Biologicals

ELISA estimating cytokine release

    1. ECVAM validated test methods

    2. Test methods under validation by ECVAM

    3. Development and optimisation of alternative methods

    4. Workshop Reports

    5. Publications

 

Background


Biologicals are products derived from biological sources, including immunobiologicals (such as vaccines and sera), hormones, antibodies and blood products. These type of products must undergo strict quality control before applied to humans or animals.

Vaccines are recognised as a highly cost effective tool for preventing infectious diseases. Their importance is likely to increase in the future given the emergence of antibiotic-resistant strains of bacteria, viral infections, as well as the high incidence of infections in large livestock industries etc.

Traditionally, laboratory animals have played a major role in quality control of vaccines. Still, many laboratory animals used in Europe are required for testing the safety and potency of batches of vaccines for veterinary and human application.

Over the last two decades, many alternative methods to classical animal tests for the quality control of vaccines have been developed and successfully implemented. New generations of vaccines have been and are being developed which are better defined and allow the use of in vitro and physico-chemical methods for their quality control, thus using less or no animals for the quality control of batches.

Moreover, the concept of vaccine quality control is changing as emphasis is being put on ensuring the consistency of production of a vaccine and not regarding each batch produced as a unique product. The characteristics of a new batch of a vaccine should be similar to those of a batch which has been shown to be safe and efficacious.

 

Regulatory framework

Being medicinal products, vaccines and other biologicals fall under the Community legislation in the area of medicinal products for human use and veterinary us and are specified in "The Rules Governing Medicinal Products in the European Union":


In addition, the European Medicines Agency and its working parties issue guidelines on the production and quality control of immunologicals.

The European Pharmacopoeia (Council of Europe) includes general and specific monographs for immunologicals for human and veterinary use setting the requirements to be met by the individual products.

 

 

Alternative test methods and approaches

 

1. ECVAM validated test methods

 

1.1 In vitro methods for pyrogenicity testing

The ESAC peer reviewed the reports of validation studies involving five in vitro methods based on the use of human monocytoid cells for pyrogenicity testing and concluded that they are scientifically validated for the detection of pyrogenicity mediated by Gram-negative endotoxins.

 

Regulatory acceptance

The European Pharmacopoeia General Method 2.6.30 Monocyte-activation test was adopted by the European Pharmacopoeia Commission in March 2009 and came into force in September 2009. It includes the five in vitro pyrogen tests.

 

References

  • Schindler S, Spreitzer I, Loeschner B, Hoffmann S, Hennes K, Halder M, Bruegger P, Frey E, Hartung T, Montag Lessing (2006) International Validation of Pyrogen Tests Based on Cyropreserved Human Primary Blood Cells. Journal of Immunological Methods 316 (1-2); 42-51
  • Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S, Mystry Y, Montag Lessing T, Spreitzer I, Loeschner B, Van Aalderen M, Bos R, Gommer M, Nibbeling R, Werner Felmayer G, Loitzl P, Jungi T, Brcic M, Bruegger P, Frey E, Bowe G, Casado Poblador J, Coecke S, De Lange J, Bogster B, Naess L, Aaberge I, Wendel A, Hartung T. (2005) International Validation of Novel Pyrogen Tests Based on Human Monocytoid Cells. Journal of Immunological Methods 298; 161-173

Outcome of the ICCVAM peer review of in vitro methods for pyrogenicity testing

 

1.2 The relevance of the target animal batch safety test for veterinary vaccines

The ESAC peer reviewed the retrospective analysis of target animal batch safety test data provided in two reports and concluded that the test is no longer relevant and should be omitted for routine batch control.

 

Regulatory acceptance

Since 2005, the European Pharmacopoeia monograph on Vaccines for veterinary use (0062) states that the target animal batch safety test can be waived when a sufficient number (e.g. 10) of batches has been found to comply with the test.

In April 2012, the European Pharmacopoeia Commission adopted the deletion of the target animal batch safety test from all monographs. This deletion will come into force on 1 April 2013.

