Production of monoclonal antibodies
1. Introduction
The following guidelines have been developed by the Animal Research Review Panel (ARRP) to assist Animal Ethics Committees (AECs) which consider applications for the production of monoclonal antibodies and to assist producers of monoclonal antibodies (mAbs).
These guidelines are not all encompassing and investigators and AECs are encouraged to seek information from the reference sources listed below as well as develop and regularly revise internal guidelines. The guidelines have been developed from information available in the published literature, as well as from information on common practices obtained during a survey on monoclonal antibody production conducted by the ARRP in 1996. Implicit in the guidelines is the principle that the ultimate decision on the conduct of animal research rests with the AEC assessing the research application.
The production of mAbs using animals (usually mice) entails procedures that have the potential to cause considerable pain and distress. These include:
- The use of irritant adjuvants such as Freund’s Complete Adjuvant (FCA) or Freund’s Incomplete Adjuvant (IFA). Freund’s creates a severe inflammatory response and reported effects include tissue necrosis, ulceration, self-trauma, hunching, decreased appetite and weight loss;
- The use of pristane as a "priming" agent in the "scale up" phase. Pristane is thought to act by causing granulomatous reactions and interfering with fluid drainage in the abdomen. Its use has been associated with weight loss, hunched appearance and inactivity;
- The production of abdominal fluid (ascites) and abdominal tumours (hybridomas). The effects can be extrapolated from the human experience which include abdominal discomfort, indigestion, difficulty breathing and difficulty walking;
- The removal of ascites fluid which may cause shock resulting from rapid fluid loss.
There are two stages in mAb production - the immunisation phase and the "scale up" phase.
Alternatives to the use of animals in the immunisation phase of monoclonal antibody production are available for specialised situations but are not in common use. Various cell culture methods to replace the use of mice in the "scale up" phase (production of ascites fluid) are available.
Alternatives to the use of animals should be used wherever possible and where this is not possible, procedures should be refined to reduce the impact on the animals used.
2. General Principles
These principles apply to the production of monoclonal antibodies in mice and should be adhered to unless compelling justification is provided by the investigator for the need to depart from them.
Immunisation phase2.1 FCA and IFA should not be used where it is possible to use no adjuvant or less irritant adjuvants.
2.2 FCA should not be used more than once in individual mice.
2.3 The volume of FCA and IFA used should not exceed 0.1ml.
2.4 Adjuvant should not be administered by intradermal, intramuscular or footpad routes.
2.5 Individual mice should not be inoculated with adjuvant more than 3 times.
2.6 Individual mice should not be bled more than twice.
2.7 Mice should not be kept more than 3 months after adjuvant/antigen inoculation.
"Scale up" phase
2.8 Mice must not be used for ascites production if effective in-vitro alternatives exist and are available.
2.9 The volume of pristane used should not exceed 0.2ml.
2.10 A priming agent should not be used in individual mice more than once.
2.11 Ascites fluid should only be harvested once at the time of euthanasia (whilst ensuring abdominal fluid build up is not excessive).
2.12 Mice should not be kept more than 4 weeks after hybridoma cell inoculation.
3. Application Assessment
The following are points to consider in preparing and assessing applications to produce monoclonal antibodies using mice. The questions are taken from the ARRP Research Application Form (Model).
3.1 Why is it necessary to use animals in this experiment?
3.1.1 Reasons that are sometimes provided for using mice instead of in-vitro alternatives include cost, technical difficulty, a lack of available in-vitro facilities, inadequate production of monoclonal antibodies and risk of contamination of a cell culture system. Such justifications for using animals need to be weighed up by AECs in the light of requirements of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (For example Clause 1.8 states Techniques that totally or partially replace the use of animals for scientific purposes must be sought and used wherever possible).
3.2 What alternatives to animals have been considered and why is it not possible to use these?
3.2.1 Alternatives to the use of animals in the immunisation phase of monoclonal antibody production are available for specialised situations but are not in common use. Nonetheless, it is reasonable to expect that an exploration of in-vitro alternatives in the immunisation phase will have been conducted.
3.2.2 Various in-vitro methods to replace the use of mice for production of ascites fluid ("scale-up" phase) exist. If an investigator wishes to use mice in this phase, a comprehensive list of which alternatives have been considered and why they are unsuitable should be provided.
3.3 Identify all factors and procedures that may have an impact on an animal’s well-being.
Describe each factor or procedure and detail how any adverse impact will be minimised.
Immunisation phase
3.3.1 The need to use FCA or IFA (rather than no adjuvant or less irritant adjuvants) should be justified by the investigator. The need to use FCA instead of IFA should also be justified.
