253
Custom monoclonal antibody development
General overview 254
High quality production under ISO 9001:2000 conditions certification 254Full confidentiality guaranteed 254Specific Pathogen Free quality 254
Promoting the ethical treatment of animals 254
Mouse monoclonal 256
Rat monoclonal 257
In vitro antibody production 258
Ascites production 259
Gene immunization 261
14EUROGENTEC 2008 Catalogue > www.eurogentec.com > [email protected]
Custom monoclonal antibody development 14
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United KingdomEurogentec Ltd.3 +44 1794 511 4115 +44 1794 522 417u [email protected]
FranceEurogentec France s.a.s.u.3 +33 2 41 73 33 735 +33 2 41 73 10 26u [email protected]
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General overview
Our monoclonal antibody development services have been offered
to the scientific community since 1996. During this time we have
focused on delivering a flexible service designed to respond to your
project goals.
A dedicated project scientist handles each monoclonal antibody
development project, available for you to discuss at any time your
ongoing project goals and project progress.
All hybridomas developed at Eurogentec are the property of the
client and we guarantee not to claim any rights on the hybridoma
nor the antibodies.
Currently we offer monoclonal development in the following hosts:
† Mouse monoclonal
† Rat monoclonal
High quality production under
ISO 9001:2000 conditions
This guarantees you:
† Constant, high quality animals for immunization
† Standard procedures for immunization and blood sampling
† Excellent animal maintenance record
† Traceability
Full confidentiality guaranteed
Eurogentec guarantees full confidentiality regarding antigen
identity and project results. Feel free to contact us to put in place a
confidential disclosure agreement (CDA).
Specific Pathogen Free quality
SPF animals are tested for exposure to various viruses, bacteria,
parasites etc as recommended by FELASA. SPF animals offer
reduced background signals and higher success rates in antibody
production. Eurogentec offers SPF animals for your custom antibody
production.
Promoting the ethical treatment
of animals
Animals are maintained in an environment that is designed to minimize
their stress and exposure to danger. Animals are provided water and
feed according to their desire, are housed in groups to promote animal
interaction, and are maintained in a climate-controlled environment.
Our animal facilities comply with the highest quality standards and to
the European Community guidelines for animal housing and in vivo
experiments. Contact us for details.
3 programmes offered
† Mouse monoclonal, custom protocol
† Rat monoclonal, custom protocol
Complete range of monoclonal services
† Anti-peptide antibodies (incl. peptide synthesis)
† Anti-protein antibodies
† DNA immunization antibodies
† In vitro antibody production
† In vivo antibody production
† Antibody labelling - enzymes, biotin, fluorescence probes …
† Affinity purification
† ELISA testing
† Assay development services
Project work flow
† Comprehension of customer’s specifications
† Project plan flowchart and cost estimation
† Project approval and service agreement
† Hybridoma development with progress reports
† Delivery of hybridomas and supernatants
† Delivery of monoclonal antibody at desired scale
† Report at the end of each completed project phase
Flexibility is our service
Starting from your project goals we can provide you with a support
team for all your production and post-production related antibody
projects. Send your contact details to [email protected] and a
specialist will be pleased to discuss your project goals with you.