 

Reference

  • AGAATI (2002). The target animal safety test - Is it still relevant? Biologicals 30, 277-287.

 

Towards international harmonisation

Despite the deletion of the target animal batch safety test in Europe, manufacturers exporting to third countries may need to carry out the test due to the requirements outside of Europe. Therefore, harmonisation is most important and, in 2008, EURL ECVAM started to work with the European Medicines Agency on a guideline towards harmonisation of requirements for waiving the target animal batch safety test at the level of the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH).

The new VICH guideline on "Harmonization of criteria to waive the target batch animal safety testing for inactivated vaccines for veterinary use" (VICH GL50) was agreed upon by the VICH Scientific Committee  at its 28th meeting (19-21 February 2013, Washington DC, USA) and finally adopted by the Committee for Medicinal Products for Veterinary Use (CVMP) on 7 March 2013 (see: pdf icon VICH GL50: Biologicals: harmonization of criteria to waive target animal batch safety testing for inactivated vaccines for veterinary use).

On behalf of the European Medicines Agency, EURL ECVAM has been topic leader for GL50. Its implementation (from 1st March 2014 onwards) is a major step towards the harmonisation of requirements for the batch release testing of veterinary vaccines in the VICH regions (Europe, North America, Japan) and will reduce the number of animals used for this purpose.

This work will be continued and a VICH guideline for "Harmonization of criteria to waive the target animal safety test for live vaccines for veterinary use" is under development.

 

1.3 Serological method (ELISA) for the batch potency testing of swine erysipelas vaccines

The study was initiated by the Paul Ehrlich Institute (PEI; Langen, Germany) and funded by the German Federal Ministry of Education and Research (BMBF project number 0311200). ECVAM has been a participating laboratory and gave advice on the study design. The report of the study was reviewed by the ESAC.

The results obtained with the ELISA procedure in the international validation study on alternative methods for the batch potency testing of inactivated swine erysipelas vaccines were reproducible, both within and among the participating laboratories that performed the tests. The ELISA procedure proved applicable to testing a diverse group of inactivated swine erysipelas vaccines, of different potencies, compositions and combinations. The concordances between the potencies derived from the in vitro serological (ELISA) data and from the in vivo data were very good. The test was able to distinguish between highly potent and less potent vaccines.

 

Regulatory acceptance

The method has been included into the European Pharmacopoeia - Monograph 064 on swine erysipelas vaccine in 2004.

 

References

  • Rosskopf-Streicher U, Johannes S, Wilhelm M, Gyra H & Cussler K. (1999). Potency testing of swine erysipelas vaccines by serology - results of a prevalidation study. ALTEX 16, 123-128.
  • Rosskopf-Streicher U, Johannes S, Wilhelm M & Cussler K. (2000). Quality control of inactivated erysipelas vaccines: Results of an international collaborative study to establish a new regulatory test. Vaccine 19, 1477-1483.

 

1.4 Two serological methods (ToBI, ELISA) for batch potency testing of human tetanus vaccines

The validation study was a joint activity of ECVAM and the European Directorate for the Quality of Medicines (EDQM; European Pharmacopoeia, Council of Europe, Strasbourg, France).

The international validation study on the ELISA and the ToBI test for batch potency testing of human tetanus vaccines showed that both methods were reproducible, both within and among the participating laboratories. They proved applicable to testing a diverse group of tetanus toxoid vaccines for human use, of different potencies, composition and combinations. The concordances between the potencies derived from the in vitro serological (ELISA, ToBI) data and from the in vivo data were very good. The ELISA and ToBI test were able to distinguish between highly potent and less potent vaccines.

 

Regulatory acceptance

Both methods have been included into the European Pharmacopoeia - General text 2.7.8 Assay of tetanus vaccine (adsorbed) and adopted by the European Pharmacopoeia Commission in March 2003.

 

Reference

  • Winsnes R & Hendriksen C. (2000). Collaborative study for the validation of serological methods for potency testing of tetanus toxoid vaccines for human use. PHARMEUROPA, BIO 2000-1, 83-124.