3.3.2 If FCA or IFA are to be used then justification for volumes in excess of 0.1ml should be provided by the investigator.
3.3.3 The need to use FCA more than once should be justified by the investigator.
3.3.4 The route of inoculation of adjuvant should be provided by the investigator. The use of intradermal, intramuscular or footpad inoculation should not be approved without compelling justification that alternative sites are not suitable.
3.3.5 The frequency of injection of adjuvant should be assessed. The need to inoculate an individual animal with adjuvant more than 2 - 3 times should be justified by the investigator (bearing in mind that in some cases the need for multiple "boosts" with adjuvant may be difficult to predict).
3.3.6 The need to bleed animals prior to sacrifice and the method of bleeding should be assessed. The need to bleed animals more than twice should be justified (bearing in mind that in some cases the number of bleeds needed may be difficult to predict).
"Scale up" phase
Pristane is the usual "priming agent" and it is usually injected into the abdomen (intraperitoneal).
3.3.7 The use of more than 0.2ml pristane should not be approved without compelling justification.
3.3.8 The use a priming agent more than once should not be approved without compelling justification.
3.3.9 Methods of harvesting ascites fluid should be assessed and the need to harvest ascites fluid more than once (at time of euthanasia) should be justified by the investigator (whilst ensuring that ascites fluid build up is not excessive).
3.4 How will animals be monitored for the duration of the project?
3.4.1 Methods of animal monitoring including signs observed, frequency of observation, frequency of measurement of weight gain or loss, methods of assessment of abdominal distension and cut-off points should be provided by the investigator.
3.5 What will be the maximum time an individual animal is held?
Immunisation phase
3.5.1 The need to keep mice more than 3 months after inoculation should be justified by the investigator.
"Scale up" phase
3.5.2 The need to keep animals more than 4 weeks after hybridoma cell inoculation should not be approved without compelling justification.
3.6 Will factors affecting animals determine the endpoint of the project?
3.6.1 Methods of monitoring abdominal fluid build up and cut-off points in the case of excessive fluid build up should be provided.
4. Facilitating the use of in-vitro systems
4.1 AECs considering applications from mAb producers should be in a position to co-ordinate investigation by the AECs’ institutions into the feasibility of providing central facilities for the in-vitro production of mAbs ("scale-up" phase).
4.2 The use of commercial companies producing mAbs via in-vitro systems may provide a means by which individual investigators can avoid the need to use mice in the "scale-up" phase.
4.3 The provision of training of mAb producers in the use of in-vitro systems should be considered. Measures to implement this could include providing opportunities for mAb producers to work with practitioners already successfully using in-vitro systems, obtaining the services of successful in-vitro producers for "in-house" workshops and investigating training provided by companies supplying in-vitro systems.
5. Information Resources
The following are documents which provide information on alternatives and refinements in the production of monoclonal antibodies.
5.1 Reference lists
Animal Welfare Information Centre Resource Series No.3 - August, 1997. Information Resources for Adjuvants and Antibody Production: Comparisons and Alternative Technologies.
Available from US Department of Agriculture, National Agricultural Library, Animal Welfare Information Centre, 10301 Baltimore Avenue, Beltsville, MD 20705, USA.
Email: awic@nal.usda.gov
Website: www.nal.usda.gov/awic/pubs/antibody
5.2 Guidelines
(* Copies available on request from NSW Department of Primary Industries Animal Welfare Branch)
* Amyx H (1987) Control of animal pain and distress in antibody production and infectious disease studies. JAVMA 191: 1287-1289
* Clark A, Befus D, O'Hashi Petal (2002) Canadian Council on Animal Care Guidelines: Antibody Production
www.ccac.ca
* DeTolla L, and Smith J (2000). Guidance document for IACUC evaluation of monoclonal antibody production protocols. Alternatives Research and Development Foundation.
* Grumstrup-Scott J and Greenhouse DD (1988) NIH intramural recommendations for the research use of complete Freund’s adjuvant. ILAR News 30 (2): 9
* Hanley WC; Taylor Bennett B and Artwohl JE (1997) Overview of Adjuvants
www.nal.usda.gov/awic/pubs/antibody/overview.htm
* Hendriksen CFM (1998) A call for a European prohibition of monoclonal antibody production by the ascites procedure in laboratory animals. ATLA 26: 523 - 540
* Jackson LR and Fox JG (1995) Institutional policies and guidelines on adjuvants and antibody production. ILAR Journal 37 (3): 141-152
http://dels.nas.edu/ilar_n/ilarjournal/37_3/37_3Institutional.shtml
* Jackson LR; Trudel LJ; Fox JG et al (1996) Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production. Journal of Immunological Methods 189: 217-231
* Jennings VM (1995) Review of selected adjuvants used in antibody production. ILAR Journal 37(3): 119-125
* Leenars M; Claassen E; Hendriksen CFM (1996) Considering the side-effects of adjuvant products in immunization procedures. Lab Animal September:40-43
* Marx U; Embleton MJ; Fischer R et al (1997) Monoclonal antibody production. The report and recommendations of ECVAM workshop 23. ATLA 25 (2): 121-135
* McGill MW and Rowan AN (1989) Refinement of monoclonal antibody production and animal well-being. ILAR News 31 (1): 7-11
http://dels.nas.edu/ilar_n/ilarjournal/31_1/31_1Refinement.shtml
* NHMRC (2000). Guidelines on monoclonal antibody production.
