Fig 1.
Schematic representation of present method for measuring rituximab in plasma.
Isolation of rituximab from plasma was conducted by anti-rituximab antibodies covalently bound to magnetic beads. Isolated rituximab was digested by papain, and then the structures of rituximab were analyzed by LC/TOF-MS.
Fig 2.
LC/TOF-MS analysis of digested rituximab in the formulation.
(A) Total ion chromatogram of the rituximab formulation that was digested by papain. The peaks at 6.8 and 7.3 min were Fc/2 and Fab fragments, respectively. (B) Overlaid extracted ion chromatograms at 1,509 m/z and 1,475 m/z, which corresponded to the Fc/2 and Fab fragments ions, respectively. (C) Typical deconvoluted chromatogram of Fc/2 fragment of the rituximab formulation. The number and pattern diagram above each peak indicate the observed molecular weight and predicted structure of attached carbohydrate chains, respectively. (D) Typical deconvoluted chromatogram of the Fab fragment of the rituximab formulation. The observed molecular weight of the Fab fragment is indicated above the peak.
Fig 3.
Detected glycoforms in rituximab and the predictive carbohydrate chains.
The carbohydrate chains were named according to the proglycan system (www.proglycan.com). Note that Fc/2 molecular weights were the mean values of three independent experiments. The accuracies of these experiments were expressed in terms of part per million (ppm).
Fig 4.
LC/TOF-MS analysis of isolated rituximab from rat plasma.
The mass spectra were overlaid to compare 1-h and day-21 samples. Black and red lines indicated the mass spectra of rituximab isolated from the samples of 1 h and day 21, respectively. The number and pattern diagram above each peak indicate observed molecular weight and predicted structure of attached carbohydrate chains, respectively. Typical deconvoluted mass spectra of Fab (A), deglycosylated Fc/2 (B) and Fc/2 (C) fragments of rituximab.
Fig 5.
Time-dependent changes in composition ratios of rituximab glycoforms in rat.
These values were calculated from peak heights of each glycoform from deconvoluted mass spectrum. Control represents isolated rituximab from plasma spiked with the formulation. (A) Each datum represents the mean of six independent replicates with standard deviation. The composition ratios of glycoforms of III, MGnF/GnMF (IV) and GnGnF (G0F, VII) (see Fig 3) are significantly different between the samples on 14 or 21 days of post-administration and controls (*: p<0.05, **: p<0.01). Additionally, the composition ratio of IX glycoform is significantly different (*p<0.05) between the samples on 21 days of post-administration and controls. (B) The composition ratios of GnGn (G0) glycoform at each time point. Each point represents the value obtained from individual plasma sample. Open symbol indicates that GnGn was not detected in these samples.
Fig 6.
CDC and ADCC activities of rituximab from rat plasma.
(A) Complement-dependent cytotoxicity (CDC) activities were determined with rituximab isolated from plasma of 1 h and 21 days of post-administration. Control represents the activity of rituximab in the formulation. (B) Antibody-dependent cellular cytotoxicity (ADCC) activities were determined with rituximab isolated from plasma of 1 h and 21 days of post-administration. Rituximab and Trastuzumab represent the activities of the rituximab and trastuzumab formulations, respectively. Each datum represents the mean of four independent replicates with standard deviation.
Fig 7.
Composition ratios of glycoforms at 1 h and on 21 days after in vitro incubation.
Rituximab was incubated at 37°C with (A) rat plasma, (B) phosphate buffer and (C) heat-inactivated rat plasma. Each datum represents the mean of five or six independent replicates with standard deviation. In rat plasma, the composition ratios of glycoforms of I, III, IV, VI and IX showed significant differences between 1 h and day 21 (*: p<0.05, **: p<0.01). In the samples of the buffer and heat-inactivated plasma, there is no significant difference between 1-h and day-21 samples.