Quality Control of Seasonal Influenza Vaccines

<jats:title>Quality Control of Seasonal Influenza Vaccines</jats:title><jats:p>The purpose of seasonal influenza vaccination is to prevent its spread. The vaccines contain strains of the influenza virus recommended and approved for a particular season. Just like any other medicinal product, all vaccines require marketing approval. Batches of approved vaccines are extensively tested by the manufacturers and additionally controlled by the approving authorities, which issue the quality control certificates. This article not only to describes the legal background of quality control, but also how control test results obtained by a Croatian official control laboratory are compared to manufacturer's results. We have found that testing results can slightly differ depending on methods/analytical procedures used in different laboratories. This investigation has also shown how important it is to test finished medicinal products, independently of testing at intermediate stages, and how retesting by control authorities ensures that marketed vaccines meet quality standards.</jats:p>

section_content: Influenza (flu) is an acute infectious disease caused by infl uenza viruses, which spreads quickly and has high morbidity. Infl uenza viruses belong to the family Orthomyxoviridae and are classifi ed according to their antigen differences (internal antigens) into types A, B, and C. Infl uenza viruses have two antigens (glycoproteins) on their surface: haemagglutinin (HA) and neuraminidase (NA). These surface antigens change rapidly and unpredictably, rendering the immune system vulnerable all over again every year. Changes are either large and are called shifts, which are rare, and minor, called drifts, which occur practically every year (1). Repeated (seasonal) vaccination is the effective way to prevent the spread of the disease. Every year, World Health Organisation (WHO) issues an overview of the epidemiological state of the infl uenza virus worldwide, and recommends which predominant strains of types A and B should be used in vaccines for the coming season. Type C infl uenza is not included because it is far less frequent than A and B types and usually causes very mild respiratory symptoms or no symptoms at all. It does not cause epidemics and does not much burden public health. 

Following the WHO recommendations, European Union (EU) experts decide which strains to use for the coming season, bearing in mind the epidemiological situation in the EU (2). For the season 2009/2010, the WHO recommended the following strain composition for the northern hemisphere: A/Brisbane/59/2007 (H1N1)-like virus, A/Brisbane/10/2007 (H3N2)like virus, and B/Brisbane/60/2008-like virus (3). Following this recommendation, vaccine manufacturers used reassorted strains that give high yields of required surface antigens (4). 

Seasonal infl uenza vaccines are sterile suspensions of strains of two types of infl uenza viruses -type A (subtypes H1N1 and H3N2) and type B. The vaccines are produced in such a way that the vaccine primarily contains the HA and NA antigens, without diminishing the antigenic characteristics of those antigens. The potency of the vaccine, i. e. the stated amount of the haemagglutinin antigen present in the vaccine per strain per dose is 15 micrograms (µg) unless clinical evidence supports the use of a different amount (4). Neither the European Pharmacopoeia (4) nor the guidelines issued by the Committee for Proprietary Medicinal Products (CPMP) require defining the content of the neuraminidase antigen (2). 

In Croatia, 2009/2010 seasonal vaccination included two types of vaccines: surface antigen, inactivated, and split virion, inactivated, both approved by the Agency for Medicinal Products and Medical Devices (HALMED). Pursuant to the Medicinal Products Act (5), the same institution performs quality control of medicinal products prior to marketing. The purpose of quality control is to establish compliance with quality standards defi ned in marketing approval. A special form of quality control for immunological medicinal products and medicinal products derived from blood or plasma is conducted by both the manufacturer and independent authority, in Croatia's case by HALMED. Immunological medicinal product is a medicinal product that is or contains vaccine, toxin, serum or allergen (5, 6). 

In the European Union (EU), quality control is conducted by the Official Medicines Control Laboratory (OMCL), which is independent from the manufacturer, to ensure safety and quality of medicinal products in use (7, 8). HALMED participates in the OMCL network, but until Croatia becomes an EU Member State, it cannot participate in the Offi cial Control Authority Batch Release (OCABR) procedure 1. The procedure ensures that the quality of immunological medicinal products is tested in only one laboratory in the EU, and the issued certifi cate is used by OMCLs in the Member States when implementing OCABR at a national level, following the requirements of Directive 2001/83/EC, as amended by Directive 2004/27/EC 2. The OMCL conducting the testing of the infl uenza vaccine is obliged to test the following pursuant to the guidelines: appearance, haemagglutinin antigen concentration/identity, bacterial endotoxin content, while purity is tested on the fi rst fi ve lots of a monovalent bulk following the introduction of a new infl uenza strain (9, 10). 

In Croatia, each batch of an immunological medicinal product is subject to special (double) quality control, regardless of whether it has the OCABR certifi cate. The applicant must submit to HALMED suffi cient quantities of samples for analysis, manufacturer's certifi cate of analysis for the batch in question, manufacturer's production documentation, and quality control and the batch release certifi cate issued by the relevant agency in manufacturer's country or the EU. If requested, the applicant should also submit a suffi cient number of samples of the original raw materials and their test reports (6). Before applying for independent quality control, the vaccine manufacturer has to perform tests on the intermediates and on the fi nished product. One of the tests on the fi nished product is potency testing, i. e. determining haemagglutinin antigen in the the vaccine fi lled in the primary containers. An other test the manufacturer performs only on the fi rst three monovalent pooled harvests from each working seed lot is the test for the presence and the type of neuraminidase (4, 10). 

