In Vitro Potency Assay for Hepatitis A Vaccines: Development of a Unique Economical Test

Biologicals (2000) 28, 247–256 doi:10.1006/biol.2001.0264, available online at http://www.idealibrary.com on

In Vitro Potency Assay for Hepatitis A Vaccines: Development of a Unique Economical Test Bertrand Poirier, Sylvie Morgeaux*, Pascale Variot and Florence Fuchs Agence Franc¸aise de Se´curite´ Sanitaire des Produits de Sante´, Direction des Laboratoires et des Controˆles, 321, Avenue Jean Jaure`s 69007 Lyon, France

Abstract. Prior to the official release of each Hepatitis A vaccine lot to the market, a quality control performed by a National Control Authority requires an in vivo or an in vitro potency assay. At the beginning of our work, no standardised in vitro test common to all hepatitis A vaccines was available for both manufacturers and National Control Laboratories. In this study, a unique polyvalent enzyme-linked immunosorbent assay (ELISA) method was developed to appraise all commercially available HAV vaccines. After comparing a direct and an indirect sandwich method with commercial antibodies, the indirect assay was selected and an evaluation of sensitivity, linearity, accuracy and precision was performed before being applied to HAV antigen determination from four different manufacturers. The results are satisfactory and incline us to use routinely this method to release Hepatitis A vaccines. Key words: Hepatitis A; Statistics; Vaccine control; ELISA.

Introduction Hepatitis A is a common infection throughout the world. The disease is caused by a pathogenic, hepatotropic picornavirus transmitted by the faecal–oral route and by person to person contact.1 Patterns of this disease vary in di#erent parts of the world. In developing countries with low socio-economic levels, the infection has a high incidence and occurs early in the life. In developed countries, hygienic and socio-economic conditions result in a decrease in the number of persons infected naturally during childhood.2 Consequently, most adults have not encountered Hepatitis A virus (HAV) and remained susceptible to the infection. Taking into account that the risk of developing fulminate hepatitis A disease is strongly correlated with increasing age,3 the need of prophylactic measures to protect adults like travellers4 with a high risk of acquiring Hepatitis A is necessary.5 In the world six manufacturers have developed inactivated Hepatitis A vaccines on MRC5 cells using various methodologies and virus strains such as: HM175,6 GBM,7 CR-336F,8 RG-SB9 and LSH/S.10 Nevertheless, the methodologies used to process *To whom correspondence should be addressed. 1045–1056/00/040247+10 $35.00/0

these vaccines and to control them are based on WHO’s recommendations and the European Pharmacopoeia monograph and guideline for batch release.11,12 The potency of the vaccine can be determined by an in vivo test comparing the amount of vaccine necessary to induce specific antibodies in mice with the amount of a reference preparation necessary to produce the same e#ect, or after a validated in vitro determination of antigen content.12 However, these requirements describe neither the tests used by the manufacturers to control the production process nor those used by the National Control Authorities (NCAs) to release each final batch of vaccine. In this context, no international standard reference and method were available. Consequently, each manufacturer developed and standardized11 in-house in vitro immunoenzymatic assays (enzyme-immunoassay or radio-immunoassay) to appraise the vaccine antigen content.6,10,13–15 Concomitantly, vaccines were formulated in di#erent arbitrary units per human dose without correspondence between each other’s.4,6,10,15,16 In the European market, four Hepatitis A vaccines are commercially available. The control and the release of the products without a standardized assay was a major issue for National Control Authorities, which were dependent on each

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manufacturer test and on their internal working references and antibodies. In this study, we developed and standardized a single in-house polyvalent enzyme-linked immunosorbent assay (ELISA) method for assessing antigenicity of di#erent HAV vaccines. Materials and methods References and vaccines were originated from four manufacturers (A, B, C and D) marketing Hepatitis A vaccines in Europe. References

Non-adsorbed inactivated references (Ref An, Ref Bn and Ref Dn) from manufacturers A, B and D were used to develop and to standardize the ELISA assay. Adsorbed inactivated references (Ref Aa, Ref Ba and Ref Ca) from manufacturers A, B and C were used to assess the antigenicity of the vaccines, to establish the control chart, the accuracy and the precision of the procedure. Vaccines

