Name: Human IgG1 (CrossMab + KiH Set) — CH1-CL Domain Crossover & Knobs-into-Holes Bispecific Expression System
SKU: AFV-35
Availability: InStock

Human IgG1 (CrossMab + KiH Set) — CH1-CL Domain Crossover & Knobs-into-Holes Bispecific Expression System

$3,980.00

The Human IgG1 (CrossMab + KiH Set) is a complete 6-vector mammalian expression system engineered for high-yield, error-free production of full-length asymmetric bispecific antibodies (bsAbs). By combining Knobs-into-Holes (KiH) in the CH3 domain for heavy-chain heterodimerization with CrossMabCH1-CL domain swapping on Arm 2, this platform prevents both heavy-chain homodimerization (>95% purity) and light-chain mispairing (>98% purity).

System Type: 6-Vector Expression Platform (Compatible with both K and λ light chains)
Host Compatibility: ExpiCHO-S™, Expi293F™, and standard mammalian transient systems
Key Features: CrossMab^CH1–CL Crossover, KiH CH3 heterodimerization, and CH3–CH3 S354C–Y349C disulfide lock

Catalog Number

AFV-35

System Type

6-Vector Expression Platform

Host Systems

ExpiCHO-S™ / Expi293F™

Applications

Asymmetric bsAb (Full IgG)

Product Overview

The Human IgG1 (CrossMab + KiH Set) is a modular 6-vector mammalian expression platform engineered for high-yield, correct assembly of asymmetric bispecific antibodies (bsAbs). By combining Knobs-into-Holes (KiH) in the CH3 domain for heavy-chain heterodimerization with CrossMabCH1-CL domain swapping on Arm 2, this platform prevents both heavy-chain homodimerization (>95% heterodimerization) and light-chain mispairing (>98% correct assembly).

Universal Isotype Compatibility: Accommodates target variable domains of either Kappa (κ) or Lambda (λ) light chains across both antigen-binding arms without re-engineering constant vector backbones.
Dual-Pairing Protection: Prevents heavy-chain homodimerization and light-chain mispairing simultaneously during four-chain co-expression.
Native Binding Kinetics: Retains parental Fab binding affinities, thermal stability, and high mammalian expression yields.

Expression Kit Setup

The kit consists of 6 optimized mammalian expression vectors allowing flexible setup for any κ or λ combination:

Human IgG1 (CrossMab +KiH Set) Engineering & Key Mutations

Engineering Architecture & Mechanism

Human IgG1 (CrossMab + KiH Set) Architecture

Engineering Mechanism Breakdown

Domain / Region	Arm 1 Modifications (Knob / Ag 1)	Arm 2 Modifications (Hole / Ag 2)	Role & Numbering Scheme
Heavy Chain CH1	Wild-Type (C220)	Swapped to Cκ or Cλ	EU Index: Native hinge cysteine on Arm 1 forms native H–L bond
Heavy Chain CH3 Disulfide	S354C	Y349C	EU Index: Inter-heavy disulfide bond (CH3–CH3) locking H1–H2
Heavy Chain CH3 Packing	T366W	T366S / L368A / Y407V	EU Index: Steric complementary packing driving heterodimerization
Light Chain Constant	Wild-Type (Cκ/Cλ)	Swapped to CH1	Converts Arm 2 light chain to universal VL-CH1 backbone

Plasmid Selection Guide

Select 4 plasmids (1 Heavy + 1 Light for Arm 1; 1 Heavy + 1 Light for Arm 2) depending on your target antibody isotypes:

Arm 1 Isotype	Arm 2 Isotype	Arm 1 Heavy	Arm 1 Light	Arm 2 Heavy	Arm 2 Light
κ	κ	pHC-IgG1-KiH-Knob	pLC-hIgk-WT	pHC-hIgG1-CrossMab-Cκ-Hole	pLC-hIg-CrossMab
κ	λ	pHC-IgG1-KiH-Knob	pLC-hIgk-WT	pHC-hIgG1-CrossMab-Cλ-Hole	pLC-hIg-CrossMab
λ	κ	pHC-IgG1-KiH-Knob	pLC-hIgλ-WT	pHC-hIgG1-CrossMab-Cκ-Hole	pLC-hIg-CrossMab
λ	λ	pHC-IgG1-KiH-Knob	pLC-hIgλ-WT	pHC-hIgG1-CrossMab-Cλ-Hole	pLC-hIg-CrossMab

Transfection & Ratio Optimization

For standard mammalian expression (ExpiCHO™ / Expi293™), start with a 1:1:1:1 equimolar ratio across all 4 selected plasmids.

Optimization Tip: If excess free light chain is observed during analytical SEC, reduce light chain DNA to a 1 : 0.8 : 1 : 0.8 (HC1 : LC1 : HC2 : LC2) mass ratio.

Storage & Handling

Plasmid DNA: Store at −20 °C.
Purified Antibody: Store at 2–8 °C for short-term (<2 weeks) or −80 °C for long-term storage.
Avoid repeated freeze–thaw cycles; use sterile handling practices.