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Production and Purification of Recombinant Antibodies from Mammalian Cells

Published: March 29, 2024

Abstract

Source: Shivange, Gururaj, et al. Analyzing Tumor and Tissue Distribution of Target Antigen-Specific Therapeutic Antibody. J. Vis. Exp.(2020).

The video explains the process of producing antibodies in mammalian host cells, from gene expression to secretion. It involves transcription, translation, signal recognition, correct folding, and eventual release into the extracellular medium via secretory vesicles.

Protocol

1. Expression and purification of antibodies Maintenance of CHO cells Grow CHO cells in FreeStyle CHO Media supplemented with commercially available 1x glutamine supplement at 37 °C shaking at 130 rpm with 5% CO2 using delong Erlenmeyer flasks, either glass or disposable. NOTE: It is highly recommended to use a baffled flask with a vented cap for increased agitation and to improve gas transfer during shaking conditions. Sig…

Disclosures

The authors have nothing to disclose.

Materials

FreeStyle CHO media Gibco Life Technologies Cat # 12651-014
Anti-Anti (100X) Gibco Life Technologies Cat # 15240-062
Anti-Clumping Agent Gibco Life Technologies Cat # 01-0057DG
BD Insulin Syringe BD BioSciences Cat #329420
Caliper IVIS Spectrum PerkinElmer Cat #124262
CHO CD EfficientFeed B Gibco Life Technologies Cat #A10240-01
Corning 500 mL DMEM (Dulbecco's Modified Eagle's Medium) Corning Cat # 10-13-CV
Corning 500 mL RPMI 1640 Corning Cat # 10-040-CV
Cy5 conjugated Anti-Human IgG (H+L) Jackson ImmunoResearch Cat # 709-175-149
GlutaMax-I (100X) Gibco Life Technologies Cat # 35050-061
HiPure Plasmid Maxiprep kit Invitrogen Cat # K21007
HiTrap MabSelect SuRe Column GE Healthcare Cat # 11-0034-93
Isoflurane, USP Covetrus Cat # 11695-6777-2
Lubricant Eye Ointment Refresh Lacri-Lube Cat #4089
PEI transfection reagent Thermo Fisher Cat # BMS1003A
Experimental Models: Cell lines
Human: OVCAR-3 American Type Culture Collection ATCC HTB-161
Human: CHO-K cells Stable transformed in our lab ATCC CCL-61
Mouse: 4T1 Kind gift from Dr. Chip Landen, UVA
Mouse: MC38 Kind gift from Dr. Suzanne Ostrand-Rosenberg, UMBC Authenticated by STR profiling

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Cite This Article
Production and Purification of Recombinant Antibodies from Mammalian Cells. J. Vis. Exp. (Pending Publication), e22082, doi: (2024).

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