Several vaccine lots supplied by each manufacturer were tested to quantify Hepatitis A antigen content per millilitre: Manufacturer A: lot numbers A1, A2, A3, A4, A5, A6, A7, A8 and A9 Manufacturer B: lot numbers B1, B2, B3, B4, B5, B6 and B7 Manufacturer C: lot numbers C1, C2, C3 and C4 Manufacturer D: lot numbers D1, D2 and D3 Direct ELISA immunocapture procedure

96-well microplates are coated (100
l/well) with an IgG1- anti-HAV antibody (Argene Biosoft, Varilhes, France) diluted 1/500 in 0·05  carbonate bu#er pH=9·6 and firstly incubated for 3 h at 37 C (in a plate incubator) followed by overnight incubation at 4 C.17 After removing the wells’ contents, a blocking bu#er (saccharose 5% w/v and BSA 0·3% w/v in 0·05  carbonate bu#er pH=9·6) is added (200
l/well) and incubated 45 min at 37 C. After removal of the blocking bu#er, wells are washed three times (300
l/well) with PBS-Tween 0·1%. Hepatitis A references diluted in PBS-Tween 0·1%– BSA 0·5%17 are dispensed in duplicate and incubated for 2 h at 37 C. After washing, Hepatitis A antigens bound to antibodies are detected after an incubation (90 min, 37 C) with a horseradish peroxidase-labelled (HROP) monoclonal

IgG3-anti-HAV (Clone 7E7, Mediagnost; Interchim, Montlucon, France) diluted 1/1000 in PBS-Tween 0·1%–BSA 0·5%. After washing, the substrate (ABTS, Roche Diagnostic, Meylan, France) diluted in ABTS bu#er (Roche Diagnostic) is dispensed and incubated 15 min at 37 C. A solution (citric acid 0·1 ) is added (50
l/well) and incubated 15 min in the dark. Optical densities are read at 405 nm wavelength. Indirect ELISA immunocapture procedure

96-well microplates are coated (100
l/well) with a human polyclonal IgA containing 6010 IU/ml of anti-Hepatitis A antibodies (Tegeline 5 g/100 ml provided by the Laboratoire Franc¸ ais du Fractionnement et des Biotechnologies, Les Illis, France) diluted 1/1000 in 0·05  carbonate bu#er pH=9·6 and incubated first for 3 h at 37 C (in a plate incubator) followed by overnight incubation at 4 C.17 After removing the wells’ contents, a blocking bu#er (saccharose 5% w/v and BSA 0·3% w/v in 0·05  carbonate bu#er pH=9·6) is added to each well (200
l/well) and incubated 45 min at 37 C. The blocking bu#er is removed and wells are washed three times (at 300
l/well) with PBS-Tween 0·1%. Hepatitis A references or vaccines diluted in PBSTween 0·1%–BSA 0·5%17 are dispensed in duplicate and incubated for 2 h at 37 C. After washing, Hepatitis A antigens bound to antibodies are incubated for 1 h at 37 C with a monoclonal IgG3 anti-HAV (Argene Biosoft) diluted 1/5000 in PBS-Tween 0·1%– BSA 0·5%. A second incubation (1 h at 37 C) is performed with a goat anti-mouse IgG3-HROP (Caltag, Le Perray en Yuelines, France) diluted 1/1000 in PBS-Tween 0·1%–BSA 0·5%. After washing, the substrate (ABTS, Roche Diagnostic) diluted in ABTS bu#er (Roche Diagnostic) is dispensed in each well and incubated for 15 min at 37 C. A solution (citric acid 0·1 ) is further added (50
l/ well) and incubated for 15 min in the dark. Optical densities are read at 405 nm wavelength. Comparison between direct and indirect method

An F-test was run to compare the variances of the methods. P values below 0·05 indicate significant di#erence between the standard deviations. Regression analysis for each reference

The relationship between optical densities (OD) of each reference and the relevant dilutions was performed by fitting a logarithmic-X model: OD=a+b*Log10(dilution). These statistical studies

In vitro potency assay for hepatitis A vaccines

required a regression Analysis of Variance.

analysis

including

an

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titres. The control limits were calculated as m0 3s0.21 m0 is the mean; s0 is the estimation of the true standard deviation of the titres’ distribution.

