Small scale screening of FVIII cysteine variants for PEGylation
| Cys mutation . | Transient expression, IU/mL . | PEGylation efficiency* . | ||
|---|---|---|---|---|
| Position . | Domain . | H-PEG . | L-PEG . | |
| Y81C | A1 | 0.36 | ++ | − |
| F129C | A1 | 0.25 | ++ | − |
| K377C | A2 | 0.11 | +++ | − |
| H378C | A2 | 0.15 | +++ | − |
| K422C | A2 | 0.28 | − | − |
| Q468C | A2 | 0.69 | + | − |
| L491C | A2 | 0.60 | +++ | − |
| L504C | A2 | 0.38 | + | − |
| K556C | A2 | 0.09 | ++ | − |
| K570C | A2 | <0.05 | − | − |
| D1795C | A3 | 0.27 | − | + |
| Q1796C | A3 | 0.29 | − | + |
| R1803C | A3 | 0.11 | − | ++ |
| K1804C | A3 | 0.18 | − | +++ |
| K1808C | A3 | 0.54 | − | ++ |
| N1810C | A3 | 0.21 | − | ++ |
| T1812C | A3 | 0.16 | − | − |
| K1813C | A3 | 0.35 | − | − |
| N1864C | A3 | 0.15 | − | ++ |
| T1911C | A3 | 0.28 | − | +++ |
| N2118C | C1 | 0.13 | − | + |
| Q2091C | C1 | 0.20 | − | ++ |
| Q2284C | C2 | 0.17 | − | + |
| L491C/K1808C | A2/A3 | 0.11 | +++ | ++ |
| L491C/K1804C | A2/A3 | 0.13 | +++ | +++ |
| BDD FVIII | NA | 0.30 | − | − |
| Cys mutation . | Transient expression, IU/mL . | PEGylation efficiency* . | ||
|---|---|---|---|---|
| Position . | Domain . | H-PEG . | L-PEG . | |
| Y81C | A1 | 0.36 | ++ | − |
| F129C | A1 | 0.25 | ++ | − |
| K377C | A2 | 0.11 | +++ | − |
| H378C | A2 | 0.15 | +++ | − |
| K422C | A2 | 0.28 | − | − |
| Q468C | A2 | 0.69 | + | − |
| L491C | A2 | 0.60 | +++ | − |
| L504C | A2 | 0.38 | + | − |
| K556C | A2 | 0.09 | ++ | − |
| K570C | A2 | <0.05 | − | − |
| D1795C | A3 | 0.27 | − | + |
| Q1796C | A3 | 0.29 | − | + |
| R1803C | A3 | 0.11 | − | ++ |
| K1804C | A3 | 0.18 | − | +++ |
| K1808C | A3 | 0.54 | − | ++ |
| N1810C | A3 | 0.21 | − | ++ |
| T1812C | A3 | 0.16 | − | − |
| K1813C | A3 | 0.35 | − | − |
| N1864C | A3 | 0.15 | − | ++ |
| T1911C | A3 | 0.28 | − | +++ |
| N2118C | C1 | 0.13 | − | + |
| Q2091C | C1 | 0.20 | − | ++ |
| Q2284C | C2 | 0.17 | − | + |
| L491C/K1808C | A2/A3 | 0.11 | +++ | ++ |
| L491C/K1804C | A2/A3 | 0.13 | +++ | +++ |
| BDD FVIII | NA | 0.30 | − | − |
The activity of FVIII Cys variants transiently expressed in HKB11 cells was measured by a chromogenic assay.
H-PEG indicates heavy-chain PEGylation; and L-PEG, light-chain PEGylation.
The FVIII variants were conjugated with 5-kDa PEG, and the PEGylation efficiency was estimated by comparing the intensity of the PEGylated band with unPEGylated band on a SDS-PAGE and Western blot. The +++ indicates approximately greater than 80% PEGylation yield, ++, approximately 30% to 70% yield; + approximately 10% to 30% yield; −, approximately less than 10% yield.