The codon-optimized gp120 gene inserts for the same four clades (A, B, C, and AE) as described above for research-grade proteins were transfected into CHO DG44 cells (Invitrogen, CA) and used to establish the master cell banks. CHO GD44 cells stably expressing each of the four gp120 were grown in 50-liter bioreactors, and the cell culture supernatants were collected after 8 to 10 days of fermentation and purified through a downstream purification process including anion-exchange, cation-exchange, and size exclusion steps, under GMP conditions. The purity of each gp120 protein was in the range of 96 to 98%, based on the release certificates. The same purified gp120 proteins are currently being tested in a phase I clinical trial (HVTN124) at six major U.S. medical centers.
Cho dg44 cells
Manufactured by Thermo Fisher Scientific
The CHO DG44 cells are a Chinese hamster ovary (CHO) cell line commonly used in the biopharmaceutical industry for the production of recombinant proteins and monoclonal antibodies. These cells are known for their ability to grow in suspension culture and their high protein expression capabilities.
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Lab products found in correlation
Pgem t easy vector, by Promega (1 mentions)
Phenol red, by Merck Group (1 mentions)
Methotrexate, by Merck Group (1 mentions)
L glutamine, by Harvard Bioscience (1 mentions)
Procho5 medium, by Lonza (1 mentions)
Macsquant tyto, by Miltenyi Biotec (1 mentions)
Vi cell xr cell counter, by Beckman Coulter (1 mentions)
Rprotein a sepharose fast flow, by GE Healthcare (1 mentions)
Endospecy es 50m kit, by Seikagaku (1 mentions)
5 protocols using cho dg44 cells
1
GMP-Grade HIV gp120 Protein Production
2
Bispecific Antibody Production in CHO Cells
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The hCXCR3 x hCCR6 BsAb was designed as a C-terminal heavy chain scFv construct for expression in CHO-DG44 cells (Invitrogen) using our “in house” pJacq expression vector. First, a (Gly4S)3 linker was inserted by PCR at the C-terminal end of the human IgG1 Fc region and the resulting PCR product was cloned in the pGem-T easy vector. The gene coding for the stabilized hX3 scFv was next amplified and cloned into the pGem-T-easy vector (Promega) containing the hIgG1-inker cassette using BamH1 and Cla1 restriction sites. The hIgG1 Fc-(Gly4S)3-hX3 scFv was then subcloned in pJacq mammalian expression vector. In a final step, hR6 VH and VL genes were cloned into pJacq. The ligation mixtures were transformed into XL-1 E. coli. DNA sequence analysis was used to confirm the correct sequence of the BsAb construct.
3
Establishment of CHO Cell Line Producing Humanized IgM Antibody
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Highly purified 2G12-IgG was a gift from Polymun Scientific (Immunbiologische Forschung GmbH, Klosterneuburg, Austria). The generation of CHO lines producing wild-type 2G12-IgM (IgM-012) as well as a control antibody (IgM-617) was described previously [16] . The germline variant of 2G12-IgM (IgM-012_GL) was designed according to standard humanization strategies for monoclonal antibodies [17] (link). Briefly, the germline genes with the highest similarity to vH and vL of IgM-012 were identified as IGHV3-21/JH3 and IGKV1-5/JK1, respectively, using the online tool IMGT/V-QUEST [18,19] and aligned with the variable regions of IgM-012 using CLUSTAL 2.0.8. Suitable replacements were then chosen based on a superimposition of an IgM-012 model with the structure of 2G12-IgG (PDB: 1OM3) using Swiss-PDBViewer [20] (link). Finally, codon-optimized sequences (Life Technologies) were cloned into bi-cistronic pIRES vectors (Clontech, # 631621) and used for transfection of CHO DG44 cells (Invitrogen, # A10999-01) to establish a CHO cell line producing IgM-012_GL, as described previously [14,16] .
Recombinant cell lines were routinely cultivated in chemically defined ProCHO5 medium (Lonza, # BE12-766Q) supplemented with 4 mM L-glutamine (Biochrom, # K0302), 15 μg/mL phenol red (Sigma-Aldrich, # P0290), 0.5 mg/mL G418 (PAA, # P11-012) and 100 nM methotrexate (Sigma-Aldrich, # M8407) [15] (link).
Recombinant cell lines were routinely cultivated in chemically defined ProCHO5 medium (Lonza, # BE12-766Q) supplemented with 4 mM L-glutamine (Biochrom, # K0302), 15 μg/mL phenol red (Sigma-Aldrich, # P0290), 0.5 mg/mL G418 (PAA, # P11-012) and 100 nM methotrexate (Sigma-Aldrich, # M8407) [15] (link).
4
Monoclonal Antibody Production in CHO Cells
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CHO DG44 cells (Invitrogen) derived from a GMP bank were cultured and transduced under serum-free conditions using lentivirus-derived vectors. After addition of lentiviral particles harboring expression constructs for hIgG1 mAbs, cells were incubated at 37°C and 5% CO2 and maintained for 20 days on average. High producer clone pre-enrichment was performed using the MACSQuant Tyto® (Miltenyi Biotec), a sterile and closed system cell sorting platform. Briefly, a mAb-specific catch reagent was attached to the cell surface of all CHO cells. Secreted mAbs were specifically bound to the catch reagent on secreting cells and subsequently labeled with two mAb-specific secondary antibodies (Miltenyi Biotec), anti-hIgkappa conjugated to phycoerythrin (PE) as detection antibody and anti-hIgG1 conjugated to allophycocyanin (APC) for positive cell selection by MACSQuant Tyto. A two-fold limiting dilution with 0.5 cells per well was used for single cell cloning from enriched high producer cell pools to ensure monoclonality. Cells were cultured at 37°C, 5% CO2, and 85% humidity for 14 to 20 days. Wells were analyzed regarding cell number, viability and expression level of the target mAb using IgG-ELISA Kit (Invitrogen) and Vi Cell XR cell counter (Beckmann Coulter). Selected clones were cryopreserved and tested accordingly to state of the art GMP guidelines.
5
Generation and Purification of Chimeric Monoclonal Antibodies
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Human-mouse chimeric MAb ch61 was generated and purified from culture supernatants as described previously [23] (link). Briefly, total RNA was extracted from mouse hybridoma cells producing MAb m61, and the variable heavy- and light-chain regions were amplified by RT-PCR with primers designed for the antibodies. The PCR products were cloned into an expression vector. Stable cell lines expressing recombinant MAb ch61 were obtained by transfection of CHO DG44 cells (Invitrogen, Carlsbad, CA). Chimeric MAbs (ch133 and ch226) specific for the Ebola virus glycoprotein were generated as control MAbs using the same methodology [23] (link). These human-mouse chimeric MAbs were purified from culture supernatants using rProtein A Sepharose Fast Flow (GE Healthcare) and EndoTrap red (Profos AG). MAb purity (>98%) and endotoxin levels (<1.0 EU/ml) were confirmed by performing SDS-PAGE and with an Endospecy ES-50M kit (Seikagaku Corporation), respectively.
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