Calculation of vaccines and reference titres

Adsorbed references were titrated vs. nonadsorbed references and adsorbed vaccines were titrated versus their homologous adsorbed references. Each titration versus a reference was calculated by a parallel-line model.18 For all assays (Table 4 and Fig. 3) suitable transformations and/or ranges of the dose–response curve were selected to fulfil the conditions of parallelism and linearity. References and vaccines potencies (HAV antigen content) were expressed in ELISA Units. The arbitrary ELISA value (EU/ml) assigned to the manufacturer’s reference was chosen as the manufacturer’s method (enzyme-immunoassay or radio-immunoassay)specific in-house value (MU/ml). Therefore, all results were expressed as EU eq MU/ml. Sensitivity of the indirect ELISA method using references

To evaluate the sensitivity of the indirect ELISA method, each reference sigmoid was fitted by a fourth order polynomial regression. Sensivities are compared to an arbitrary value of 1 for the OD which was chosen to obtain the corresponding reference dilution by using the calculated equation. Precision and accuracy of the indirect ELISA method19,20

In order to obtain a normal distribution of the data, a logarithmic transformation was applied to the titres. The experimental design common to accuracy and precision consisted in five groups of assays complying with conditions of intermediate precision (assays performed independently using the same method, in the same laboratory on di#erent days). Within each group, four assays were carried out under conditions ensuring repeatability (assays performed independently using the same method, in the same laboratory, with the same equipment, on one day). The homogeneity of within-tests variances was verified by a Cochran’s test. Concerning the accuracy, the 95% confidence interval of the recovery percentage which must include the 100% value (230 EU eq MU/ml) was calculated. To evaluate the repeatability and intermediate precision variances, an Analysis of Variance was performed. Control chart

In order to obtain a normal distribution of data, a logarithmic transformation was applied to the

Results Comparison between direct and indirect ELISA immunocapture methods with non-absorbed references

Non-adsorbed references (An, Bn and Dn) from three manufacturers were tested with the direct ELISA immunocapture method (Fig. 1A, B and C). The upper asymptotes of the sigmoid curves were comparable for references An and Dn but were 2·8 times lower by extrapolation for reference Bn. In order to increase the sensitivity of the direct test, an indirect ELISA immunocapture method was performed (Fig. 1). This indirect method allowed to increase the sensitivity of the three references: for an OD=1, the sensitivity of the antigen detection was enhanced 1·2-fold for ref. An, 1·6-fold for ref. Bn and 1·6-fold for ref. Dn. For the three non-adsorbed references, standard deviations and geometric coefficients of variation were calculated for the direct and indirect method. Results were obtained for each reference concentration. The standard deviations between the direct and indirect method are not statistically di#erent (Table 1). Moreover, the geometric coe#icients of variation are acceptable (<20%). Assessment of the indirect method with adsorbed references

Vaccines titration required the use of reference comparable to the vaccines. For three manufacturers, the titration of vaccines required inactivated alum-adjuvanted references (ref. Aa, ref. Ba and ref. Ca), and for one manufacturer a non-adsorbed reference that we previously tested by direct and indirect method (ref. Dn). For adsorbed references Aa, Ba and Ca, a desorption of antigen was required prior to antigen ELISA assay. A common desorption procedure is not available for the three products, therefore a specific desorption method was necessary. The maximal OD values (Fig. 2A) of adsorbed references Aa and Ba were comparable to those found with the indirect method for non-adsorbed references An and Bn (3 OD and 2 OD respectively). The sensitivity was assessed for the four references. The dilutions were 1:18 and 1:16 for references Aa and Dn respectively. For references Ca and Ba, dilutions were 1:5 and 1:3.

