Fig 1.
The recovery of the affinity purification of MAB1.
The recovery rate of the affinity purification of MAB1 was >99.5% while <0.5% total MAB1 was detected in the flow-through. The calculation of percent recovery rate was described in the Materials and Methods section. A known amount of high molecular-weight standard (shown as “Upper Marker”) was run with each sample as an internal normalization control. The ratio of the MAB1 peak area to the Upper Marker peak area in each sample was calculated to correct for run-to-run variability.
Fig 2.
Example MS/MS spectra of peptide identification (A) and example extracted ion chromatograms for peptide peak integration and PTM quantitation (B). (A) The MS/MS spectrum of an example Met oxidized (Met site 1) peptide, DTLMISR (top panel) and the MS/MS spectrum of the example wild-type peptide, DTLMISR (bottom panel). (B) Extracted ion chromatograph of the Met oxidized peptide, DTLMISR (top panel) and the wild-type peptide, DTLMISR (bottom panel).
Fig 3.
The relative abundance of deamidation at each of the three Asn sites in the Fc region of MAB1 from the single-dose PK study (A) and the multiple-dose PK study (B). (A) In the single-dose study, the relative abundance of deamidation at each of the three Asn sites increased over time at different rates following the first-order kinetic equation: . Using non-linear regression, the in vivo deamidation rate constants at Asn site 1, 2, and 3 were determined to be 0.003523% day-1, 0.5394% day-1, and 0.1546% day-1, respectively. (B) In the multiple-dose study, the relative abundance of deamidation at each of the three Asn sites increased over time during each dosing interval following the first-order kinetic equation, but decreased sharply following each subsequent dose due to dilution with newly administrated unmodified MAB1, exhibiting an upward trending saw-tooth pattern. Each dosing time is indicated with an arrow “↑”.
Table 1.
The best-fit parameter values in the modification rate equations from the single-dose PK study.
Fig 4.
The subject exposure to total MAB1 from the single-dose PK study (A) and the subject exposure to MAB1 with a PQA from the single-dose study (B). (A) The MAB1 serum concentration-time curve (black line) described by the two-compartment pharmacokinetic model equation as C(t) = Ae−αt+Be−βt was fitted to the ELISA measured MAB1 serum concentrations (black dots). The subject exposure to total MAB1 over the course of 56 days, represented by the AUC of C(t) was determined as 4302.0 μg/mL∙day. (B) The serum concentration-time curve of MAB1 with deamidation is described as CPQA(t) = C(t)∙PPQA(t), The AUC of the CPQA(t) curve corresponds to the subject exposure to MAB1 with the PQA. The subject exposure to MAB1 with deamidation at Asn site 2 over 56 days was determined as 426.7 μg/mL∙day (solid line). The subject exposure to MAB1 with deamidation at Asn site 3 over 56 days was determined as 152.5 μg/mL∙day (hyphenated line). The subject exposure to MAB1 with N-terminal pyroglutamate over 56 days was determined as 191.5 μg/mL∙day (dot line).
Table 2.
The best-fit parameters values in the MAB1 serum concentration-time equations from the single-dose PK study and the first dose interval of the multiple-dose PK study.
Fig 5.
Model predictions and experiment measurements of the serum concentration of total MAB1 and MAB1 with deamidation at Asn site 2 or Asn site 3 from the multiple-dose PK study (A) and model predictions and experimental measurements of the relative abundances of deamidation at Asn site 2 or Asn site 3 (B). (A) The predicted pre-dose and post-dose serum concentrations of total MAB1 and MAB1 with deamidation at Asn site 2 or Asn site 3 are in good agreement with the experimental measurements. The pre-dose and post-dose concentrations of total MAB1 and MAB1 with deamidation at Asn site 2 or 3 approach the steady-state levels following an extended period of dosing. (B) The predicted levels are in good agreement with the experimental values. The pre-dose and post-dose relative abundances of deamidation at Asn site 2 or Asn site 3 approach the steady-state levels following an extended period of dosing. Each dosing time is indicated with an arrow “↑”.
Fig 6.
Model predictions and experiment measurements of the serum concentration of MAB1 with N-terminal pyroglutamate from the multiple-dose PK study (A) and model predictions and experimental measurements of the relative abundances of N-terminal pyroglutamate (B). (A) The predicted pre-dose and post-dose serum concentrations of MAB1 with N-terminal pyroglutamate are in good agreement with the experimental measurements. The pre-dose and post-dose concentrations of MAB1 with N-terminal pyroglutamate approach the steady-state levels following an extended period of dosing. (B) The predicted levels are in good agreement with the experimental values. The pre-dose and post-dose relative abundances of N-terminal pyroglutamate approach the steady-state levels following an extended period of dosing. Each dosing time is indicated with an arrow “↑”.
Fig 7.
Modeling the subject exposures to a hypothetical CDR deamidation with an in vivo deamidation rate of 2.5% per day-1 and initial deamidation levels at 0%, 10%, and 20% in the single-dose study (A) and the multiple-dose study (B). (A) The subject exposure to the hypothetical CDR deamidated variants with 0%, 10%, and 20% initial deamidation over 56 days in the single-dose study are 1385 μg/mL∙day, 1653 μg/mL∙day, and 1921 μg/mL∙day, respectively, consisting of 32.5%, 38.8%, and 45.1% subject exposure to the total mAb, respectively. (B) The subject exposure to the hypothetical CDR deamidated variants with 0%, 10%, and 20% initial deamidation over 5 doses (56 days) in the multiple-dose study are 1086 μg/mL∙day, 1478 μg/mL∙day, and 1871 μg/mL∙day, respectively, consisting of 21.7%, 29.5%, and 37.3% subject exposure to the total mAb, respectively Each dosing time is indicated with an arrow “↑”.