www.nhmrc.gov.au/publications/synopses/monosyn.htm
Schulhof J (editor) (1999) Small-Scale Monoclonal Antibody Production Lab Animal Autumn: 1 - 35
* Workman P; Twentyman P; Balkwill F et al (1997) UKCCCR guidelines for the welfare of animals in experimental neoplasia (Second Edition) (UKCCCR: PO Box 123, Lincoln’s Inn Fields, London WC2A 3PX)
www.ncrn.org.uk/csg/publications.htm
* Workshop - (1997) Conclusions and recommendations prepared following "Alternatives and Monoclonal Antibody Production" - The John Hopkins Centre and National Institutes for Health.
http://altweb.jhsph.edu/meetings/mab/proceedings.htm
6. Some suppliers of in-vitro systems
This information is for the assistance of AECs and investigators and does not imply endorsement of a company or product.
Australia
John Morris Scientific Pty Ltd
PO Box 447
Willoughby NSW 2068
Telephone: (02) 9417 8877
Fax: (02) 9417 8855
Free Call: 1800 251 799
www.johnmorris.com.au/ssl/store/
(Celligen Plus and others)
Radiometer Pacific
45 Booralie Road
Terrey Hills NSW 2084
Telephone: (02) 9450 2822
Facsimile: (02) 9450 1572
Free Call: 1800 723 405
www.radiometer.com.au/
(miniPERM)
Invitrogen Australia Pty Limited
2A / 14 Lionel Road
Mount Waverley VIC 3149
Phone: (03) 8542 7400
Facsimile: (03) 9544 5622
Free Call: 1800 331 627
www.invitrogen.com
Overseas
Integra Biosciences
Northumbria Biologicals Ltd
Nelson Industrial Estate, Cramlington
Northumberland NE23 9BL UK
Telephone: (0) 670 732 992
Facsimile: (0) 670 732 537
www.integra-biosciences.com/
(Tecnomouse)
Serotec Ltd
22 Bankside
Station Approach
Kidlington
Oxford OX5 1JE
UK
Telephone: + 44 (0) 1865 852 700
Facsimile: + 44 (0) 1865 852 739
www.serotec.co.uk
(Harvest Mouse)
(Australian outlet: Australian Laboratory Services
Unit 48, 378 Parramatta Road
Homebush NSW 2140
Telephone: (02) 9764 4055
Facsimile: (02) 9764 3533
Toll Free: 1800 252 286
www.austlabservices.com.au/)
TCS Biologicals Ltd
Botolph Claydon
Buckingham MK18 2LR
UK
Telephone: 01296 714 222
Facsimile: 01296 714 806
www.tcsbiosciences.co.uk/
7. Commercial producers using in-vitro systems
This information is for the assistance of AECs and investigators and does not imply endorsement of a company or product.
Agenix Ltd
PO Box 391
Acacia Ridge
Brisbane QLD 4110
Telephone: (07) 3370 6300
Facsimile: (07) 3370 6370
www.agen.com.au/
Serotec
(see above)
Dr George Lovrecz
Head - Fermentation Group
Parkville Laboratories
343 Royal Parade
PARKVILLE VIC 3052
Telephone: 03 9662 7100
Facsimile: 03 9662 7101
email: George.Lovrecz@csiro.au
www.csiro.au/mht
Zenyth Therapeutics (formerly Amrad Corporation)
576 Swan Street
Richmond VICTORIA 3121
Telephone: 03 9208 4000
Facsimile: 03 9208 4350
www.zenyth.com.au/
8. Users of in-vitro systems and alternative adjuvants
Volunteers needed!
Comments on these guidelines and requests for inclusion on the address lists are welcomed and should be sent to: Lynette Chave, Senior Veterinary Officer, Animal Welfare Branch, PO Box 100, Beecroft NSW 2119; Telephone: (02) 9872 0570 Facsimile: (02) 9871 6938 email: lynette.chave@dpi.nsw.gov.au
Animal Research Review Panel Guideline 13