• search ORA
  • cwru.edu
close button

For graduate school admissions

Visit the Office of Graduate Education for info on our many doctoral programs and the graduate education application process at the School of Medicine.


For medical school admissions

Visit the Office of Admission for info on application process, financial aid, and student life.

close button

Give online to the Biomedical Sciences PhD Fund

Use our secure online giving form to make your gift to Case Western Reserve University now.

Make a difference

Find out how you can Take Three Steps to make a difference at Case Western Reserve University.

close button

Welcome to campus

Visit Case Western Reserve University's uniquely urban campus, located in the heart of Cleveland's cultural hub, University Circle. Plan your trip:

Policy on the Production of Monoclonal Antibodies

It is the policy of the Case IACUC to require justification for the production of monoclonal antibodies using the ascites method because of the potential for pain in distress to the host animal. The CWRU IACUC strongly discourages propagation of monoclonal antibodies using ascites tumors but recognizes that there are circumstances where the continued use of ascites production is warranted. The justification for the continued use of the ascites method must be provided at the time of submission or renewal of protocols. Such circumstances might include but are not limited to:

  • Antibody production by the hybridoma has been lost in vitro,
  • Failure of a hybridoma to grow in vitro,
  • Supernatant concentration of in vitro-produced antibody falls below an acceptable level,
  • More than 5 mg of monoclonal antibody are needed from five or more hybridomas cell lines simultaneously,
  • When more than 50 mg of a functional monoclonal antibody are required AND previous experience indicates that in vitro techniques are inappropriate for meeting this amount
  • As a means of "de-contaminating" a hybridoma line that has become infected with a contaminating micro-organism.

Monoclonal Antibody Production:

  1. Introduction
  2. In vitro production
  3. Antibody production using the BD CELLine device
  4. Protein G purification/concentration of antibodies
  5. Ascites production in mice

I. Introduction

Small-scale monoclonal antibody (mAb) production in research laboratories has traditionally been accomplished by injection of mAb-producing hybridomas into the abdomen of mice, and subsequently collecting the ascites that develops over the next seven to 10 days. This technique, while still in common use, has fallen into disfavor for a number of reasons including the following:

  • Stress and/or suffering of the mice,
  • Contamination of ascites with rodent plasma proteins, immunoglobulins, infectious agents and bioreactive cytokines,
  • Cost, support services, and the need for frequent monitoring of the mice,
  • Failure of some hybridomas (for example,human) to grow in rodents,
  • Failure to produce ascites due to premature death, development of solid tumors, or failure to establish in vivo hybridoma growth,
  • Variation in batches of ascites in quality and quantity.

II. In vitro production

While there are now many systems commercially available for in vitro production of Mabs, most are prohibitively expensive, particularly for those laboratories that produce Mabs infrequently. The most simple and cost-effective method for in vitro production of Mabs is standard tissue culture in either large flasks or roller bottles. These tissue culture methods are faster than ascites production, require less labor, avoid the variability experienced with ascites production, and depending on the desired amount and purity of the Mab, can be less expensive. The production of Mab by hybridomas in tissue culture is hybridoma-dependent and can vary between 1 - 100 µg/ml. Therefore, it is often desirable to concentrate Mab from supernatant. However, this step, which also removes non-antibody proteins is often required when using Mab generated by ascites as well.

The following information outlines the basic protocol for generating antibody in large flasks and roller bottles. Descriptions, resources, and references for other in vitro production systems are described on the USDA web site at http://www.nalusda.gov. Additional information is available in "Monoclonal Antibody Production", an online book published by the Institue for Laboratory Animal Research and National Academy Press in 1999.

