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Applications | Function/gene | Mechanism of action | Reference |
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(1) Protein engineering |
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Disulfide bond introduction | Cysteine engineering | Stabilizing FVIIIa | [46] |
Stabilizing scFV | [47] |
N-linked site introduction | Half-life extension of EPO | Reducing renal clearance | [48] |
IgG framework/CDR | Improving solubility | [49, 50] |
Poly O-linked sites | Fusion of CTP of gonadotropin | half-life extension | [51, 52] |
Engineering N-terminal Gln | Forming pyroglutamate | Preventing N-terminal degradation | [53] |
Protease resistant mutant | Mutating Arg336 & 562 of FVIII | Eliminating two APC sites | [54] |
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(2) Cell-line engineering |
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| Overexpressing GnTIV, GnTV, ST3GalIV, and ST6GalI | Increasing sialic acid content | See text |
Half-life extension | CMP-sialic acid transporter | | [55, 56] |
CMP-sialic acid synthase, GNE | | [57, 58] |
| Knockdown sialidase |
Preventing sialic acid degradation | [59, 60] |
Reducing immunogenicity | knockdown CMP-sialic acid hydroxylase | Preventing conversion of Neu5Ac to Neu5Gc | [61] |
E-selectin interaction | Overexpressing C2GnT, Fut6 Knockdown ST3Gal | Enhancing sLex formation | [62, 63] |
Modulating effector function | Knockout or knockdown | Fucose removal | [64, 65] |
Fut8, GMD, overexpressing GnTIII | | [66, 67] |
Lysosomal targeting | Inactivating GnTI | Oligomannose formation | [68, 69] |
Improving ADCC | Overexpressing sialidase | Reducing sialylation | [70] |
Improving carboxylation | Overexpressing VKOR | Facilitating vitamin K reduction | [71] |
VKOR ↑ calumenin ↓ | Increasing carboxylation of FVII | [72] |
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(3) Process engineering |
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Modulating glycosylation | Serum content, pH, dissolved oxygen, Temperatures, ammonium, salt concentration | Bioprocessing parameters | See text |
Glucosamine/uridine | Enhancing glycan antennarity | Elevating intracellular UDP-GlcNAc | [73] |
Lipid supplement | Maintaining site occupancy | | [74] |
Decreasing temperature/adding butyrate | Enhancing glycan occupancy | Increasing site exposure time to OST | [75] |
Addition of ManNAc | Improving sialylation | CMP-Neu5Ac precursor | [76] |
Adding manganese/iron | Enhancing glycan occupancy | Increasing OST activity | [75] |
Addition of kifunensine | Oligomannose formation | -mannosidase I inhibitor | [77] |
Lowering glucose | Reducing heterogeneity | Reducing glycation | [78] |
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