Table 3. Identification and evaluation of Mab1 Asn deamidation and Asp isomerization sites using accelerated degradation conditions and quantitative UPLC-MS (Procedure B).
| Storage Duration/Temperature | RM/80°C | 1 M/25°C | 2 M/25°C | 7 d/40°C | 1 M/40°C | 2 M/40°C |
| LC-Asn-30 | ||||||
| LC-Asu-30 | n.q. | n.q. | n.q. | n.q. | n.q. | n.q. |
| LC-deamid-30 | n.q. | n.q. | n.q. | n.q. | n.q. | n.q. |
| (LysC digestion) | ||||||
| LC-Asp-56 | ||||||
| LC-Asu-56 | n.q. | n.q. | n.q. | n.q. | n.q. | n.q. |
| LC-isoAsp-56 | n.q. | n.q. | n.q. | n.q. | n.q. | n.q. |
| (LysC digestion) | ||||||
| HC-Asp-99/101 | ||||||
| HC-Asu-99/101 | 9.1 (±1.1) | 16.2 (±1.4) | 19.8 (±1.3) | 19.9 (±0.5) | 32.0 (±0.6) | 38.2 (±0.4) |
| HC-isoAsp-99/101 | n.q. | n.q. | n.q. | n.q. | n.q. | n.q. |
| (LysC digestion) | ||||||
| LC-Asn-30 | ||||||
| LC-Asu-30 | 0.3 (±0.1) | 0.3 (±0.1) | 0.4 (±0.1) | 0.5 (±0.1) | 0.7 (±0.1) | 0.9 (±0.1) |
| LC-deamid-30 | 3.4 (±1.4) | 3.1 (±0.9) | 3.2 (±1.0) | 3.1 (±1.5) | 3.4 (±2.0) | 4.2 (±1.4) |
| (Trypsin digestion) | ||||||
| LC-Asp-56 | ||||||
| LC-Asu-56 | 6.2 (±0.8) | 8.5 (±1.1) | 9.4 (±1.5) | 9.5 (±0.7) | 11.9 (±0.3) | 12.6 (±0.6) |
| LC-isoAsp-56 | 2.3 (±0.4) | 3.9 (±1.1) | 5.5 (±1.3) | 4.5 (±1.2) | 9.0 (±1.2) | 11.3 (±1.3) |
| (Trypsin digestion) | ||||||
| HC-Asp-99/101 | ||||||
| HC-Asu-99/101 | 2.9 (±0.1) | 4.7 (±0.4) | 5.1 (±0.4) | 4.9 (±0.6) | 6.0 (±0.6) | 6.7 (±0.6) |
| HC-isoAsp-99/101 | 9.4 (±0.1) | 17.6 (±0.3) | 22.1 (±0.1) | 21.4 (±0.1) | 40.0 (±0.3) | 55.6 (±0.8) |
| (Trypsin digestion) | ||||||
| HC-Asn-389,394,395 | ||||||
| HC-Asu-389,394,395 | 0.8 (±0.1) | 0.8 (±0.1) | 0.8 (±0.1) | 0.9 (±0.1) | 0.9 (±0.1) | 0.9 (±0.1) |
| HC-deamid-389,394,395 | 2.1 (±0.2) | 2.1 (±0.2) | 2.3 (±0.1) | 3.2 (±0.1) | 3.2 (±0.1) | 4.3 (±0.1) |
| (LysC digestion) | ||||||
| CEC | ||||||
| % Acidic | 18.6 | 17.1 | 19.1 | 16.4 | 17.6 | 22.7 |
| % Main | 59.7 | 45.3 | 36.9 | 37.7 | 21.1 | 17.3 |
| % Basic | 21.7 | 37.6 | 44.0 | 45.9 | 61.3 | 60.0 |
| SEC | ||||||
| % Fragment | <0.1 | <0.3 | 0.3 | <0.3 | 0.5 | 5.4 |
| % Monomer | 98.7 | 98.5 | 98.4 | 98.6 | 98.1 | 92.7 |
| % Aggregate | 1.3 | 1.3 | 1.3 | 1.2 | 1.4 | 1.9 |
| SPR | ||||||
| % Target Binding | 100 (±2) | 90 (±2) | 83 (±1) | 85 (±2) | 53 (±2) | 22 (±1) |
Relative quantification (in %) was conducted by specific ion current chromatogram analysis of proteolytic peptides (LysC or trypsin) using the quantification software GRAMS/32™ (n = 2, mean ± S.D). MAB1 charge variants were monitored by cation-exchange chromatography (CEC). Formation of fragments and aggregates was monitored by size-exclusion chromatography (SEC) and target binding activity was assessed by SPR-analysis. deamid, total Asp/iso-Asp; n.q., not quantifiable; RM, Reference material.