Production of disulfide-rich cow knob domain antibodies: picobodies™
Background
- About 10% of cow antibodies have exceptionally long heavy chain CDR3 regions, including a stalk and knob region .
- The knob region of CDR H3 is about 4-6 kDa in size with a highly diverse sequence content and multiple disulfide bonding patterns: these are known as Picobodies™.
- Picobodies can be used for inhaled therapy because of their small size and stability.
Challenge
Because of their small size Picobodies can be independently genetically engineered and developed to therapeutically target crevices, pores, or other protein epitopes that larger antibodies cannot. However, Picobodies with complex disulfide bonding patterns can be difficult to produce.
Solution
The Pfenex Expression Technology® platform, with its extensive toolbox of genetic elements, fusion partners and modified hosts strains, was utilized to quickly identify expression strains producing high titer and high quality Picobodies for further preclinical development.
Key Results
SDS-CGE Analysis of Soluble Picobodies (90 min/plate)
SDS-capillary gel electrophoresis (SDS-CGE) used to assess expression and solubility.
SDS-CGE detected induced bands of soluble Picobody fusion proteins (green) up to ~2g/L at 96-well expression scale.
Cleaved Picobody Maintains Binding to SARS CoV-2 Spike Protein
RP-UPLC and LC-MS confirm expected cleavage of Picobody from fusion protein. ELISA analysis confirmed binding to SARS CoV-2 spike protein (ICSO = 19 nM).
BLI Activity Analysis (30-60 min per plate)
Product quantity and quality assessed via biolayer interferometry (BLI). Strains producting highly active soluble Picobody fusion proteins (green) identified by binding to SARS CoV-2 spike protein compared to wild-type strain (red
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