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Antibody Reformatting

Apart from canonical antibodies, which are around 150 kDa and composed of antigen-binding fragment (Fab) and Fc region, a wide range of antibody formats can be produced through recombinant expression technology. These reformatted antibodies can benefit certain applications by improving solubility, thermostability, immunogenicity, etc. For instance, scFv is used in medical imaging due to its increased tumor penetration. Bispecific antibodies, on the other hand, enable the binding of two target antigens at once, which has a great potential in cancer immunotherapy and drug delivery.

As an expert in producing recombinant antibodies, Bon Opus Biosciences offers a broad range of antibody reformatting services including scFv, Fab, bispecific. A variety of different antibody formats are available and can be tailored to customer requirements.

Canonical Antibody

Fab

scFv

Bispecific

(with or without Fc)

Antibody Fragments Expression and Purification 

ab fragment
Antibody fragment image.jpg

scFv (~25kDa)

•Mammalian cell: secretion/intracellular expression

•E. coli: soluble/refolded

•Tag: His, Fc, tag-free

•Linker design

Case Study: Mammalian-expressed, scFv, His-tag

fab
Antibody fragment image.jpg

Fab (~50kDa)

•Mammalian cell: secretion expression

•His-tag and tag-free

Case Study: Mammalian-expressed, Fab, His-tag

Antibody fragment image.jpg

Nanobody/VHH (~15kDa)

•Mammalian secretion expression

•E. coli: soluble or refolded

•Tag: His and Fc

Case Study: VHH, His-tag (left) VHH, Fc-tag (right)

bispecific

Bispecific Antibody Expression and Purification 

Bispecific antibody is produced by assembling two Fab regions. It can be manufactured in several formats (with or without Fc) using different approaches.

  1. Gene synthesis and codon optimization of target Fab regions

  2. Cloning into expression vectors

  3. Transient expression in Expi293, ExpiCHO

  4. Purification

  5. Endotoxin removal as low as 0.1EU/mg (optional) 

  6. QC analysis: SDS-PAGE, SEC-HPLC, endotoxin level test

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Deliverable:

- Purified bispecific antibodies with customized QC standard

Turnaround time:

4 weeks plus shipping

Case Studies

scfv case study

100mg ScFv Expression and Purification​​

  • Expressed by Expi293 with VH-(GGGGS)3-VL-6x His and purified by Ni-NTA

SDS-PAGE and Anti-His WB image

M: Molecular weight marker

Me: Culture medium

FT: Flow-through

W: Washes

E: Elution

  • Final Sample

SDS-PAGE

SEC-HPLC

  • Deliverable: 100mg purified antibody, >SEC-HPLC 99% purity, endotoxin < 0.1EU/mg

bsab case study

Bispecific Antibody Expression and Purification​​

Antibody properties: Human IgG4, Human Kappa Light Chain

  • Gene synthesis of two VH and one VL regions

  • Cloning into premade Fc vectors (contains one chimeric Fc*)

  • Co-expression with three constructs in Expi293

  • Purification by MabSelect SuRe LX and pH gradient elution

1st Mab Select Sure LX Purification

Analytic SEC-HPLC (Elution sample)

2nd CIEX Purification

QC of final sample (desired heterodimer)​​

  • Deliverable: Purified bispecific antibody, SEC-HPLC >99% purity

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