EUROGENTEC 2008 Catalogue > www.eurogentec.com > [email protected]
255
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United KingdomEurogentec Ltd.3 +44 1794 511 4115 +44 1794 522 417u [email protected]
FranceEurogentec France s.a.s.u.3 +33 2 41 73 33 735 +33 2 41 73 10 26u [email protected]
Germany • AustriaEurogentec Deutschland GmbH3 +49 221 258 94 555 +49 221 258 94 54u [email protected]
The NetherlandsEurogentec Nederland b.v.3 +31 43 352 06 985 +31 43 354 19 65u [email protected]
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In vitroMab production
EUROGENTEC 2008 Catalogue > www.eurogentec.com > [email protected]
Monoclonal development flowchart
Definition of Project Plan
Phase IImmunization (11 weeks)
Customer sends Antigen Eurogentec Produces Antigen
Report IELISA against antigen
Phase IIFusion (2-3 weeks)
Phase IIIScreen antigen
Screen cross-reactants(5-7 weeks)
Phase IVCloning/Subcloning
& Isotyping
In vivoMab production
Report to Customer
Report to Customer
Report to Customer
Report to Customer
Low titre
No titre
No hybridoma
No clones
Stop
Stop
Stop
Custom monoclonal antibody development 14
Belgium• LuxembourgEurogentec Headquarters3 +32 4 372 74 005 +32 4 372 75 00u [email protected]
United KingdomEurogentec Ltd.3 +44 1794 511 4115 +44 1794 522 417u [email protected]
FranceEurogentec France s.a.s.u.3 +33 2 41 73 33 735 +33 2 41 73 10 26u [email protected]
Germany • AustriaEurogentec Deutschland GmbH3 +49 221 258 94 555 +49 221 258 94 54u [email protected]
The NetherlandsEurogentec Nederland b.v.3 +31 43 352 06 985 +31 43 354 19 65u [email protected]
SwitzerlandEurogentec s.a.succursale de Genève3 FR: +32 4 372 74 00 DE: +49 221 258 94 555 FR: +32 4 372 75 00 DE: +49 221 258 94 54
Mouse monoclonalYou send us
† 250 µg of antigen per mouse (4 mouse minimum)
† May be sent lyophilized or in solution (1 ml, 250 µg/ml)
† Your ELISA protocol
You receive
† ELISA results of the polyclonal sera for mouse selection
† Fusion report – cell count of fused cells
† Screening report
- ELISA results of hybridomas against the antigen, and customer
supplied cross-reactants
- 10 ml of supernatant from up to 20 clones for customer to test
in desired application
† Cloning and isotyping report
- 10 ml of supernatant for each desired clone
† In vitro / In vivo production report
- Delivery of monoclonal antibody to desired scale
Phase I: Immunization
† A minimum of four mice (6–8 weeks old) are used for
immunization. Preimmune serum is taken on day 0.
† A total of four injections are administered (50 µg antigen per animal
per injection). The first injection on day 0 with equal parts (v/v)
of complete Freund’s adjuvant and antigen. The following three
boosts are carried out with equal parts (v/v) incomplete Freund’s
adjuvant and antigen, with three weeks between boosts.
† Selection of the best responding mouse 10 days after the last
injection using the client’s ELISA test.
Mouse monoclonal phases
Phase Reference
Phase I AS-ACMC-IM
Phase II AS-ACMC-FU08
Phase III AS-ACMC-AN08S
Phase IV AS-ACMC-CIFH
In vitro production
Mab production Reference
Roller bottle (10 mg) AS-ACMC-ROLLB
CL1000 (20-100 mg) AS-ACMC-CL1000
CP100-BR1 (100-300 mg) AS-ACMC-CP100
BR19 (> 300 mg) AS-ACMC-BR19
BR35 (> 1 g) AS-ACMC-BR35
CP2500 (> 10 g) AS-ACMC-CP2500
† If the antibody titre is too low after three booster injections, you
may request additional injections. If the ELISA report remains
negative, the immunization should be ended because the
probability of success is too low.
† If the antigen has a low immunogenicity, it can be coupled to an
appropriate protein carrier.
† Duration of this standard program: 11 weeks.
Phase II: Fusion
† The splenic lymphocytes of the best responding animal will be
fused with the cell line Sp2/O-Ag-14 using PEG (polyethylene
glycol), and the resulting hybridomas will be seeded in 96-well
plates in HAT medium. If any fusion cocktail remains, it will be
stored in liquid nitrogen.
† Duration: 2–3 weeks
Phase III: Screening and amplification of hybridomas
† IgG producing hybridomas will be screened against the target antigen
and two vials of every positive hybridoma stored in liquid nitrogen.
† The positive colonies are expanded and tested again for antibody
production with the antigen used for immunization.
† At this point, positive colonies can also be screened against
additional antigen for cross-reactivity or against a region of the
antigen of interest.
† Other screening tests can be used such as Western Blots or
immunofluorescence. Please contact us for a price offer.
† In addition, 5–10 ml supernatant of the 20 most positive
hybridomas will be sent to you for screening confirmation and
additional tests. This will ensure that the positive ELISA results
identify antibodies useful for your purpose.
† Duration: 5–7 weeks
Phase IV: Cloning and isotyping
† The specific hybridomas you have selected at the end of Phase
III will be cloned by limit dilution. The assay can be carried out as
in Phase III (screening for the antigen used for immunization and
additional screening of positive clones with additional antigens).
After cloning, three vials of every cloned hybridoma are stored
in liquid nitrogen. We send you 15–20 ml of every hybridoma
supernatant together with the frozen hybridomas.
† Subsequently, the subclass of every monoclonal antibody will
be determined by ELISA testing.
