Table 1. Summary of the mutations, heterodimerization yields, production yields, and heterodimer-favoring and homodimer-disfavoring interactions of the isolated heterodimeric Fc variants from LibA libraries.

	Paired mutations a		Yield (scFv-FcCH3A/FcCH3B system)		Main interactions d
Variant	CH3A chain	CH3B chain	Heterodimer (%) b	Production (%) c	Favoring CH3A-CH3B	Disfavoring CH3A-CH3A	Disfavoring CH3B-CH3B
W-VT	K409W	D399V/F405T	60.8 ± 3.0	102 ± 31.1	K409WCH3A-D399V/F405TCH3B complementary hydrophobic interaction	F405CH3A-K409WCH3A steric clash	K392ECH3B unpaired charged residue K409CH3B unpaired charged residue
Variants from the LibA1 library constructed using a W-VT template with mutation pairs K409W CH3A -D399V CH3B /F405T CH3B
A107	K370E/K409W	E357N/D399V/F405T	93.4 ± 1.1 (78.2 ± 4.2)	108 ± 51.1 (103 ± 49.0)	K370ECH3A-E357NCH3B hydrogen bond K370ECH3A- S364CH3B hydrogen bond Y349CH3A-E357NCH3B hydrogen bond K370ECH3B-K409CH3B electrostatic interaction	E357CH3A-K370ECH3A electrostatic repulsion	–
A108	K370E/K409W	E357I/S364T/D399V/F405T	70.5 ± 3.3	125 ± 78.1	K370ECH3A-K409CH3B electrostatic interaction	E357CH3A-K370ECH3A electrostatic repulsion	–
A109	K370M/K409W	E357M/S364W/D399V/F405T	90.5 ± 2.7 (61.6 ± 4.5)	102 ± 27.2 (86.9 ± 2.1)	K370MCH3A-E357M/S364WCH3B complementary hydrophobic interaction	E357CH3A unpaired charged residue	K370CH3B-S364WCH3B steric clash
A146	K370D/K409W	E357M/D399V/F405T	74.5 ± 3.4 (71.0 ± 3.7)d	127 ±22.7 (89.5 ± 13.5)	K370DCH3A-K409CH3B electrostatic interaction K370DCH3A-S364CH3B hydrogen bond	K370DCH3A-E357CH3A electrostatic repulsion	–
Variants from the LibA2 library constructed using a A107 w/oW-VT template with mutation pairs K370E CH3A -E357N CH3B
A205	E357D/S364W/K370E	E357N/K370R	88.8 ± 2.2	118 ± 5.0	S364WCH3A-K370RCH3B cation-π	E357DCH3A-S364WCH3A anion-π repulsion K370ECH3A-E357DCH3A electrostatic repulsion	Hole-hole interface
A210	E357A/S364Y/K370E	E357N/K370H	80.8 ± 5.8	104 ± 9.2	S364YCH3A-K370HCH3B π-π, S364YCH3A-K370HCH3B hydrogen bond	S364YCH3A-K370ECH3A anion-π repulsion	Hole-hole interface
A216	E357G/S364W/K370E	E357N	80.3 ± 4.6	92.1 ± 13.1	S364WCH3A-K370CH3B cation-π	S364WCH3A-K370ECH3A anion-π repulsion	–
A241	E357N/S364W/K370E	E357N	81.0 ± 3.9	150 ± 49.6	S364WCH3A-K370CH3B cation-π	S364WCH3A-K370ECH3A anion-π repulsion	–

^a Newly introduced mutations in the CH3A or CH3B domain of the isolated Fc variants are highlighted in bold. Other mutations were present in the template variant.

^b Heterodimer yield (mean ± SD of three independent experiments) was determined by SDS-PAGE analyses under non-reducing conditions of the purified proteins after coexpression of the scFv-Fc_CH3A/Fc_CH3B proteins carrying the indicated CH3 variant pair in HEK293F cells as described in the text.

^c The values represent relative purification levels (mean ± SD of three independent experiments) of Fc variants from HEK923 cells coexpressing scFv-Fc_CH3A/Fc_CH3B proteins for 6 days compared with the purification yield of the EW-RVT variant (3.1 ± 0.7 mg/100 mL culture).

In (^b and ^c), the values in parenthesis are the heterodimerization and purification yields of the A107_{w/o W-VT}, A109_{w/o W-VT}, A146_{w/o W-VT}, and B168_{w/o W-VT} Fc variants, in which the W-VT mutation pairs were back-mutated to the corresponding wild-type residues.

^d The heterodimer-favoring and homodimer-disfavoring interactions are those involving the newly introduced mutations on the CH3A or CH3B domain of the isolated heterodimeric Fc variants. Thus, the heterodimer-favoring and homodimer-disfavoring interactions from the parent template Fc variant should also be considered. The hole-hole interface means the absence of favorable intermolecular interactions at the CH3 domain interfaces due to less packing of amino acids with small-sized side chains. The minus “-” means that there are no particular repulsive interactions disfavoring CH3B-CH3B homodimerization, except for loss of the wild-type homodimer-favoring electrostatic interactions.