Monoclonal Antibody Production and Purification

lymphocyte and antibodies

  • 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
WilsonWolf Manufacturing: 1.5L bioreactors
Cat# 82000S
New Brighton, MN 55112
Tel# (651) 6289259

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

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