Laboratory - Οrganization
Purpose: As stated in "About Us", the design of BReMeL in full operation will include 3 Departments: Production- Research / Diagnosis - Education / Information. We give priority to the activation of the Production Department and the "APIs Production" Sector. Initially, the laboratory will deal with the production of two bio-peptides in purified form - insulin and HBsAg antigen - first in laboratory and then in industrial scale. The two biomolecules have no structural and physiological affinity, but existing literature suggests the possibility of biosynthetic production by both using the same technique.
Lab structure: The initial structure of the laboratory will support two processes: a) The small-scale production of the two peptides and b) the applied method of production. In fact, three laboratory units will be set in order of operation: Laboratory of Molecular Biology (Genetics Engineering) - laboratory of Cell Biology - laboratory of Analytical Chemistry.
The process of recombinant insulin / recombinant HBsAg production in purified crystalline form involves the following complex stages:
1. Synthesis of the insulin or HBsAg gene.
2. Synthesis of the recombinant plasmid-vector of the corresponding gene.
3. Transformation: Incorporation of plasmids carrying the insulin or HBsAg gene into the chromosomes of the microorganisms to be cultured.
4. Selection of the recombinant cellular clone containing the optimal copy number of the plasmid gene vector.
5. Primary cultivation of the recombinant strains of microorganisms in nutritional medium of specific composition.
6. Integrated fermentation of recombinant microorganisms into controlled concentration media for optimal gene expression.
7. Collection of cells by continuous centrifugation.
8. Isolation and purification of the insulin precursor secreted into the culture medium and transforming it into insulin (HLPC chromatography - biotransformation with transpeptidation and deprotection -crystallisation).
9. Obtaining final product in sterile lyophilized form after multiple purification steps, including high
pressure chromatography to remove trace impurities.
10. Insulin / HBsAg identification by mass chromatography.
11. Purity control, and other tests according to specifications of the corresponding monograph of the European Pharmacopoeia VIII.
Of the above eleven steps, ten steps are the same for obtaining both substances. They differ only in step number eight because - unlike to proinsulin - the HBsAg antigen is a highly hydrophobic protein, resulting in inadequate secretion in the culture fluid and its detection at high intracellular concentrations.
Equipment: Sophisticated equipment and technologies are used for the production of human recombinant insulin. From Cell Biology, we will apply cell culture techniques (traditional cell cultures - fermentation). From Molecular Biology, the technique of recombinant DNA. Consequently, the required laboratory equipment will be proportionate to the method applied.
All the manipulations involving: cells and materials - fermentation of genetically modified microorganisms - isolation and modification of the extracellular product will take place in a sterile environment.
During the initial phase of production (laboratory scale), multi-surface or large flasks with 1-10lit capacity are used, while constantly stirring the culture fluid. Upon switching to a higher production scale, bio-reactors of varying capacities of 10-1000 litters are used while at the highest industrial scale more than 20,000 liters are used ... more
A list of the basic equipment of BReMeL laboratory with the main instruments will include ...