† Duration: 4 weeks
257
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Rat monoclonal
Phase 0 – Test phase for antigenicity† One rat is immunized in an 18-day protocol and titre is determined
by ELISA to evaluate antigenicity
† Duration 3 weeks
Phase I – Immunizations† Two rats are immunized in our 31-day protocol. Pre-immune
serum from day 0 is drawn and saved.
† 3 injections are administered
† Selection of the best responding rat for the fusion is done by
ELISA to evaluate antegenicity
† Duration 4 weeks
Phase II – Fusion† Cells from the best responding rat are fused to rat myeloma
IR983F and a selection is made using HAT medium
† Duration 2-3 weeks
Rat monoclonal phases
Phase Reference
Test AS-RAMC-IMTEST • 1 rat
Phase I & II AS-RAMC-FU088 plates • 2 rats
Phase III AS-RAMC-AN08S • 8 plates
Phase IV AS-RAMC-CIFH • 1 clone
In vitro production
Mab production Reference
Roller bottle (10 mg) AS-RAMC-ROLLB
CL1000 (20-100 mg) AS-RAMC-CL1000
CP100-BR1 (100-300 mg) AS-RAMC-CP100
BR19 (> 300 mg) AS-RAMC-BR19
BR35 (> 1 g) AS-RAMC-BR35
CP2500 (> 10 g) AS-RAMC-CP2500
Phase III – Screening and amplification of hybridomas† Hybridomas will be screened against the target antigen and two
vials of every positive hybridoma stored in liquid nitrogen.
† The positive colonies are expanded and tested again for antibody
production with the antigen used for immunization
† At this point, positive colonies can also be screened against
additional antigens for cross-reactivy or against a region of the
antigen of interest
† Other screening tests can be used such as WB or
immunofluorescence. Please contact us for a price.
† In addition, 5-10 ml supernatant of the 20 most positive
hybridomas will be sent to you for screening confirmation and
additional test. This will ensure that the positive ELISA results
identify antibodies useful for your application
† Duration 5-7 weeks
Phase IV – Cloning and isotyping† The specific hybridomas you have selected at the end of Phase III will
be cloned by limit dilution. After cloning, three vials of every cloned
hybridomas are stored in liquid nitrogen. We send you 15-20 ml of
every hybridoma supernatant together with the frozen hybridomas.
† Subclasses of every monoclonal antibody is determined by
ELISA testing
† Duration 4 weeks
You receive† A report at the end of each phase.
† Supernatants both before and after the cloning of the hybridomas,
in order to select the best monoclonal antibodies.
† The cloned hybridoma(s) with corresponding isotype(s).
Additional services are also offered, such as ascites purification,
antibody coupling and cell line storage. Please inquire for additional
information.
Mouse monoclonal additional service
Phase I : Immunization Reference
Per additional month and per mouse (price includes maintenance, immunization,bleedings and ELISA tests (minimum 4 mice)
AS-ACMC-IMADDM
Animal housing after mouse selection (if requested) price / month
AS-ACMC-HOUS
Phase II : Fusion Reference
Twelve 96-well plates AS-ACMC-FU12
Twenty 96-well plates AS-ACMC-FU20
Phase III : Analysis Reference
Additional screening of the positive wells. Per cross-reactant
AS-ACMC-AN08A
Additional screening of the positive wells. Per cross-reactant
AS-ACMC-AN12A
Screening - 12 plates fusion AS-ACMC-AN12S
Screening with 2 antigens (used for immunization + other one)
AS-ACMC-AN20S
Additional screening of the positive wells. Per cross-reactant
AS-ACMC-AN20A
Cell line storage (per year and cell line)
AS-ACMC-STOCK
Thawing - freezing (per clone)
AS-ACMC-DEFREE
Phase IV : Cloning and isotyping
Reference
Cloning and isotyping of each additional hybridoma
AS-ACMC-CIAH
Custom monoclonal antibody development 14
Belgium• LuxembourgEurogentec Headquarters3 +32 4 372 74 005 +32 4 372 75 00u [email protected]
United KingdomEurogentec Ltd.3 +44 1794 511 4115 +44 1794 522 417u [email protected]
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Germany • AustriaEurogentec Deutschland GmbH3 +49 221 258 94 555 +49 221 258 94 54u [email protected]
The NetherlandsEurogentec Nederland b.v.3 +31 43 352 06 985 +31 43 354 19 65u [email protected]
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In vitro antibody production
Phase I: Evaluation
Since different cell lines differ significantly in levels of antibody
production, we evaluate your hybridoma before starting the
procedure. The following parameters will be tested:
† Growth
† Mycoplasma status
† Production potential
† Immunochemical characterization
This will allow us to establish the most efficient method of growing
your hybridomas and to give you a reasonable estimate of delivery
time and price.