III. Antibody production using the BD CELLine device

An easy method to produce large quantities of highly concentrated monoclonal antibody has been developed using a CELLine flask made by Becton Dickinson (catalog # 353137). This system yields antibody concentrations approaching that of ascites fluid, thus reducing the need for mice in the propagation of large amounts of antibody. The basic protocol for antibody production using this method is as follows:

  1. Culture hybridoma cells as usual in the established growth media. When cells are in optimal growth phase, gradually wean them onto BD Quantum Yield Basal medium (catalog # 220511) supplemented with 10% FBS of good quality. When cells are growing rapidly in 100% Quantum Yield media, proceed to the next step. Stocks of cells conditioned to this media can be frozen down for future antibody production.
  2. Seed 2x10 6 /ml in 15 ml Quantum Yield /FBS media into the cell compartment of a CELLine flask. Fill the nutrient compartment of the CELLine flask with 50% Quantum Yield /FBS and 50% BD Cell Serum-Free medium (catalog # 220509). Add the 50/50 mixture to bring the volume in the nutrient compartment to 1000ml.
  3. Place the flask into a 5% CO2 incubator for 48 hours. After 48 hr, sample the cells from the cell compartment for cell density and viability assessment using a hemacytometer.
  4. If cells are growing normally, change the media in the nutrient compartment to 100% BD Serum-Free media. If cells are growing slowly, wait 24 hours then change the media to 75% BD Serum-Free / 25% Quantum Yield with 10% FBS. Continue to incubate cells in the incubator.
  5. Harvest the cells at day 14 by removing the 15 ml cells in the cell compartment. Centrifuge the cells 5 minutes at 1000 rpm. Remove the antibody-containing cell supernatant.
  6. Reseed the cell compartment with cells at a density of 2x10 6 cells/ml in 15 ml 100% Serum-Free media.
  7. Continue in this fashion, harvesting cells every 7 to 14 days with a nutrient compartment media change every 21 days. For fast growing cell lines, it may be best to harvest every 7 days. The cells in the cell compartment over time will decrease in viability, when this happens consider them exhausted and stop antibody production.
  8. The antibody from the supernatants can be purified or used directly depending on need. For best results store antibody at -80ºC and avoid freeze-thaw.

In general, the CELLine system:

  • Produces antibody in concentrations of 1 to 5 mg/ml.
  • Produces approximately 15 ml supernatant yielding between 30 and 150 mg of antibody every two weeks
  • Produces antibody in concentration and amount equivalent to that of 12 mice.

Denice Major (dxm7@po.cwru.edu), in Vance Lemmon’s lab (vance@cwru.edu), has used this system to make large amounts of antibody from two different monoclonal lines with very little difficulty. It is far easier than hollow fiber devices we have used in the past. Using defined media, eliminated purification problems from contaminating bovine Ig using protein G

IV. Protein G purification/concentration of antibodies

Protein G can be used for the isolation of IgG from serum, ascites, or hybridoma supernatants. Although this specific protocol is geared for large volumes of hybridoma supernatants, the buffers and principles are applicable to other sources of IgG. Most volumes of supernatant can be readily handled using 5 ml of Protein G-Sepharose. This can cost several hundred dollars but can be used for years as long as the column is cleaned and re-equilibrated properly.

Important considerations:

  • Concentration/purification of antibody is typically performed by affinity chromatography using Protein A- or Protein G-Sepharose beads. These proteins bind to the Fc region of many mammalian species but only for IgG. Therefore, if your hybridoma secretes IgA or IgM this method will not be applicable for purification or concentration.
  • While Protein A and Protein G both bind the IgG Fc of many mammalian species, Protein A does not bind very well to the IgG1 subclass of mice. Therefore, to avoid any potential problems it is best to use Protein G.
  • Affinity purification of IgG from tissue culture supernatant will co-purify bovine IgG from fetal calf serum. Therefore, if purity is important it is best to use low IgG fetal calf serum or even serum-free media to grow your hybridomas. Serum-free media is also practical since high protein concentrations can make filtering tedious.

