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:
I. Introduction
II. In
vitro production
III. Antibody
production using the BD CELLine device
IV. Protein
G purification/concentration of antibodies
V. 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.
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. "Monoclonal
Antibody Production" is also available as an Acrobat
pdf file for download.
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
i. 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.
ii. Some workers have reported that retired breeders
(i.e., old males) are particularly good for ascites
production. Follow the protocol as suggested.
iii. 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.
iv. 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.
