ was published in AAPSJ in 2015 [11]. In this Perspective, we provide a brief summary of this White Paper’s recommendations, and we have also provided some additional thoughts and dis- cussions of general interest that arose during the drafting of the White Paper. These discussions are intended to clarify some of the areas where the validation of PrD- LCMS methods may differ from those of the LBA and/
www.future-science.com 1393
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107 |
Page 108 |
Page 109 |
Page 110 |
Page 111 |
Page 112 |
Page 113 |
Page 114 |
Page 115 |
Page 116 |
Page 117 |
Page 118 |
Page 119 |
Page 120 |
Page 121 |
Page 122 |
Page 123 |
Page 124 |
Page 125 |
Page 126 |
Page 127 |
Page 128 |
Page 129 |
Page 130 |
Page 131 |
Page 132 |
Page 133 |
Page 134 |
Page 135 |
Page 136 |
Page 137 |
Page 138 |
Page 139 |
Page 140 |
Page 141 |
Page 142 |
Page 143 |
Page 144 |
Page 145 |
Page 146 |
Page 147 |
Page 148 |
Page 149 |
Page 150 |
Page 151 |
Page 152 |
Page 153 |
Page 154