Transient transfection of mammalian cell lines is being implemented by the pharmaceutical industry for more rapid research and development of drug candidates. Transient transfected cells produce therapeutic protein candidates based on cloned genes and gene variants of interest. The ability to produce the therapeutic protein candidates with the transient transfection process is very rapid compared to previous technology allowing large numbers of drug candidates to be screened and studied. However, high throughput automated transient transfection is required to cope with the dramatically increased sample load.
High throughput transient transfection requires sample formatting and sample processing that are conducive to automated workflow. To implement high throughput transient transfection, each step of sample preparation leading to the automated transient transfection process needs to be planned and integrated. This includes automating all the processes leading to preparing the plasmids containing various genes and gene variants that are used to transient transfect the cells. The automated process also facilitates the automated production and purification of expressed therapeutic protein candidates from the transient transfected cells.
Researchers select genes that are related to a molecule of interest and submit DNA sequences. The sequences are cloned into plasmids using PCR cloning. Researchers may request a library of archived genes for screening against a target. These genes are available from various sources as plasmids. A protein engineering campaign is carried out to create protein variants, perform affinity maturation or design a novel fold and function. These variants are generated through site-directed or random mutagenesis of a parental plasmid carrying the cloned gene of interest for produce plasmids and cloned using PCR cloning.
The PCR cloned or archived plasmids (which are only available in small mass quantities) are transformed with automation into a bacterial host cell to grow larger quantities of plasmid. Transformation of these plasmids is a form of quality assessment. Heat shock is performed using automatable heated deck position. No incubation neither cooling with ice nor shaking for growth is needed. The cloned genes and gene variants require transformation to become useable plasmids. Antibiotic resistance is used to select for positive clones. The transformed bacteria with plasmids are plated onto agar plates supplemented with the appropriate positive selection marker such as an antibiotic. This process may also be automated. Successful clones survive and grow into colonies.
Once the colonies reach an optimal size, a colony picking robot picks these colonies to inoculate larger cultures. Colony Picking may be automated with 96 well or 24 well growths. Picker software adjustment parameters include colony radius, amplitude, elongation, and separation from other colonies.
Growth is automated and performed in a 96 or 24 well format with deep well plates. The cells are pelletized and then automated plasmid purification is performed directly from the cell pellet using PhyNexus Lysate direction purification technology with chaotropic reagents and silica based columns. Depending on the system mini and maxi plasmid purifications are performed. Complete walk away automation plasmid purification is performed in a robotic system.
The cells are lysed with addition of reagent by a robotic pipette tip and plasmid is released into a mixture of liquid and debris. The suspension is precipitated with the addition by robotic pipette tip of a chaotropic reagent. A PhyTip column is lowered into the sample mixture of released plasmid and debris. Sample is processed by back and forth flow into the column. The debris may enter the column but does not constrict or plug the column because of large pore frits and large resin particles. The column is washed with several steps and then dried in a drying block to remove residual liquid
The PhyTip column is moved to an eluting solvent and pure, concentrated plasmid is eluted from the column into the well. The plasmids are endotoxin free suitable for Transient Transfection.
The PhyTip column chemistry produces a pure plasmid: no high salt matrix, no endotoxin – unique to PhyNexus. PhyTip Tip Concentrating Effect produces high concentration purified proteins and plasmids from limited sample (grown in 96 well format) – outperforms all other technologies.
Candidate sequences and be process and automated transient transfection can be performed in automated 96 well format. Transfected mammalian cell cultures are produced, maintained and kept alive to generate the therapeutic protein candidates of interest again in 96 well format.
Because proteins are expressed in 96 well format, they may be purified from expression in automated format. PhyTip column technology enables automation. The automation processes is segmented: automation can be introduced as needed. R&D productivity of drug candidate examination of express proteins is increased several fold. Qualified drug candidates of interest are transfected in stable cells and undergo further research. Proteins expressed from these cells may also be processed through automation with PhyTip columns and are assayed.