In addition to other tests mentioned earlier, the control laboratory does the potency testing in the fi nished product, pursuant to the OCABR guidelines for infl uenza vaccines (10). HALMED follows the same quality control procedure. However, time requirements are tight. Whenever WHO recommends a new strain to be included in the vaccine, manufacturing process for the new strain has to be revalidated and report submitted to regulatory authorities. In addition, the manufacturer has to obtain an international reagent specifi c to the selected strain to measure the concentration of each HA antigen (11). 

The objective of this article is to present special (double) quality control procedure and compare control test results to manufacturer's to see if there are and what are the causes of discrepancies.

section_name: MATERIALS AND METHODS

section_content: Samples of seasonal fl u vaccine (2009/2010) were tested in four laboratories. HALMED laboratory (Lab 1) and EU OMCL laboratory (Lab 2) tested two batches of the fi nished product (batches A and B) produced from the same fi nal bulk (FB) 3 and samples of that fi nal bulk. The manufacturer of the fi nished product (Lab 3) tested the same samples as Lab 1 and 2, while the manufacturer of the fi nal bulk (Lab 4) tested only the batch of the fi nal bulk All test results were delivered to HALMED.
section_name: Samples

section_content: Table 1 shows all materials used for testing. The content of haemagglutinin antigens in infl uenza vaccines was tested using the single radial immunodiffusion (SRID) method, as described in the European Pharmacopeia (4) 4. This method is not affected by purifi cation of surface antigens or virus dissociation used in vaccine production. Specifi city is achieved with specifi c antiserums, and can serve to compare two antigen preparations (14). The method is based on passive diffusion of antigens into an agarose gel containing uniformly distributed antibodies. Upon the fi rst contact of the antigen with an antibody soluble complexes are formed, while with further penetration of the antigen through the agar, the concentration of the antigen drops. When the optimum concentration ratio of antigens and antibodies is obtained, an insoluble precipitation ring is formed, which is round in shape (15). This ring confirms the identity of vaccine antigen and it is measured to calculate (and therefore quantify) the content of HA in the vaccine. Each laboratory has its own standard operating procedure for the SRID method. Lab 1 followed the procedure of the manufacturer of the fi nished product. The agarose solution was prepared by adding an optimal quantity of specifi c antiserum in the volume necessary to precipitate individual virus strains 5. The agarose/antiserum mixture was poured onto glass plates and allowed to cool. The plates were placed in a humid, dark chamber and left at +4 o C until the following day. Wells 4 mm in diameter were punched into the resulting gel for inoculation with 20 µL of the diluted reference antigen, i. e. vaccine sample. 

Reference antigens were dissolved according to the NIBSC instructions and/or vaccine manufacturer's qualification report. A series of vaccine sample solutions and reference substances were prepared using Zwittergent 3-14 detergent and PBS in the appropriate dilutions for vaccine samples and reference antigens (1:1, 3:4, 1:2, and 1:4). The tested solutions were inoculated in triplicate and the reference substances in duplicate. The prepared plates were stored in a humid, dark chamber at room temperature for 24 hours to allow for diffusion of the antigen and reaction between the antigens and antibodies. The reaction was stopped by sinking the plate into a prepared 5 The quantity of each antiserum is stated on the antiserum certifi cate. The quantity of the necessary specifi c antiserum for obtaining sharp precipitation rings is established by the vaccine manufacturer in a series of preliminary tests for the fl u vaccine for the 2009/2010 season. Lab 2 tested the same batches of the fi nished product and of the fi nal bulk as did Labs 1 and 3. Table 3 compares haemagglutinin antigen content for the Type A (H3N2) virus, strain A/Uruguay/716/2007 NYMCX-175C. The results are represented as individual values and their means. There is a good agreement, with minor deviations, between Labs 1, 2, and 3. Lab 1 and 3 got the results that did not meet specifi cation and Lab 2 results were bordering on the lower limit for both the fi nal bulk and the fi nished product (Figure 1 ). 

Considering that the tests were conducted on immunological medicinal products, whose active substances are of biological origin, the results may show variations under different testing conditions, even if the same methods/analytical procedures are applied. The test results confi rm the importance of testing the fi nished product (fi nal lot) and not just the fi nal bulk, and the importance of testing batch samples by a control laboratory before marketing.
section_name: Reagents and methods

section_name: ACKNOWLEDGEMENTS

section_content: The authors would like to acknowledge Ms Laila Stefanini Orešić for critical review of the manuscript.
section_name: ACKNOWLEDGEMENTS

section_name: RESULTS AND DISCUSSION

section_content: solution, and the plates were left to dry. After drying, the plates were stained to make precipitation rings visible and destaining of the background made the rings appropriate for reading. 

The precipitation rings were measured using a glass magnifi er which magnifi es 7 times and antigen content was obtained following the procedure described by Williams (13). The content of haemagglutinin antigen is calculated using the software program CombiStats v4. 0, EDQM (16).

section_content: The specifi cations for haemagglutinin antigen content for all three strains of the infl uenza virus were taken from the European Pharmacopoeia. The declared content per strain for the fi nished product (fi nal lot) is 15 µg/dose and the lower limit is ≥12 µg/dose (4). 

Table 2 The repeated control testing of the fi nished product in Lab 1 confi rmed the low content of haemagglutinin for Type A (H3N2), strain A/Uruguay/716/2007 NYMCX-175C, which is why Lab 1 performed additional quality control of the fi nal bulk.
section_name: RESULTS AND DISCUSSION