Antibody development and production by HybriFree TechnologyHybriFree publication in BMC Biotechnology. Kivi et al. 2016
Development of monoclonal antibodies (MAbs) using HybriFree Technology is attractive for generation of immunomolecules from species other than mice. There are several technologies described for the development of recombinant monoclonal antibodies including phage display, single B-cell cloning, proteomics, NGS (Next Generation Sequencing) based etc. However, these all have drawbacks: using bacterial or yeast expression system in phage display, selected MAbs may not be functional; problems with robustness (single B-cell manipulations and/or sterile cultivation of B-cells) may occur; or expensive devices or patented technologies are needed.
We have worked out HybriFree Technology for the development of monoclonal antibodies from different species such as chicken and rabbits. It can also be used for cloning antibodies from existing hybridomas.
The process contains following steps:
- Isolation of PBMCs from the animal spleen. Enrichment of B-cells expressing antigen-specific antibodies from the PBMC population
- RNA isolation, generation of cDNA library of VH/VL combinations and construction of full IgG or ScFv-Fc expression library
- High-throughput screening of the VH/VL combinatory library expressed in CHO cells. Identification of high-affinity VH/VL combinations by ELISA/Octet K2 followed by sequencing and reconstruction if necessary.
- Production of immunomolecules (full antibodies, fragments, chimeric molecules, etc) by QMCF expresssion technology in mammalian (CHO) cells
The HybriFree Technology has several advantages:
- Fast- sequence information of the antigen-binding VH/VL pair can be obtained in 6-7 weeks starting from the immunization, and purified antibodies can be delivered in as fast as 8-9 weeks
- Cost efficient- antigen-recognizing enrichment of B-cell population reduces significantly size of antibody library
- Manufacturability- B-cells are used for the in vivo development, and CHO cells are used for the screening of the full IgG or ScFv-Fc libraries
- Universal - antibodies can be
reconstructed into different subtypes, origins and structures
- QMCF mammalian expression system can be used to produce antigens, as well as milligrams to grams amounts of developed antibodies