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1

10 100 EU eq MU/ml

1000

10 000

reference sigmoid curve was selected from four and five dilutions of, respectively, reference curves Aa and Dn and from three dilutions for reference curves Ba and Ca (Fig. 2A). A statistical description of the relationship between OD of each reference and its dilutions was performed (Table 2, Fig. 2B). For the four references, a statistically significant relationship was shown. An evaluation of precision and accuracy of the method was done with the adsorbed reference Aa. The precision results (Table 3) showed acceptable repeatability (within-tests variance) and intermediate precision (between-tests variance). The accuracy was tested based from the Aa reference (data not shown). Cochran’s test showed that the variances of the five groups (assays) were homogeneous (P=0·580) for the reference batch. The 100% value (230 EU eq MU/ml) was included in the confidence interval of the recovery percentage (97·8–100·1%). Including all manufacturers’ results, the determination of antigen content in vaccines showed (Table 4) that this method could quantify the antigen in the mean range of 81% to 95% of the quantities assigned by the manufacturers.

1

10 100 EU eq MU/ml

1000

10 000

Setting up of a control chart

10 100 EU eq MU/ml

1000

4 A Absorbance at 405 nm

3.5 3 2.5 2 1.5 1 0.5 0 0.1 4 B Absorbance at 405 nm

3.5 3 2.5 2 1.5 1 0.5 0 0.1 4 C Absorbance at 405 nm

3.5 3 2.5 2 1.5 1 0.5 0 0.1

1

10 000

Figure 1. Titration of HAV references with direct and indirect ELISA methods. To compare the two methods, the data are expressed as the meanSD of OD values, obtained for 12, 4 and 11 assays for the direct method () and 8, 13 and 9 assays for the indirect method ( ) respectively for the references. An (A), Bn (B) and DN (C).

Precision and accuracy of the indirect method

To titrate vaccines vs. references, the dose– response curves were selected to fulfil the tests of validity (non-parallelism, linear regression, etc.). As shown in Figure 2B, the linear part of each

After a desorption step, the absorbed reference Aa was titrated vs. non-adsorbed reference An. The percentage of desorption recovery was higher than 70% (data not shown). A control chart of the reference Aa (34 results) was set up to analyse the ELISA method consistency (Fig. 3). The centre line was around 200 EU eq MU/ml. The upper and the lower control limits were respectively of 240 and 166 EU eq MU/ml. Despite a statistically significant slight trend (data not shown) was underlined, only one point was outside the control limits. Moreover, the established value (230 EU eq MU/ml) is inside the control limits. More titrations would be necessary to confirm or refute this trend. Discussion In 1997, since National Control Laboratories (NCLs) were without a standardized method or reference available to determine the antigen content in Hepatitis A vaccines, the in vivo potency test was the unique release test. In order to reduce potency tests on animals, we decided to develop a polyvalent immunocapture ELISA method for assessing antigenicity of di#erently formulated vaccines. For each Hepatitis A vaccine, NCLs rely on





 0·242 0·212 0·759 No

 17 16 — —

gCV

16 17 — —

gCV

17·9

0·213 0·229 0·972 No



0·174 0·113 0·261 No

 15 13 — —

gCV

3·59

0·145 0·108 0·448 No

 14 13 — —

gCV

7·19

0·129 0·109 0·665 No

 13 13 — —

gCV

14·38

0·116 0·092 0·559 No

 13 12 — —

gCV

28·75

19 15 — —

gCV 0·253 0·242 0·786 No

 18 17 — —

gCV

31·2

0·208 0·167 0·501 No

 16 15 — —

gCV

62·5

0·168 0·143 0·593 No

 15 14 — —

gCV

125

0·146 0·131 0·677 No



14 14 — —

gCV

250

0·135 0·117 0·700 No

 14 13 — —

gCV

35·7

0·147 0·118 0·553 No

 14 13 — —

gCV

71·5

0·121 0·116 0·912 No



13 13 — —

gCV

143

0·121 0·104 0·664 No



13 13 — —

gCV

286

0·114 0·097 0·687 No



13 12 — —

gCV

572

Concentration of HAV antigen expressed in EU eq MU/ml

0·281 0·188 0·345 No



15·6

14 12 — —

gCV

57·5

0·143 0·071 0·745 No



Concentration of HAV antigen expressed in EU eq MU/ml

14 14 — —

gCV

7·81

0·136 0·131 0·926 No



1·80

: Standard deviation. gCV: geometric coe#icient of variation (%). No: No significant di#erence between standard deviations. ND: Not done.