Phase II: Production
Antibody production will start according to the time schedule defined
with the client.
Quality Assurance
For this procedure, all reagents and culture media are obtained from
Biowhittaker-USA and its derived culture media.
We perform a rigorous Quality Control procedure with detailed product
batch characterization including HPLC, PAGE and IEF profiles.
All procedures are performed under tightly controlled conditions to
ensure a constant quality.
Different purification techniques can be used after discussion with
the client.
The client is guaranteed absolute confidentiality and security of his
cell lines and projects.
Comparison study
A one-year comparison study has been performed using different
bioreactors: culture flasks, Tecnomouse (BioIntegra), Mini-Perm
(Heraeus) and Cell Pharm System (Unisyn). Our study has clearly
shown that the most cost-effective production in the scale from
about 200 mg up to several grams of antibodies is obtained with the
highly automated Cell Pharm System (Unisyn).
Hollow-fibre technology
The Cell Pharm System makes effective use of hollow-fibre
technology. In this production method, the hybridoma cells are
placed in a bioreactor containing hollow fibres throµgh which fresh
culture medium flows. The capillaries are porous and the nutrients
can pass freely to the cells while the secreted antibodies and the
cells are excluded from the intracapillary space. Oxygen content and
medium pH are automatically regulated by a computer, ensuring that
all important parameters are controlled and available online.
In order to avoid nutrient gradients, which normally would develop,
a second pump also circulates the extracapillary medium, which
ensures a uniform nutrient distribution to all the hybridoma cells and
therefore optimal growth and production.
This system has an additional advantage in that the operator
can continually remove the secreted antibodies using the
Autoharvester, which continually samples the extracapillary medium
and automatically replaces it with fresh. The sampled antibody-
containing medium can then be processed by the purification unit
if necessary.
Closed system
As a consequence, the whole system remains closed and any risk of
contamination by bacteria or yeast is minimized. This type of reactor
can be run over months with minimal human intervention, except for
medium stock replacement.
Moreover, the system has been designed for flexibility in terms
of production quantities. Different reactor sizes are available for
optimum production of the needed quantities. This type of reactor
can easily be adapted to GMP production, as all the important
production parameters are controlled by the computer.
If you are interested in in vitro antibody production, feel free to
discuss with us all the relevant parameters of the project, (i.e. culture
conditions, antibody secretion yield in flask culture etc.), to make
sure that we can fulfill your wishes in terms of production quantities
and time frame.
Custom mouse monoclonal antibody
Phase V : In vitro production Reference
Roller bottles AS-ACMC-ROLLB
CL1000 AS-ACMC-CL1000
CP100-BR1 AS-ACMC-CP100
BR19 AS-ACMC-BR19
BR35 AS-ACMC-BR35
CP2500 AS-ACMC-CP2500
259
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United KingdomEurogentec Ltd.3 +44 1794 511 4115 +44 1794 522 417u [email protected]
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Germany • AustriaEurogentec Deutschland GmbH3 +49 221 258 94 555 +49 221 258 94 54u [email protected]
The NetherlandsEurogentec Nederland b.v.3 +31 43 352 06 985 +31 43 354 19 65u [email protected]
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Ascites production
On a case by case basis where in vitro production methods fail,
Eurogentec offers ascites production.
Full confidentiality guaranteed
Antibody production can be done under the terms of a “secrecy
agreement” if requested. Eurogentec guarantees full confidentiality
regarding the hybridomas and projects.
Eurogentec will never claim any property rights on either the
hybridomas or the monoclonal antibodies. These will remain the
property of the customer.
You send us
† Completed ascites production request form.
† We need a description of the myeloma used in the fusion
process to ascertain the compatibility of the hybridoma with
Balb/c mouse. In case of non-compatibility, it is possible to
produce ascites fluid in Nude mice.
† We need approximately 2–2.5 x 106 cells per animal. A minimum
of three mice will be used per hybridoma.
† As soon as we receive your order, we will plan a date to pick up
the hybridoma at your laboratory.
We provide
† Upon arrival, the cells are washed, resuspended in a physiological
buffer and injected into pristanised Balb/c or nude mice.
† 10–15 days later, we will start the recovery of the ascites fluid
(approximately 5–10 ml per mouse).