Solutions:

20 mM Phosphate Buffer, pH 8.0
0.05 M NaAcetate, pH 3.0
1.0 M NaAcetate, pH 2.5
1.0 M Tris Base, pH 10.0
20 mM Phosphate Buffer, pH 8.0; 0.05% NaN3

Preparation of cell supernatant:

  1. Remove flasks or roller bottles from incubator and pellet the hybridoma cells by centrifugation at 5000g for 15 min.
  2. Transfer the supernatant to a clean container and pre-filter using a fluted funnel filter.
  3. Filter supernatant through 0.8 micron bottle top filter. It is critical to place a pre-filter disc on the 0.8 micron membrane to delay clogging as long as possible. If flow stops, discontinue the vacuum and replace the pre-filter disc. Re-apply the vacuum and complete filtering.
  4. Repeat using a 0.45 micron bottle top filter (remember to use pre-filter discs).
  5. Store supernatant at 4°C until ready for protein G chromatography.

Affinity purification of lgG:

  1. Wash a 5 ml protein G-Sepharose column with 30 ml PBS.
  2. Apply the cell supernatant to the column by setting up a reservoir to allow for continuous flow. Remember slower is better. Shoot for one drop every five seconds. Collect and save all the effluent in case a second application is desired. The reservoir can be as large as several liters. The volume is only limited by the potential saturation of the Protein G column. For example, 1 ml of Protein G Agarose from Gibco-BRL (Rockville, MD) can bind up to 18 mg of human IgG. Therefore a 5 ml gel bed will bind between 90 - 100 mg IgG. Even at the high end of antibody concentration, a 5 ml column should easily handle one liter of hybridoma supernatant.
  3. When all the media has passed over the column, wash with excess PBS (about 30-40 ml).
  4. Prepare tubes for collecting the eluted IgG. Set up 10 boroscilicate glass tubes. Place 25 µL Tris solution into tube #l and 70 µL Tris solution into tubes #2-10.
  5. Elute the lgG by adding 20 ml 0.05 M NaAcetate, pH 3.0 to the column. Immediately begin to collect the drops in your glass tubes. Collect 1 ml in tube #l and then 2 ml into each of tubes 2-10.
  6. Strip the column by applying 20 ml of 1.0 ml I M Na Acetate, pH 2.5 (collect into a waste container).
  7. Re-equilibrate the column in PBS with sodium azide.

IV. Quantification of IgG

***The following analysis is designed for assessing the yield for one or more 2 liter roller bottle flasks. Smaller volumes of supernatant may require the user to scale down the fraction size and/or dilution for measuring O.D.

  1. Dilute an aliquot of each fraction 1:20 in PBS.
  2. Determine the O.D. of each diluted aliquot at 280 nm.
  3. Calculate concentration of IgG in each of the original tubes.
    Ex: [(O.D.) x (dilution factor of 20)]/(extinction coefficient of 1.5)
  4. Pool the fractions you are interested in keeping based upon the protein concentrations.
  5. Dialyze versus PBS (or whatever buffer you require).
  6. Remove from dialysis and determine the protein concentration again by diluting an aliquot 1:20 and measuring the O.D. 280 nm.

V. Ascites production in mice

Ascitic fluid (also called ascites) is an intraperitoneal fluid extracted from mice that have developed a peritoneal tumor. For antibody production, the tumor is induced by injecting hybridoma cells into the peritoneum, which serves as a growth chamber for the cells. The hybridoma cells grow to high densities and continue to secrete the antibody of interest, thus creating a high-titered solution of antibodies for collection. Antibody concentrations will typically range between 1 and 10 mg/ml.

The production of monoclonal antibodies using the ascites method requires appropriate justification because of the potential for pain in distress to the host animal. The CWRU IACUC strongly discourages propagation of monoclonal antibodies using ascites tumors but recognizes that there are circumstances where the continued use of ascites production will be warranted. The justification for the continued use of the ascites method must be provided at the time of submission or renewal of protocols. Such circumstances might include but are not limited to:

  • Antibody production by the hybridoma has been lost in vitro,
  • Failure of a hybridoma to grow in vitro,
  • Supernatant concentration of in vitro-produced antibody falls below an acceptable level,
  • More than 5 mg of monoclonal antibody are needed from five or more hybridomas cell lines simultaneously,
  • When more than 50 mg of a functional monoclonal antibody are required AND previous experience indicates that in vitro techniques are inappropriate for meeting this amount,
  • As a means of "de-contaminating" a hybridoma line that has become infected with a contaminating micro-organism.