— 12 — —

gCV

8·94

Direct ND Indirect 0·072 P value — Significant di#erence —

Reference Dn

22 — — —

gCV

3·91

Direct 0·342 Indirect ND P value — Significant di#erence —

Reference Bn

— 15 — —

gCV

0·898

Direct ND Indirect 0·186 P value — Significant di#erence —

Reference An

Concentration of HAV antigen expressed in EU eq MU/ml

0·086 0·057 0·381 No



12 11 — —

gCV

13 13 — —

gCV

1144

0·129 0·127 0·825 No



11 — — —

gCV

500

0·051 ND — —



115

Table 1. Comparison of standard deviations and geometric coe#icients of variation for each non-adsorbed reference

0·061 ND —




12 — —


gCV

10 — — —

gCV

2288

0·000 ND — —



230

In vitro potency assay for hepatitis A vaccines 251

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B. Poirier et al.

Absorbance at 405 nm

3.5 3

A

2.5 2 1.5 1 0.5 0

1/18

1/ 8 1/ 5 1/ 4 1/ 2. 5 1/ 2 1/ 1. 25

1/ 25 6 1/ 12 8 1/ 80 1/ 64 1/ 40 1/ 32 1/ 20 1/ 16 1/ 10

–0.5

1/16

1/4.8

1/3

Reference dilutions Absorbance at 405 nm

4

B

3 2 1 0 –1 25

2 1/

1. 1/

5 2.

4

Reference dilutions

1/

1/

5

8

1/

1/

10

16

1/

1/

20

32

1/

1/

1/

40

–2

Figure 2. Comparison of sensitivity (A) and linearity (B) of indirect method for each reference. The data are the mean of OD values of 45, 8, 6 and 9 assays for references Aa (), Ba (), Ca () and Dn ( ), respectively.

manufacturers’ reagents and methodologies and are obliged to perform product-specific in vitro potency tests. In these conditions, antigen units are arbitrary and the di#erence in the design of vaccines available on the market does not allow a direct comparison of antigen content. A direct sandwich method was performed using a monoclonal IgG1- anti-HAV for catching the antigen and a monoclonal IgG3-HRPO anti-HAV for developing the reaction. After an optimisation step we obtained a complete sigmoid curve for references An and Dn with significant OD values, but for reference Bn the sigmoid curve was not optimal and ODs were very low. In this condition for this reference it was obvious that the direct ELISA method was not sensitive enough to titrate vaccines. Furthermore, for the same OD, the quantities of HAV antigen content expressed in EU eq MU/ml were totally di#erent depending on HAV strain and methodology used for vaccine production. The a#inity of monoclonal antibodies for the reference seems to be dependent on Hepatitis A virus strains: two monoclonal antibodies had a similar high

a#inity for the GBM and RG-SB strains, but the same antibodies had weaker a#inity for the two other strains (HM175 and CR336F). These results showed that monoclonal antibodies were too selective and that the method was not sensitive enough to determine the antigenic content in various Hepatitis A vaccines. In order to detect all antigenic epitopes from the various virus strains and to increase the sensitivity of the test we used a human polyclonal IgA containing anti-Hepatitis A antibodies to coat the plates and a monoclonal IgG3- anti-HAV and an antimouse IgG3-HROP to reveal the assay. After optimisation, the sensitivity of the detection was slightly enhanced for each reference and the increase factor was dependent on reference (factor 1·2 to 1·6). The upper asymptote of the sigmoid curve was not reached for reference Bn, nevertheless a mean of the OD equal to 2 was obtained for the undiluted sample which allowed us to consider that an antigen determination was feasible with this indirect ELISA method. Nevertheless, additional assays were further performed before using this method to titrate HAV vaccines. In potency assays, the reference used in the test should be similar to that of the vaccines under test. For Hepatitis A vaccines, three references are adsorbed on aluminium hydroxide and one is nonadsorbed. Consequently, before choosing the indirect ELISA method to determine the antigenicity of the vaccines, assays were performed with adsorbed references Aa, Ba, Ca and with the non-adsorbed Dn reference. A desorption step was a preliminary requirement to allow the epitopes accessibility to antibodies. For this purpose we used each manufacturer–specific methodology. Similar OD values were obtained with adsorbed references Aa and Ba, and with non-absorbed references, which proved a non-alteration of epitopes during desorption treatments. Two groups of sensitivity could be distinguished: a high sensitivity for references Aa and Dn and a low sensitivity for references Ba and Ca (Fig. 2A). Actually, the IgG3- monoclonal antibody used to reveal the assay recognised an immunodominant epitope and a neutralising site located on VP3 capsid protein of HAV.22 According to the various virus strains used for reference production, this monoclonal antibody seems to have a high a#inity and specificity for two of the strains but a lower a#inity and specificity for the two others. These results could be due to the specificity of the monoclonal antibody linked to the virus strain used

