IcoCell platform is a well characterized proprietary host cell line platform to efficiently generate GMP-compliant stable CHO cell lines for production of recombinant proteins and monoclonal antibodies.
For IcoCell cell line development the DNA is integrated to the chromosome using our proprietary technology that facilitates integration of the expression cassette into transcriptionally active regions of the chromatin and ensures stable expression of recombinant protein. Our optimized production conditions support high viable cell densities and protein yields. In addition, the production process can be customized, depending on the product of interest.
A cell line development project is divided into three main stages:
1. DNA expression vector construction and transfection into CHO cells
During the initial stage of the project, the codon optimized coding sequence of transgene is inserted into the DNA expression vector. The proprietary DNA expression vector that is used for transfection, is specifically designed to Icosagen’s CHO cells. The expression cassettes are optimized by carefully selected regulatory elements (promoters, introns, polyadenylation) and the secretion signals for enhanced secretion as well as additional unique elements promoting the integration of transgene into transcriptionally active region of CHO genome. The linearized DNA vector is transfected into parental CHO cell line. The initial stage of the project takes 4 weeks.
2. Cell minipool generation and identification of top producing minipools
days after transfection, the secreted expression of a desired antibody or
antigen is confirmed by Western Blot analysis, and metabolic selection is
initiated by seeding few thousand transfected cells per one well of 5x 96-well
plates. This means that each 96-well is an individual selection unit called “minipool”. After the initial selection and recovery of
minipools, which lasts around 2 weeks, the first round of
ELISA screening is performed to test expression titers of each minipool. 72
best minipools with the highest protein expression levels are then selected, gradually
expanded to 6-well plates and tested second time with normalized ELISA. It
means that minipools are seeded to 6-well plates at equal cell densities and
allowed to grow for three days prior to ELISA screening. Top 10–15 minipools by highest
titers are thereafter scaled up to 125mL flasks for 1) cryopreservation (3-vial
pre-RCB) and 2) further productivity analysis in a small-scale full fed-batch
production mode in shake flasks. Based on the fed-batch productivity determined
by ELISA or BLI (Octet), up to three best minipools are selected for single cell cloning.
3. Clonally-derived cell line generationThe lead cell minipools are subjected to single cell cloning procedure. Single cells are seeded into five 96-well plates. To obtain clonally derived cell line, the VIPS instrument (Solentim) is adopted into our CLD workflow that couples single-cell printing method with high resolution imaging system to provide assurance of clonal derivation. This can be divided into two main steps:
1. Single cell detection in a droplet while depositing. VIPS tries 16 times to gently dispense cells in 1 nL droplets into a well. Crucially, the cell is imaged in the bottom of the dry well, as an evidence of seeding. Automated imaging in multiple tight z-stack layers of the whole droplet commences with intelligence-based image analysis confirming the existence of a single cell. Thereafter, the well is then filled automatically with media.
2. Whole well imaging is performed 3 hours after the cell droplet is dispensed into the well. After that, colony outgrowths are photo-documented in the next seven days.
A Chassis for Enhanced Mammalian Cell Line Development and Engineering
Biologic drugs ($160+ billion market) provide better long-term health outcomes with fewer side effects than many traditional drugs. However, due to high development and production costs, these drugs are 20x more expensive, on average, limiting the access to these therapies and burdening healthcare systems. To a great extent, this is because traditionally flexible and creative biotech SMEs currently lack rational tools for mammalian cell line development (most of the biologic drugs are produced in mammalian cell lines), meaning that large investment in terms of capital and time is required to make a production cell line.
ICF, an established player in the biotechnology industry, develops a technology for more efficient development of mammalian cell lines for high-yield protein production. It reduces the need for robotic systems and high-throughput screening facilities (€900 000 -> €200 000), the time (6-12 months -> 3) and cost (€100 000 -> €50 000) of cell line development. These advantages stem from specific landing platforms, predefined integration sites, recombination-dependent insertion and amplification of expression units. The technology will enable biotech SMEs to develop the process of production of new biologic drugs at lower costs and faster, eventually enabling biologic drugs to penetrate more cost-sensitive indications and target groups.
IcoCell project is aimed at optimizing the landing platform, generating cell lines compatible with process of development of industrial cell factories, generating regulatory documentation, preparing standard operating procedures, demonstrating the production of different types of proteins and implementing initial exploitation activities.
Cell line development market is expected to grow at CAGR of 12.8% (2014-2022) and the market for cell line development to produce therapeutic proteins exceeds €900M. Commercialization of IcoCell technology will mean breakthrough for ICF, increasing revenues by an estimated €26M in 2021
The opportunity to focus on the production of biopharmaceuticals is often taken from SMEs since huge amount of resources is required for the development of high-producer cell lines. We envision that our parental cell factories will increase the efficiency of cell line engineering and will effectively reduce the time-to-market for mammalian cell-based products. It allows SMEs to leverage their traditional strengths of adaptability and creativity, and to develop novel products for biotechnology industries.
Project period: May 1, 2016 - April 30, 2018.