Biologicals are products derived from biological sources, including immunologicals (such as vaccines and sera), hormones, antibodies and blood products.
These types of products undergo strict quality control before applied to humans or animals.
Vaccines are recognised as a highly cost effective tool for preventing infectious diseases. They are derived from biological sources and due to the complexity of composition and heterogeneity of products, vaccine lots undergo legally required quality control before they are released.
Traditionally, laboratory animals have played a major role in quality control of vaccines, and still, many animals are used in Europe for this purpose.
Over the last decades, 3Rs methods to classical animal tests have been developed by control authorities, academia and vaccine manufacturers, successfully validated and implemented.
New generations of vaccines have been and are being developed which are better defined and allow the use of non-animal methods for their quality control.
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 use:
- Legal framework for medicinal products for human use
- Legal framework for medicinal products for veterinary use
In addition, the European Medicines Agency and its working parties issue guidelines regarding safety and efficacy testing as well as quality control of immunologicals.
The European Pharmacopoeia issued by the European Directorate for the Quality of Medicines & HealthCare (EDQM; Council of Europe) includes – amongst others - general and specific monographs for immunologicals for human and veterinary use setting the requirements to be met by the individual products.
Most of the validation studies on alternative methods for the quality control of immunologicals are carried out under the umbrella of EDQM's Biological Standardisation Programme (BSP) which is co-sponsored by the European Commission.
More information on the BSP, its background and work programme is available here.
Alternative test methods and approaches
[collapsed] EURL ECVAM released in 2016 a report on the Replacement, Reduction and Refinement of Animal Testing in the Quality Control of Human Vaccines to inform its stakeholders on ongoing activities in development and validation of 3Rs methods for the quality control of vaccines for human use.
The focus of the report is on methods for lot release testing (e.g. safety, pyrogenicity, potency) and projects related to the implementation of the consistency approach to established vaccines such as diphtheria, tetanus, pertussis and rabies vaccines.
EURL ECVAM validated test methods
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.
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 pyrogenicity tests.
The outcome of the ICCVAM peer review of in vitro methods for pyrogenicity testing is available here.
See also TSAR: TM2002-01 (EU), TM2002-02 (EU), TM2002-03 (EU), TM2002-04 (EU) and TM2002-05 (EU).
2. General batch safety tests for veterinary vaccines
The target animal batch safety (TABST)
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.
In April 2012, the European Pharmacopoeia Commission adopted the deletion of the target animal batch safety test from all monographs. This deletion came into force on 1 April 2013.
Since 2005, the target animal batch safety test could be waived when a sufficient number (e.g. 10) of batches had been found to comply with the test.
See also TSAR: TM1997-01 (EU)
The laboratory animal batch safety test (LABST)
The LABST, better known in Europe as the 'abnormal toxicity test', has been deleted from the European Pharmacopoeia monographs in 1997 due to its lack of scientific and regulatory relevance.
The TABST and the LABSTmay still be required outside of Europe, European manufacturers may need to carry out these tests when exporting to third countries.
Collaboration with VICH
From 2008 - 2020, EURL ECVAM worked on behalf of the European Medicines Agency with experts at the level of the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) towards the development of guidelines on harmonisation of criteria to waive the general batch safety tests for veterinary vaccines.
- Two VICH guidelines on harmonisation of criteria to waive target animal batch safety testing for inactivated (VICH GL50R) and live (VICH GL55) vaccines for veterinary use are in force since in May 2017. An earlier version of VICH GL50R had been in force since 2013.
- One VICH guideline harmonisation of criteria to waive laboratory animal batch safety testing for vaccines for veterinary use (VICH GL59) is in force since November 2020 and should be implemented by 2021.
By meeting the requirements outlined in VICH GL50R, 55, 59 vaccine manufacturers can now apply for a waiver of the TABST / LABST at their national competent authorities.
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 method has been included into the European Pharmacopoeia - Monograph 064 on swine erysipelas vaccine in 2004.
See also TSAR: TM2001-01 (EU)
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 report of the study was reviewed by the ESAC.
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.
See also TSAR: ToBI - TM1999-05 (EU) and ELISA TM1999-04 (EU)
5. In vitro generation and production of monoclonal antibodies
Based on the recommendations of ECVAM Workshop Report 23 and a report compiled on request of the ESAC (Hendriksen, 1998), the ESAC concluded that in vitro methods should be used for the production of monoclonal antibodies.
See also TSAR: TM1998-04 (EU)
Test methods under validation by EURL ECVAM
At present there is no test method related to the quality control of (immuno)biologicals under validation at the EURL ECVAM.
Development and optimisation of alternative methods
IMI2 VAC2VAC project
VAC2VAC is a collaborative five-year research project (2016-2021) funded under the Innovative Medicines Initiative, a joint undertaking of the EU Horizon 2020 Research and Innovation Programme and the European Federation of Pharmaceutical Industries and Associations (EFPIA). It started in 2016 as a five-year project and following a one-year, no-cost extension will now end in 2022. The project brings together 22 public and private partners including the JRC represented by EURL ECVAM.