In vitro potency assay for hepatitis A vaccines

253

Table 2. Regression analysis for the references Aa, Ba, Ca and Dn Regression description Reference Aa Reference Ba Reference Ca Reference Dn Analysis of variance of the regression

Source

Parameter

Estimate

P value

Intercept Slope Intercept Slope Intercept Slope Intercept Slope


1·40 2·21
4·10 2·17
1·07 2·10
3·62 2·20

0·000 0·000 0·000 0·000 0·000 0·000 0·000 0·000

Sum of squares

Df

Mean square

F-ratio

P value

Reference Aa

Model 99·61 Residual 17·16 Total 116·77 Correlation coe#icient=0·924

1 99·61 178 0·0964 179 r-squared=85%

1033

0·000

Reference Ba

Model 6·81 Residual 1·47 Total 8·28 Correlation coe#icient=0·907

1 6·81 22 0·0667 23 r-squared=82%

102

0·000

Reference Ca

Model 4·806 Residual 0·095 Total 4·901 Correlation coe#icient=0·990

1 4·806 16 0·006 17 r-squared=98%

806

0·000

Reference Dn

Model 33·18 Residual 5·03 Total 38·21 Correlation coe#icient=0·932

1 33·18 39 0·129 40 r-squared=87%

257

0·000

Df: Degrees of freedom.

Table 3. Within-tests and between-tests analysis of variance Source Between-tests Within-tests Total(corrected)

Sum of squares

Df

0·0073 0·0069 0·0142

4 15 19

for reference preparation, or to the intrinsic content of Hepatitis A antigen in each reference. The monoclonal IgG3- was obtained with the CF53 virus strain which represents the genotype II whereas virus strains for vaccines production were genotype I.23 However, a unique antigenic specificity has been associated with HAV; significant antigenic variation has not been identified among di#erent HAV strains and epitopes recognised by

Mean square 0·0018 0·0005

F-ratio

P value

3·93

0·0225

the monoclonal antibodies appear to be highly conserved among various HAV strains.24 Consequently, the two groups of sensitivity studied among the references were more probably due to the intrinsic content of Hepatitis A antigen in each reference. If the quantity of Hepatitis A in each reference was expressed in the same unit, it could be proven that the intrinsic content of Hepatitis A antigen would di#er from one manufacturer to another.

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Table 4. Antigen determination and percentage of HAV quantities found in vaccines from four di#erent manufacturers using the same indirect ELISA method

Manufacturer

Vaccine lot number

Number of assays

Titre: EU eq Mu/ml Mean

Lower limit

Upper limit

A

A1 2 330 A2 4 266 A3 8 232 A4 8 220 A5 8 263 A6 8 274 A7 8 312 A8 8 221 A9 8 242 Recovery percentage for manufacturer A vaccines

270 228 208 196 224 244 241 192 208 81%

404 311 258 246 307 308 402 253 283

B

B1 3 836 B2 3 1548 B3 3 1398 B4 3 1454 B5 3 1221 B6 3 526 B7 3 1137 Recovery percentage for manufacturer B vaccines

800 1457 1287 1344 1130 474 1039 94%

873 1641 1519 1574 1320 584 1244

C

C1 3 59 C2 3 60 C3 3 60 C4 3 50 Recovery percentage for manufacturer C vaccines

54 57 56 47 95%

65 63 64 53

D

D1 4 369 D2 7 1175 D3 7 967 Recovery percentage for manufacturer D vaccines

247 1077 884 84%

392 1282 1057

The titres for vaccines A, B and C where calculated vs. the homologous adsorbed references after a desorption treatment, whereas the titres for vaccines D were obtained vs. the non-adsorbed homologous reference.

