Antibody Production and Purification

• Antibodies are produced in disposable bioreactors
• Hybridomas are grown in media containing Fetal Clone Serum (Hyclone) depleted of IgG
• Average yield approximately 25mg/500ml prep
• Yield is hybridoma dependent
• Prep sizes starting at 500ml
• Antibody Request Form
• Approximate delivery: four to five weeks

Equipment:

Lampire Biological Products:  500ml bioreactors

Cat# 7800101

PO Box 270

Piperville, PA 18947

Ph: (215)7952838

email: info@lampire.com

Basic Protocol:

• 1-2 x 10⁸ hybridoma cells/500ml (growth medium) in log phase growth are pelleted and inoculated into bioreactor (bioreactor medium)
• Bioreactor placed in incubator (5% CO₂ and air)
• Bioreactor checked after 14 days
• Medium harvested when cell viability reaches 10% live cells
• Prep clarified by centrifugation

IMDM Media, 10% FC1: (Growth Medium)

• Iscove's MDM (Invitrogen 12440053)
• 10% Fetal Clone Serum (Hyclone)
• 6 x 10^-5 M β-mercaptoethanol
• 292 mg/L L-glutamine

IMDM Media, 5% dFC1: (Bioreactor Medium)

• Iscove's MDM
• 5% Fetal Clone Serum IgG depleted
• 6 x 10^-5 M β-mercaptoethanol
• 292 mg/L L-glutamine
• Gentamycin
• Nystatin

Monoclonal Antibody Purification:

• Antibody purified from harvest using affinity chromatography on recombinant Protein G columns (Gamma-Bind Plus, GE Biomedical)
• HPLC: Shimadzu Automated Preparative/Analytical System

Basic Protocol:

• Bioreactor product sterile filtered
• Column washed with 0.01 M sodium phosphate buffer, pH 7.0, 0.15M sodium chloride and 0.01 M EDTA
• Prep loaded on column
• Antibody eluted with 0.5M acetic acid
• Antibody peak adjusted to pH 7.0 using 2M Trizma base