Table 2.

Biopharmaceutical applications of therapeutic protein modifications.

Applications	Function/gene	Mechanism of action	Reference
(1) Protein engineering
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]
(2) Cell-line engineering
	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]
(3) Process engineering
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	α-mannosidase I inhibitor	[77]
Lowering glucose	Reducing heterogeneity	Reducing glycation	[78]