The objective of the statistical analysis was to demonstrate that the procedure was precise and accurate. Statistically, in accordance with the European Pharmacopoeia recommendations, assays’ parallelism and linearity were demonstrated. Moreover, the correlation coe#icients indicated a relatively strong relationship (Table 2). Concerning the precision of the technique (Table 3), as expected, because of the uniformity of the experimental conditions, the within-tests results showed a good homogeneity, with a small within-tests variation. The intermediate precision was acceptable and the accuracy demonstrated for absorbed vaccines. The control chart implemented with the reference Aa confirmed this. The parallelism, linearity and control chart are criteria which must be veri-

fied to validate or invalidate the assay in routine testing. To check the vaccines’ production consistencies, the quantity of HAV antigen in several vaccine lots was determined (Table 4). The results proved that this single indirect ELISA method could be used to assess antigen determination in di#erent vaccine formulations. Concerning manufacturer D’s vaccines, it was obvious that the variability was high, due to a lower reproducibility of the HAV content determination. On the contrary, the results obtained with the homologous reference Dn were homogeneous. This phenomenon is explained by the fact that the homologous reference Dn and the manufacturer D vaccine have a di#erent formulation.

In vitro potency assay for hepatitis A vaccines

Titre:EU eq MU/ml (Log10)

2.6 2.5 2.4 2.3 2.2 2.1 2

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 Number of assays

Figure 3. Control chart of the reference Aa. The results of the reference titrations were taken into account if the percentage of desorption recovery was higher than 70%. These results were obtained independently using the same method, in the same laboratory on di#erent days. The mean (- - - -) was around 200 EU eq MU/ml. The upper (– – – –) and the lower (——) control limits were respectively of 240 and 166 EU eq MU/ml.

Prior to the titrations of adsorbed references Aa and Ba, a desorption treatment was necessary to remove the alum hydroxyl which masked epitopes towards antibodies. For both references the recovery percentage was estimated and we considered that after a desorption treatment a percentage around 80% was satisfactory and expected for an ELISA assay. In 1998, a joint World Health Organisation/ European Department for the Quality of Medicines collaborative study was organised to assess inactivated Hepatitis A vaccine antigenicity based on product-specific methodologies and immunogenicity.25 This study provides information on the correlation between antigenicity and immunogenicity of the candidate reference materials. A common standard antigenicity assay was proposed to be performed in parallel with participants’ in-house antigenicity assays. Our laboratory took part in this study and performed the standard antigenicity assay in parallel with our in-house antigenicity assay. It appeared that our in-house assay is not statistically di#erent from the standard one: results are very close to others (data not shown) and the relationship between the immunogenicity assay with the common and in-house antigenicity assays is statistically significant (data not shown). Taking into account these results, it appears that our in-house ELISA assay to determine Hepatitis A antigenicity seems to be a good candidate for a common standard assay. Its advantage is its low

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cost compared to the assay proposed in the collaborative study based on commercially available kits, which is approximately ten times more expensive. Consequently, this assay is suitable for manufacturer large-scale control runs as well as simple and applicable for NCAs involved in the quality control of various products. Moreover, the second aim of the collaborative study was to obtain an international standard reference and to assign international units to the antigen content for each marketed vaccine. To strengthen the validation of our in-house ELISA assay a second step will be to determine the antigen content of Hepatitis A vaccine marketed in France vs. the international standard reference and to compare international units to the values assigned by each manufacturer. Acknowledgements The authors are grateful to Doctor Martine Langlois and Mrs Geraldine Lloveras for their helpful suggestions.  2000 Government of France

