27
method transfer across labs, analysts, and multiple sites – which is common in the pharma/CRO relationship.
Ultra-high-sensitivity quantification via affinity purification: Infliximab
With the infliximab US patent expiration date of 2017 drawing ever closer [1], both CROs and biosimilar research labs are increasing focus on this biotherapeutic. Using a single, universal sample prep method (e.g., Waters®
ProteinWorks™
eXpress Digest Kit), an LLOQ of 10 ng/mL infliximab was achieved. (Figure 1).
Figure 2: Chromatogram showing 10 ng/mL of infliximab in rat plasma, as compared to blank rat plasma. Infliximab is quantified using the unique peptide SINSATHYAESVK.
Table 1: Linear dynamic range and standard curve statistics for signature peptides used to quantify infliximab in rat plasma.
Peptide
DILLTQSPAILSVSPGER* SINSATHYAESVK* DSTYSLSSTLTLSK
*Unique signature peptide Figure 1: Infliximab (Remicade) protein structure Experimental: Infliximab
Infliximab was first immuno-purified from 35 µL rat plasma using a 96-well Protein A agarose-based plate. Samples were then prepared for LC-MS analysis using a commercially available digestion kit and protocol from Waters Corporation. Finally, signature peptides were cleaned-up using a second commercially available kit from Waters based on strong cation exchange of tryptic peptides using SPE [2].
Infliximab samples were affinity purified, digested, and peptides extracted using SPE in less than 6 hours total. This enabled us to begin the LC-MS/MS assay and acquire data the same day, with several 96-well plates being run by the next business morning. (See Appendix for analytical method conditions).
Multiple unique signature peptides as well as a generic human peptide were simultaneously monitored for use in quantification. The best sensitivity was achieved using the unique peptide SINSATHYAESVK from the heavy chain, while additional unique (DILLTQSPAILSVSPGER,
light chain) and generic (DSTYSLSSTLTLSK, light chain) infliximab peptides were monitored for confirmation. A unique peptide (SVSELPIMHQDWLNGK) from a common murine mAb standard (Waters
Corp, p/n 186006552) was used as the internal standard.
Using the optimised protocol [2] and reagents provided in the kit, only 35 µL of plasma was needed to achieve a detection
Table 2: Statistics for QC samples from all infliximab peptides used for quantification. Peptide
QC conc (µg/mL)
SINSATHYAESVK*
0.035 0.350 3.500
35.000 350.000 DILLTQSPAILSVSPGER*
0.035 0.350 3.500
DSTYSLSSTLTLSK
0.035 0.350 3.500
35.000 350.000 *Unique signature peptide.
Mean cal. conc (µg/mL)
0.036 0.331 3.330
38.287 -
-
0.359 3.210
-
0.333 3.271
36.256 369.975
Std. d ev.
0.001 0.003 0.105 1.168 -
-
0.015 0.026
-
0.010 0.186 1.999 7.432
Std. curve range (µg/mL)
0.05–250 0.01–100 0.10–500
Weighting 1/X
1/X2 1/X2
Linear fit r2
0.998 0.995 0.997
Mean % accuracy of all points
100.00 98.47 99.34
%CV Mean accuracy
2.78 0.80 3.15 3.05 -
-
4.10 0.81
-
2.85 5.70 5.51 2.01
103.1 94.5 95.1
109.4 -
-
102.6 91.7
-
95.3 93.5
103.6 105.7
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