Table 1.

HC and LC variants to direct the assembly of anti-HER2/CD3 BsIgG₁ in a single host cell. The anti-HER2 and anti-CD3 HC and κ-LC variants were co-transfected into Expi293F cells. The resultant BsIgG₁-containing mixture was purified from the cell culture harvest fluid by protein A chromatography and then analyzed by liquid chromatography-EMR Orbitrap mass spectrometry. The percentage of BsIgG₁ was calculated by quantifying all IgG₁ species present (excluding very small amounts of half antibodies and HC homodimers). Statistical estimation of the isobaric mixture containing BsIgG and double LC scrambled IgG₁ was performed as previously described.¹⁹ The anti-HER2 HC variants additionally contains the knob mutation (T366W) whereas the anti-CD3 HC variants contains the hole mutations (T366S:S368A:Y407V) as previously described.⁵ yt65H refers to the C_H1 mutations A141I:F170S:S181M:S183A:V185A and yt65L refers to the C_L mutations F116A:L135V:S174A:S176F:T178V. Data shown are for optimized LC ratios. The top designs, v10 and v11, are highlighted in bold.

Orthogonal pairing	Anti-HER2 HC		Anti-HER2 LC		Anti-CD3 HC		Anti-CD3 LC		BsIgG1
variant	VH	CH1	VL	CL	VH	CH1	VL	CL	(%)
Parent									24.6
v1	Q39E		Q38K		Q39K		Q38E		55.7
v2						S183E		V133K	46.5
v3		S183K		V133E					24.9
v4		yt65H		yt65L					59.5
v5		S183K		V133E		S183E		V133K	46.4
v6		yt65H		yt65L		S183E		V133K	87.0
v7	Q39E		Q38K		Q39K	S183E	Q38E	V133K	81.2
v8	Q39E	S183K	Q38K	V133E	Q39K		Q38E		63.0
v9	Q39E	yt65H	Q38K	yt65L	Q39K		Q38E		84.4
v10	Q39E	S183K	Q38K	V133E	Q39K	S183E	Q38E	V133K	91.7
v11	Q39E	yt65H	Q38K	yt65L	Q39K	S183E	Q38E	V133K	100.0
v12		yt65H		yt65L	Q39K	S183E	Q38E	V133K	98.2