The overall objective is to demonstrate proof-of-concept of the consistency approach for batch release testing of established vaccines. For this purpose, non-animal methods are being developed, standardised and validated for the quality control of vaccines for human use (e.g., diphtheria, tetanus, acellular pertussis, tick-borne encephalitis) and veterinary use (e.g., tetanus, rabies, clostridial, leptospiral, infectious bronchitis). Several method developers started to transfer their assays to partner laboratories (e.g., monocyte activation test, ELISA for tetanus, diphtheria and tick-borne encephalitis, clostridial vaccines) and small-scale. A small-scale collaborative studies to assess assay transferability and reproducibility are planned to take place during 2021.
EURL ECVAM is participating in the project as leader of the work package related to validation, and will also support project activities related to international dissemination, harmonisation and regulatory acceptance of the consistency approach.
One activity aims at developing guidance on how to move from the classical in vivo test-based quality control strategy to a consistency test-based strategy relying on non-animal based assays. Initial discussions with international experts (e.g., VAC2VAC, EDQM, WHO, national control authorities) took place in 2020 and will be continued in 2021.
EURL ECVAM and EPAA projects related to quality control of biologicals
How the consistency approach could be applied to established vaccines has been the topic of several workshops organised by EURL ECVAM (Metz et al., 2007; Hendriksen et al., 2008) and co-organised with the European Partnership for Alternative Approaches to Animal Testing - EPAA (De Mattia et al., 2011).
As a follow-up of the latter, EPAA launched in 2011 a project to develop the consistency approach for established human and veterinary vaccines. De Mattia et al. (2015) provide a general description of the project, summaries of meetings, workshops and discussions on possibilities to implement the consistency approach for the four priority vaccine groups (diphtheria, tetanus and acellular pertussis vaccines; human rabies vaccines; veterinary rabies vaccines and clostridial vaccines).
EPAA decided in 2016 to close the overarching Vaccines Consistency Approach project and continue the two sub-projects on clostridial vaccines and human rabies vaccines as individual projects.
The work on implementation of the consistency approach, in general, and in particular for diphtheria, tetanus, acellular pertussis vaccines and veterinary rabies vaccines is continued in the IMI2 VAC2VAC project (see above).
EPAA - Clostridial vaccine project
The project aims at the validation of in vitro assays using Vero cells to replace the in vivo Minimum Lethal Dose and Total Combining Power assays required for in-process control of Clostridium septicum vaccines.
The collaborative study has been carried out in collaboration with the EDQM BSP. The final results demonstrated that the optimised in vitro tests are very consistent, with intra- and inter-laboratory variations far lower than those for the analogous in vivo tests (Dass et al., 2020; Behr-Gross et al., 2021). This indicates that the non-animal, cell line-based assays for in-process toxicity and antigenicity testing of C. septicum vaccines outperform the animal-based methods. This will allow full advantage to be taken of the superior sensitivity and accuracy of the in vitro MLD and TCP tests when manufacturers implement these alternatives into the current in-process controls.
In light of these results, the European Pharmacopoeia (Ph. Eur.) monographs for vaccines based on cytotoxic Clostridial antigens were revised to introduce Vero cell line-based methods and will be implemented from April 2022.
EPAA - Human rabies vaccine project
Based on the outcome of discussions held at an EPAA workshop in 2012, the EPAA human rabies vaccines group organised a collaborative study to identify the most suitable ELISA for quantitation of glycoprotein-G in rabies vaccines and possible replacement of the current in vivo test for potency testing of human rabies vaccines.
The results of the study and possible follow up have been discussed at a workshop in May 2015 (Morgeaux et al., 2017). One out of the three ELISAs correctly quantified the antigen content of all vaccine samples (including degraded samples).
After additional work carried out during 2016, this ELISA was presented to and accepted by the EDQM BSP for full validation.
Phase 1 of the study, the transfer of the assay and protocol to study participants and relevant regulatory agencies has been completed successfully.
Phase 2, which includes an inter-laboratory comparison of different vaccines, is ongoing (see the EPAA project platform website)
EPAA – Harmonisation on Biologicals
The project aims at progressing harmonisation of requirements for batch testing of vaccines and other biological products at a global level.
Due to evident differences in the current regional requirements, manufacturers may need to carry out animal tests which are no longer required in Europe, if they want to market their products outside of Europe.
In an EPAA workshop on Modern science for better quality control of medicinal products: Towards global harmonisation of 3Rs in biologicals the major recommendation – agreed by all participants – was the deletion of general safety tests, e.g. abnormal toxicity test, target animal batch safety, from regulatory requirements at a global level.
The project continues to (a) encourage deletion of these in vivo tests. In addition, the project now also includes pyrogenicity testing which is relevant to a wide range of products including vaccines, chemicals and blood products (see the EPAA project platform website).