Leveraging the speed and agility of eukaryotic
cell-free expression protein expression.
expressing proteins in a single tube using microsome cell free expression technology.
Cell-free protein expression (CFPE) systems derived from crude cell extracts have been used for decades as a research tool and have introduced many attractive advantages to protein expression technology. However, a critical barrier to the adoption of CFPE systems as alternatives to cell-based approaches has been the problem of relatively low protein yields.
To address this problem, Bombi has taken advantage of plant cell biology and developed a novel eukaryotic cell-free expression system: MICE.
Our MICE-T utilizes tobacco cell lysates, depleted of vacuoles. Compared to other cell-free expression systems, the key differentiator of MICE-T is that its intact protein production machinery reflects that of a living cell. It has built-in active energy storage and regeneration and includes everything necessary for in-vitro transcription (RNA polymerases, NTPs), translation reactions (ribosomes, translation initiation/elongation factors, tRNA, etc.), protein folding, disulfide bonding, and glycosylation.
Using a simple protocol, both the RNA transcription and translation take place in a single tube (combined transcription/translation). Reactions are started by simply mixing plasmid DNA with a proprietary reaction mix. The outstanding protein yields are based on highly efficient protein expression over the duration of the reaction which takes approximately 48 hours.
continuous growing of tobacco by-2 cells
Nicotiana tabacum BY-2 cells are growing continuously in a specially designed rocking wave bioreactor at a constant packed cell volume and a doubling time of around 35 hours to ensure a reproducible supply of homogeneous cell material.
The cells are harvested during the exponential growth phase and treated with a proprietary enzyme mixture to digest the cell wall to generate protoplasts.
Low-density vacuoles contain a great part of undesirable enzymes, including proteases and ribonucleases that can reduce protein expression. They are removed by density-gradient centrifugation which yields high-density evacuolated protoplasts.
The recovered evacuolated protoplasts are washed, treated with a protease inhibitor mixture and lysed by nitrogen cavitation to protect labile components from oxidation. The nuclei and non-disrupted cells are removed by centrifugation. The resulting reproducible cell-free homogenates are generated after nuclease treatment and nuclease deactivation.
The system contains all factors required for in vitro transcription (RNA polymerases, NTPs) and translation reactions (ribosomes, translation initiation/elongation factors, tRNA, etc.). Both the RNA transcription and translation take place in a single tube (coupled transcription/translation). Protein expression takes place during the reaction which approximately takes 48 hours.
The isolated and extracted recombinant protein from the coupled cell-free lysate undergoes industry standard protein purification before finishing the biologic in its final form.
From gene sequence to protein production in as little as 7 days
Designed to accurately match the gene of interest
Ability to produce different proteins at the same time
The platform is linear scalable just by growing more BY-2 cells
With increasing commercial opportunities for biotherapeutics across global markets, demands are as high as ever for technologies that can support production of complex glycoproteins. N-linked glycosylation is a common post-translational modification to many proteins that may influence biological activity, protein conformation, stability, solubility, secretion, pharmacokinetics, and antigenicity.
Consequently, the ability to control and tailor N-glycosylation is critical for today’s antibody engineering. Bombi’s MICE platform provides in-situ glycan engineering, supported by multi-attribute analytical methods such as mass spectrometry to elucidate and quantify precise glycoforms on recombinant proteins.
why glycan engineering?
Bombi's MICE platform delivers products with inherently greater N-linked glycosylation homogeneity versus competing platforms:
Bacteria do not glycosylate. Yeast hyperglycosylate. Chinese hamster ovary [CHO] cell lines do not precisely mimic human glycosylation patterns and intellectual property barriers limit access to glycosylation controls in CHO.
N. tabacum BY-2 cells do not attach α1,6-fucose, terminal β1,4-galactose residues or any sialic acid residues, leading to simpler, more homogeneous N-linked glycosylation patterns than other eukaryotic expression platforms. It adds α1,3-fucose.