From 40 subclones, 20 leading ones were selected and assessed in terms of overall productivity

From 40 subclones, 20 leading ones were selected and assessed in terms of overall productivity. The contents of the isolated plasmid DNA from your producing bacterial clones were confirmed by restriction analysis. For transfection of the highly pure (transfection grade) isolated plasmid DNA, we used the Plasmid Maxi kit (QIAGEN, USA). Proper assembly of the manifestation vector was verified by restriction analysis. Lubiprostone The nucleotide sequences of both the genes and the adjoining areas were verified by sequencing. Culturing the CHO-S and CHO-DG44 cell lines Cell ethnicities were carried out in 125?mL Erlenmeyer DDPAC flasks inside a CO2 Multitron Cell shaker-incubator (Infors HT, Switzerland) operating at a rate of 125?rpm in an atmosphere of 5?% CO2, at a heat of 37?C and 95?% moisture. Reseeding was performed every 3C4?days to a denseness of 0.3C0.5??106 cells/mL. We used CD DG-44 (Existence systems, USA) and PowerCHO 2CD (Lonza, Switzerland) serum-free press supplemented with 8?mM L-glutamin. Cell counts and viability analysis were performed after staining with trypan blue (Panreac, Spain) using an automatic cell counter TC10 (Bio-Rad, USA). Transfection of CHO-DG44 and CHO-S cell lines Transfection was performed using the following combination of manifestation vectors: pcDNA3.3 LC Adalimumab?+?pOptiVec HC Adalimumab and pOptiVec LC Adalimumab?+?pcDNA3.3 HC Adalimumab, using the lipophilic agent FreeStyle Maximum (Invitrogen, USA). One day prior to transfection, the cells were re-plated to a denseness of 0.5C0.6??106?cells/mL. On the day of transfection, cell denseness was determined, and the cells were pelleted by centrifugation at 200for 10?min at room heat in an Allegra 25-R centrifuge (Beckman, Germany). The supernatant was eliminated by decantation, and the cells were suspended in FreeStyle? CHO Manifestation Medium, comprising 8?mM alanyl-glutamine (both reagents were from Invitrogen, USA) to a final density of 1 1.2C1.5??106 cells/mL. Further transfection was performed in 6-well plates, according to the manufacturers instructions (FreeStyle CHO-DG44 Cells, Invitrogen, USA). Transfection Lubiprostone effectiveness was assessed by fluorescent microscopy of cells with the pEYFP Lubiprostone plasmid and a blue color filter. The transfection effectiveness was evaluated visually using a CKX41 microscope (Olympus, Japan). According to the manufacturers instructions, subsequent selection of the transfected clones was performed as demonstrated in the schematic representation below (Fig.?1). Open in a separate windows Fig.?1 Clone selection scheme (modified from User guide for Freedom? DG44 Kit) and development of stable cell lines for protein production Selection of individual clones Limiting dilutions were used to select individual clones. After transfection for 24?h, the cells were suspended in CD OptiCHO Medium (Life systems, USA) containing 500?g/mL solution of G418 (Lonza, Switzerland) and 10?nM MTX at a denseness of 10,000, 5000 or 1000?cells/well. Hundred microliter of the cell suspension was added to every well of a 96-well plate, using 30C40 plates for each dilution. The plates were cultured inside a CO2 incubator at 5?% CO2 at 37?C and 95?% moisture Lubiprostone for 14C20?days. After 12?days, the growth of cells in the wells was controlled under a microscope, registering the wells that were experiencing cell growth and division. Upon reaching 80C100?% confluence, the individual mini-pools were transferred into 24-well plates. After 5?days, the samples were analyzed for the manifestation level of the prospective antibody using the IgG-ELISA-BEST kit (Vector-Best, Russia). The selected pools with the highest productivity were subcultured into 6-well plates, and then the positive swimming pools were re-selected for cell denseness and productivity by ELISA. Then the leading clones were transferred into T-75 flasks, and further into 125?mL Erlenmeyer flasks in two media in parallel: CD OptiCHO Medium (Existence technologies, USA) and ActiCHO SM (PAA, Austria) supplemented with 8?mM alanyl-glutamine, 25?nM MTX and 500?g/mL G418. At every step the number of clones was reduced, basing on growth of cells, viability, and productivity (Fig.?2). Open in a Lubiprostone separate windows Fig.?2 Construct of pOptiVEC-HC adalimumab, containing the sequence of adalimumab weighty chain. HC adalimumab, synthetic gene of weighty chain mAb for adalimumab, codon optimized sequence; CMV promoter, early.