Regardless of the rationale for the production of ascites, IACUC approval is always required and now undergoes heightened scrutiny. Where the ascites method is required, investigators must conform to the standards of practice proposed by the NIH. The NIH Standards have been incorporated into the following protocol.

  1. Prime adult female mice (at least 6 weeks old) of the same genetic background as your hybridoma by injecting 0.5 ml of pristane (2,6,10,14-tetramethyldecanoic acid) or incomplete Freund's adjuvant into the peritoneum. These solutions will act as irritants to the mice, which respond by secreting nutrients and recruiting monocyte and lymphoid cells into the area. This creates a good environment for the growth of the hybridoma cells. In some case 0.1 - 0.2 ml of Pristane has been found to be sufficient. Since it does act as an irritant efforts should be made to use as small a volume as is practical.
  2. After 7-14 days, inject 5 x 105 to 5 x 106 hybridoma cells in a volume of 0.1 - 0.5 ml by intraperitoneal injection. Prior to injection, the cells should be growing rapidly. Centrifuge the cells and wash once in PBS or some other physiologic solution.
  3. Ascitic fluid may begin to accumulate within 1-2 weeks following the injection of cells. Tap the fluid when the mouse is noticeably large, but before the mouse has difficulty moving. To relieve the pressure and collect the ascitic fluid insert a syringe needle (18 - 22 gauge) into the lower abdomen and collect the ascites in a tube as it drips. Always use aseptic technique. (Anesthetizing the mice will make the collection of the ascites fluid easier. Consult your local officials for the best methods.)
    • Monitor mice following the tap for possible signs of shock due to fluid withdrawal (pale eyes, ears, and muzzle; difficulty breathing).
    • Shock may be prevented by subcutaneous injection of 2 - 3 ml warm saline or lactated Ringers solution.
  4. Many mice will continue to produce ascites, allowing for subsequent taps. However, no more than three survival taps should be performed. A final 4th tap can be performed at termination.
  5. Mice should be monitored daily, 7 days a week by personnel familiar with clinical signs associated with ascites production and circulatory shock.
  6. Mice should be euthanized appropriately before the final tap or at any point if there is evidence of debilitation, pain or distress. Signs of distress include hunched posture, rough haircoat, reduced food consumption, emaciation, inactivity, difficulty in ambulation, respiratory problems, and solid tumor growth.
  7. Incubate the ascites fluid at 37°C for 1 hr. Transfer to 4°C overnight.
  8. Spin the fluid at 3000g for 10 min. If there is an lipid layer, remove this first and discard. Carefully remove the supernatant from the cell pellet. Spin again if necessary.

Notes

  1. Between 2 and 10% of the antibodies from ascitic fluid will be from the mouse's current antibody repertoire and not from the hybridoma. This can be eliminated by using immunodeficient nude mice to generate ascites.
  2. Some workers have reported that retired breeders (i.e., old males) are particularly good for ascites production. Follow the protocol as suggested.
  3. Some cells will preferentially form solid tumors in the peritoneum rather than the soft-fluid tumors that yield good antibody titers. If these develop, switch the type of priming injection you are using (i.e., pristane to incomplete Freund's adjuvant or reverse) and inject the hybridoma cells into multiple sites ip. Even with these changes, this problem often cannot be overcome.
  4. Ascites tumors may be passaged serially from one mouse to a second primed mouse or set of mice. Transfer approximately 105 to 5 x 105 cells directly from the ascites fluid into the second mouse. Such serial passage may be used to convert a line that predominately produces solid tumors to a line that grows as ascites. Wash the peritoneal cavity of a mouse bearing a solid tumor with 2 ml of PBS and transfer to a primed mouse.