References 1. Hollinger FB, Ticehurst JR. Hepatitis A virus. In: Fields BN, Knipe DM (eds) Virology. New York, Raven Press, 1990: pp. 631–667. 2. Gust ID. Epidemiological patterns of hepatitis A in di#erent parts of the world. Vaccine 1992; 10: 56–58. 3. Forbes JR, Williams R. Increasing age. An important adverse prognostic factor in hepatitis A virus infection. J R Coll Physicians 1988; 22: 237–239. 4. Reuman PD, Kubilis P, Hurni W et al. The e#ect of age and weight on the response to formalin inactivated, alum-adjuvanted hepatitis A vaccine in healthy adults. Vaccine 1997; 15: 1157–1161. 5. Goubau P, Van Gerven V, Safary A et al. E#ect of virus strain and antigen dose on immunogenicity and reactogenicity of an inactivated hepatitis A vaccine. Vaccine 1992; 10: S114–118. 6. Andre´ FE, Hepburn A, D’Hondt E. Inactivated candidate vaccines for hepatitis A. Prog Med Virol 1990; 37: 72–95. 7. Flehmig B, Haage A, Pfisterer M. Immunogenicity of a hepatitis A virus vaccine. J Med Virol 1987; 22: 7–16. 8. Armstrong ME, Giesa PA, Davide JP, Redner F, Waterbury JA, Rhoad AE, Keys RD, Provost PJ, Lewis JA. Development of the formalin-inactivated hepatitis A vaccine, VAQTA from the live attenuated virus strain CR326F. J Hepatol 1993; 18: S51–S55. 9. Glu¨ ck R, Mischler R, Brantschen S et al. Immunopotentiating reconstituted influenza virus virosome vaccine delivery system for immunization against hepatitis A. J Clin Invest 1992; 90: 2491–2495.

256

B. Poirier et al.

10. Pellegrini V, Finschi N, Matteucci G et al. Preparation and immunogenicity of an inactivated hepatitis A vaccine. Vaccine 1993; 11: 383–387. 11. World Health Organisation. Technical Report Series 1995; 858: 39–65. 12. European Pharmacopoeia. Vaccinum hepatitidis A inactivatum adsorbatum. 1998; 1107. 13. Provost PJ, Hugues JV, Miller WJ et al. An inactivated hepatitis A viral vaccine of cell culture origin. J Med Virol 1986; 19: 23–31. 14. Burkardt F, Glu¨ ck R, Finkel-Jimenez B et al. Hepatitis A vaccines: a comparison between three methods of antigen determination. Dev Biol Stand 1996; 86: 129–135. 15. Fisch A, Cadilhac P, Vidor E et al. Immunogenicity and safety of a new inactivated hepatitis A vaccine: a clinical trial with comparison of administration route. Vaccine 1996; 14: 1132–1136. 16. Poovorawan Y, Theamboonlers A, Chumdermpadetsuk S et al. Safety, immunogenicity, and kinetics of the immune response to a single dose of virosomeformulated hepatitis A vaccine in Thais. Vaccine 1995; 13: 891–893. 17. Perrin P, Lafon M, Sureau P. Enzyme linked immunosorbent assay (ELISA) for the determination of glycoprotein content of rabies vaccines. In: Meslin F-X, Kaplan M-M, Koprowski H (eds) Laboratories Techniques in Rabies. Gene´ va, WHO, 1996: pp. 383–388. 18. European Pharmacopoeia. Chapter 5.3, Statistical analysis of results of biological assays and tests.

19. ICH Topic Q2A (CPMP/ICH/381/95). Validation of analytical methods: Definitions and Terminology. 20. ICH Topic Q2B (CPMP/ICH/281/95). Validation of analytical procedures: Methodology. 21. AFNOR—NF X 06-031-1. Application de la statistique, Cartes de controˆ le, Partie 1: Cartes de de controˆ le de Shewhart aux mesures. 22. Crevat D, Crance J-M, Chevrinais A-M et al. Monoclonal antibodies against an immunodominant and neutralising epitope on hepatitis A virus antigen. Arch Virol 1990; 113: 95–98. 23. Robertson BH, Jansen RW, Khanna B et al. Genetic relatedness of hepatitis A virus strains recovered from di#erent geographical regions. J Gen Virol 1992; 73: 1365–1377. 24. Stapleton JT, Lemon SM. Neutralisation escape mutants define a dominant immunogenic neutralisation site on hepatitis A virus. J Virol 1987; 61: 491–498. 25. Wood D, Sands D, Heath A. Collaborative study for the establishment of three product specific European pharmacopoeia biological reference preparations for inactivated adsorbed hepatitis A vaccines. Pharmeuropa Special issue BIO 2000-1; August 2000: 53–81.

Received for publication 3 April 2000; accepted 8 January 2001