Table 2. Free cysteine determined at different digestion pHs for rituximab and RNA-mediated mAb.
| pH 8.0 | pH 6.8 | pH 2.0 | ||||
|---|---|---|---|---|---|---|
| Rituximab (%) | RNAi-mediated (%) | Rituximab (%) | RNAi-mediated (%) | Rituximab (%) | RNAi-mediated (%) | |
| T12H (C148) | N.D. | N.D. | 5.4 ± 0.9 | 0.4 ± 0.0 | 1.0 | 0.2 |
| T13H (C204) | N.D. | N.D. | 1.3 ± 0.3 | N.D. | 0.2 | 0.1 |
| T20H (C265) | N.D. | N.D. | 3.0 ± 0.3 | 0.2 ± 0.0 | 3.4 | 1.2 |
| T26H (C325) | – | — | — | — | 3.1 | 0.8 |
| T34H (C371) | N.D. | N.D. | 10.7 ± 1.8 | 0.2 ± 0.1 | 1.6 | N.D. |
| T39H (C429) | N.D. | N.D. | 4.7 ± 0.3 | 0.1 ± 0.0 | 1.0 | N.D. |
| T2L (C23) | N.D. | N.D. | 0.4 ± 0.2 | 0.0 ± 0.0 | N.D. | N.D. |
| T5L (C87) | N.D. | N.D. | 0.2 ± 0.0 | 0.0 ± 0.0 | N.D. | N.D. |
| T9L (C133) | N.D. | N.D. | 1.7 ± 0.2 | 0.1 ± 0.0 | N.D. | N.D. |
| T16L (C193) | N.D. | N.D. | 1.1 ± 0.1 | 0.1 ± 0.0 | 1.0 | N.D. |
| T17L (C213) | — | — | — | — | 0.2 | 0.1 |
The percent of free cysteine at pH 8.0 and 6.8 was obtained by the measured amount of the free cysteine form divided by the total cysteines on the specific tryptic peptide, assuming each cysteine is alkylated. The percent of free cysteine at pH 2 was obtained by the ratio of the observed free cysteines divided by the total cysteines after reduction with TCEP. “N.D.” represents not detectable (the values is too low to be observed), and “–” means not measurable due to the tryptic peptide length (too short to retain in the LC chromatogram).