Custom Bio-Manufacturing Services

Creative Biolabs offers a full range of custom protein expression and bio-manufacturing services that cover gene synthesis, vector construction, pilot expression and purification, process development and process optimization. We have an established track record in delivering custom proteins in both small research and large preparative scale. In particular, we are very experienced in producing human protein in bacterial and mammalian systems in soluble forms of biological activities. Process development and process optimization services for therapeutic protein production are provided using both bacterial and mammalian cell expression systems.

We, at Creative Biolabs [Protein expression services], together with our sister division, Creative BioStructure [Protein crystallography] and Creative BioMart [catalogue proteins], have bio-manufactured a large number of recombinant proteins of distinct properties in various expression systems. Importantly, our integrated expertise in bio-manufacturing allows us efficiently producing almost any recombinant proteins in large scales using either soluble or inclusion body expression in bacterial cells and either transient or stable transfection in mammalian cells. In particular, we have also established a solid platform for industrial scale refolding of recombinant proteins in bacterial cells.
 

Bio-manufacturing in Bacteria cells

Our home-made bacterial expression vector pCD-Bac is a temperature-inducible prokaryotic expression vector, which contains a PRPL promoter, cIts857 gene [temperature-sensitive CI repressor], multiple cloning sites and two strong transcription terminators. With genetic codons optimized into that bacterial preference, this vector enables high-yield protein expression of most proteins in the host bacterial strain Escherichia coli DH5α when our propriety fermentation protocol is employed using NLF-22 fermentor (Bioengineering). Our fermentation process is a batch process, not a continuous process.

We have extensive experience in refolding proteins from inclusion bodies which is evidenced by the large list of our catalogue proteins at Creative BioMart, among which over 200 were expressed, refolded and purified into a soluble and biologically active form. Also, using the same skills, we are providing X-ray crystallography work at Creative BioStructure, in which protein refolding is the key. We are able to set up a series of inclusion body solubilisation and recombinant protein refolding buffers and a set of refolding strategies are tested for each protein target. Importantly, we are using a wide range of protein purification skills such as ultrafiltration, Hydrophobic Interaction Chromatography, Ion exchange Chromatography and Size Exclusion Chromatography in purifying the refolded protein. Finally, for batch release, the following assays were performed sequentially: protein bioactivity test [usually using relevant cell lines], protein concentration [Lowry method], protein purity [SDS-PAGE, reduced and non-reduced, SEC-HPLC] protein MW [SDS-PAGE], Endotoxin [LAL test] and protein sequence [the first five N-terminal amino acids]. From a fermentation of 100L, 6g purified protein is regularly produced.
 

Bio-manufacturing in Mammalian cells

Stable Cell Line Creation:

To create stable cell lines is a key method in reducing the production costs of biopharmaceuticals. We use a modified FRT/Flp-In recombinase system to produce human proteins in CHO cells and 293 cells for stable expression. We have pre-selected CHO cell lines [and 293 cell lines] that contain a single stably integrated FRT site at a transcriptionally active genomic locus and we have proven that they can produce high level expressers of recombinant proteins.

We have also developed a unique series of high-yield vector system, in which two GC-rich regions flanking the gene expression cassette makes the promoter and enhancer continuously exposed to transcriptional machinery by preventing chromatin condensing. This revolutionary ground-breaking technology allows the available highest yields in all common mammalian cell protein production host cell lines, such as 293, CHO and NSO. Antibody yields of up to 2.0- 5.5 grams/liter [80-120pg/cell/day] have been achieved using this system in chemically defined, animal component-free medium. The selection strategy has been designed to identify cell lines that are highly productive and have the ability to grow well in suspension culture in a state-of-the-art, fed-batch fermentation process, in which cell density can reach 1.5×10⁷/mL.

In the end of production, the recombinant proteins are usually three-step purified to a purity >99.99% and endotoxin level <0.5 EU/mg. The cell lines will be archived for a period of 2 years free of charge to clients in case future production is needed. Note that our head in charge of this CHO production business received a Ph.D. from MIT and had over 6 years industrial experience at Merck Research Lab at West Point.

Process Development:

Following creation of a new cell line, or receipt of an existing cell line, to establish a process that is suitable for transfer to the manufacturing facility is the next step. Typical studies at this stage would include selection of a culture medium, definition of an inoculum expansion and production process, including feeding strategies, and cell line stability studies. We have extensive experience in developing a wide range of scalable processes for CHO, 293, hybridoma, and a variety of engineered cell lines. In particular, we use patented Perfusion Bioreactors that rely on a novel oxygen transfer method, enabling extremely high-cell density in culture. Together with the unique expression vector system, this bioreactor technology significantly increases the yield of bio-manufacturing, thus lowering the cost. Our current bio-manufacturing capacity reaches 1000L.

Biologicals manufactured:

In addition to the large number of research-use proteins marketed at Creative BioMart, we have produced the following biologicals: IL-18BP-FC-IL-1RA, TNFR2-Fc-IL-1ra, TNFR2-Fc-IL-ra, GLP-1-Fc-Leptin, PYY-Fc-Leptin, Symlin-Fc-IL-1ra, FC-Leptin, VEGF trap, IGF, PYY-Fc-laptin, GLP1-Fc-IL-1ra Cyminlin-Fc-IL-1ra, Interferon α2b, G-CSF, PEG-GCSF, and GM-CSF.
 

Currently, we are able to offer GLP compliant protein manufacturing work in both bacterial and mammalian cells.