Other information
We will keep no samples after the end of the procedure. No samples
will be transferred to any third party or be commercially used. You
will remain the sole owner of the hybridoma. Moreover, we will not
disclose to any third party that we have used your material for any
manipulation or work.
In the case where we provide ascites production with your own
hybridoma, we can not guarantee high quality ascites production if
the antibody production from your hybridoma is too low.
You receive
An average of 5–10 ml of ascites fluid per animal will be sent to
you by express courier service. Shipment costs from and to your
laboratory are included in the price per mouse as indicated in our
price list.
Custom monoclonal antibody
Ascites production # mice Reference
Balb/c* about 5 ml per animal for SP2 derived hybridomas
3 to 5 mice6 to 20 mice
› 20 mice
AS-CBAL-B
Nude* about 5 ml per animal 3 to 5 mice6 to 20 mice
› 20 mice
AS-CNUD-E
(*) depends on each hybridoma
Custom monoclonal antibody development 14
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Germany • AustriaEurogentec Deutschland GmbH3 +49 221 258 94 555 +49 221 258 94 54u [email protected]
The NetherlandsEurogentec Nederland b.v.3 +31 43 352 06 985 +31 43 354 19 65u [email protected]
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Ascites production flowchart
Discuss with us all aspects of the project:
- small or large production scale
- Balb/c or nude mice
- additional services such as purification and labelling
For ascites production, we will choose a date to pick up the hybridoma in your laboratory.
We need approximately 5 x 106 cells per animal (minimum 3 mice per hybridoma)
Cells are washed and injected into pristanized mice. 10–15 days later, the recovery of ascites fluid will start
Affinity chromatography purification using
Protein A - Sepharose 2 weeks
Antibody labelling 2 weeks
You receive the remaining ascites fluid, affinity purified IgG and the corresponding quality control data sheet (SDS-PAGE) You will receive the labelled antibody with the corresponding Quality Control datasheet (enzymatic activity, HLPC/SDS-PAGE)
Large scale antibody production
STOP
Receive an average of 5–10 ml ascites fluid per animal
STOP
You receive the remaining ascites fluid, affinity purified IgG and the corresponding Quality Control data sheet (SDS-PAGE)
261
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Germany • AustriaEurogentec Deutschland GmbH3 +49 221 258 94 555 +49 221 258 94 54u [email protected]
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SwitzerlandEurogentec s.a.succursale de Genève3 FR: +32 4 372 74 00 DE: +49 221 258 94 555 FR: +32 4 372 75 00 DE: +49 221 258 94 54
Gene immunization
Antibodies against your protein by direct immunization with your DNA
plasmid expression vector
How does it work ?
Plasmids harboring genes to be expressed are transferred directly to the
tissues of animal using needle injection or particle bombardment. These
plasmid vectors, containing all elements required for expression in higher
organisms then allow the in situ expression of antigenic proteins and the
immune response is induced. From this starting point, many applications
can be envisioned relating either to vaccine design and development,
basic research in immunology or polyclonal antibody production against
your protein.
What Eurogentec offers
Eurogentec has gained a wide experience in polyclonal antibody
production and has developed its own gene immunization procedure.
Simply provide us with your plasmid expression vector and we will send
you the corresponding serum at the end of each month. The animals
available for this procedure are rabbits and mice.
Uses for gene immunization
† A protein is difficult to purify or its structure is destroyed by
purification.
† Antibodies produced by genetic immunization are themselves to be
used to purify the protein.
† Antibodies need to be produced against mutated forms of the
protein.
† Proteins of poor antigenicity can be made more immunogenic by
fusing their gene to that of a highly immunogenic protein.
Immunization procedure
† In order to improve DNA uptake, we induce a cycle of muscle
degeneration and regeneration in the animal.
† The DNA is injected into the regenerating muscle. We need at least
200 µg of vector per mouse (minimum 4 mice).
† Blood sampling is performed 4 weeks later and sera are sent to you
for testing.
† You receive 10–50 µl serum per mouse (300 µl for the final bleed) or
20 ml serum per rabbit (50 ml for the final bleed).
† You decide to continue the procedure or to stop at the end of each
month according to your needs. If you continue, additional bleeds
and boosts can be performed.
† A modification of this protocol is always possible. Please contact us.
Please noteThe choice of the expression plasmid and the DNA purification procedure
are crucial. Our scientists are at your disposal to discuss this.
Gene immunization
DNA immunization Reference
4 mice/2 months AS-ADN1-MO
Additional month AS-ADN2-MO
Additional injection AS-ADN2-IN