Table 2. Assessment of HER2 Asn deamidation and Asp isomerization 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 | 2.6 (±0.2) | 9.0 (±0.3) | 11.0 (±0.8) | 12.5 (±0.3) | 23.4 (±1.8) | 24.8 (±1.7) |
| LC-Asp-30 | 0.4 (±0.1) | 2.1 (±0.1) | 3.1 (±0.1) | 3.2 (±0.2) | 4.7 (±0.1) | 7.0 (±0.6) |
| LC-isoAsp-30 | 7.5 (±0.7) | 10.9 (±0.4) | 15.3 (±1.1) | 11.1 (±1.1) | 18.8 (±2.6) | 26.5 (±0.2) |
| HC-Asn-55 | ||||||
| HC-Asu-55 | 5.6 (±0.4) | 5.5 (±0.7) | 5.1 (±0.9) | 5.5 (±0.4) | 5.2 (±0.4) | 5.4 (±0.6) |
| HC-deamid-55 | 3.6 (±0.4) | 4.1 (±0.1) | 3.8 (±0.6) | 4.3 (±0.1) | 5.4 (±0.5) | 8.9 (±1.1) |
| HC-Asp-102 | ||||||
| HC-Asu-102 | 2.1 (±0.2) | 2.8 (±0.1) | 2.8 (±0.1) | 3.4 (±0.1) | 3.1 (±0.4) | 3.8 (±0.1) |
| HC-isoAsp-102 | 2.5 (±0.4) | 9.0 (±0.5) | 15.9 (±0.6) | 14.6 (±0.2) | 25.9 (±1.2) | 46.1 (±1.0) |
| HC-Asp-283 | ||||||
| HC-Asu-283 | 4.8 (±0.6) | 5.2 (±0.7) | 5.7 (±1.0) | 5.9 (±1.3) | 5.8 (±0.1) | 5.6 (±0.5) |
| HC-isoAsp-283 | 0.9 (±0.1) | 1.0 (±0.1) | 1.3 (±0.3) | 1.3 (±0.2) | 2.1 (±0.1) | 3.4 (±0.1) |
| HC-Asn-387,392,393 | ||||||
| HC-Asu-387,392,393 | 0.7 (±0.1) | 0.7 (±0.1) | 1.0 (±0.1) | 0.8 (±0.1) | 0.9 (±0.1) | 0.9 (±0.1) |
| HC-deamid-387,392,393 | 0.3 (±0.1) | 0.8 (±0.1) | 1.4 (±0.4) | 1.0 (±0.1) | 1.8 (±0.1) | 3.8 (±0.1) |
| CEC | ||||||
| % Acidic | 27.7 | 32.7 | 39.8 | 33.7 | 46.7 | n.q. |
| % Main | 63.5 | 51.3 | 38.5 | 45.0 | 26.0 | n.q |
| % Basic | 8.8 | 16.0 | 21.6 | 21.3 | 27.3 | n.q |
| SEC | ||||||
| % Fragment | <0.1 | 0.1 | 0.2 | 0.2 | 0.4 | 1.0 |
| % Monomer | 99.8 | 99.6 | 99.4 | 99.4 | 99.0 | 98.2 |
| % Aggregate | 0.2 | 0.3 | 0.4 | 0.4 | 0.6 | 0.8 |
| SPR | ||||||
| % Target Binding | 100 (±2) | 93 (±2) | 89 (±2) | 90 (±2) | 79 (±2) | 59 (±3) |
Relative quantification (in %) was conducted by specific ion current chromatogram analysis of proteolytic LysC peptides using the quantification software GRAMS/32™ (n = 2, mean ± S.D). HER2 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.