 


1.5 In vitro production of monoclonal antibodies

Based on the recommendations of ECVAM Workshop Report 23 and a report compiled on request of the ESAC (Hendriksen, 1998, ATLA 26, 523-540), the ESAC concluded that in vitro methods should be used for the production of monoclonal antibodies:

 

Reference

  • Hendriksen, C.F.M. (1998). A call for a European prohibition of monoclonal antibody (mAb) production by the ascites procedure in laboratory animals. ATLA 26, 523-540.


2. Test methods under validation by ECVAM

 

At present there is no test method related to the quality control of (immuno)biologicals under validation at the EURL ECVAM.

 

3. Development and optimisation of alternative methods

 

3.1 EPAA project "Application of alternative methods through the consistency approach for improved vaccine quality control"

EURL ECVAM is a member of the steering committee of EPAA project "Application of alternative methods through the consistency approach for improved vaccine quality control". The project was launched in 2011 as follow-up of the ECVAM/EPAA Workshop on "The Consistency Approach for Quality Control of Vaccines – a 3Rs opportunity" held on 11-12 January 2010 in Brussels (see Final report).

The consistency approach is based upon the principle that the quality of a vaccine is the consequence of the strict application of a quality system and of a consistent production of batches. Through the application of this principle, which has been implemented for some novel human vaccines, agreed product characteristics can be tested in vitro during the manufacturing process of a batch and shown to be similar to those of batches demonstrated to be safe and effective in clinical trials. The concept of consistency may therefore be applied to conventional vaccine production in order to replace in vivo tests with in vitro tests indicative of the quality and quantity of the product whilst maintaining the highest quality and safety standards.

The focus of the project is on the development/standardisation of methods for several vaccine groups, e.g. rabies vaccines for human and veterinary use, diphtheria, tetanus, acellular pertussis vaccines, clostridial vaccines. During 2012, a series of workshops was held to tackle these issues. 

Flashreports are available on 

 

4. Workshop reports

 

Quality control of vaccines


Quality control of hormones 

Pyrogenicity testing

Production of antibodies



5. Publications

  • Von Hunolstein C, Gomez Miguel M, Pezella C, Scopetti F, Behr-Gross M, Halder M, Hoffmann S, Levels L, Van Der Gun J, Hendriksen C. (2008) Evaluation of Two Serological Methods for Potency Testing of Whole Cell Pertussis Vaccines - Potency Testing of Whole Cell Pertussis Vaccines, Pharmeuropa Bio 1; 7-18
  • Halder, M., Hartung T. (2008). European Centre for the Validation of Alternative Methods (ECVAM): its role and contribution. In: Proceedings of the International Symposium, Dubrovnik, Croatia, pp 23-32, EDQM, Council of Europe, Strasbourg.
  • Balls, M. and Halder, M. (2002). Progress in applying the Three R's of Russell & Burch to the testing of biological products. In: Advancing Science and Elimination of the Use of Laboratory Animals for Development and Control of Vaccines and Hormones, 111, 3-13. F. Brown and C. Hendricksen (Eds.), Karger (Publs.).
  • Halder, M. and Balls, M. (2002). Implementation of Three R's alternatives in regulatory testing: possibilities and obstacles. The view of the validator. In: Advancing Science and Elimination of the Use of Laboratory Animals for Development and Control of Vaccines and Hormones, 111 (2002), 203-210. F. Brown and C. Hendriksen (Eds.), Karger (Publs.).
  • Halder, M., Embleton, J., Fischer, R., de Geus, B., Hendriksen, C.F.M., de Leeuw, W.A., Marx, U.  & Balls, M. (1998). Comments on appendix C of the NIH response to the petition of the American Anti-Vivisection Society (AAVS) to prohibit the use of animals in the production of monoclonal anitbodies. ATLA 26, 549-554.


Photo: ELISA estimating cytokine release. Copyright EU 2003.