Physical, chemical and immunological stability of CHO-derived hepatitis B surface antigen (HBsAg) particles

Vaccine 18 (2000) 3±17

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Physical, chemical and immunological stability of CHO-derived hepatitis B surface antigen (HBsAg) particles Dvorah Diminsky a, Neomi Moav b, Marian Gorecki b, Yechezkel Barenholz a,* a

Department of Biochemistry, The Hebrew University-Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel b BioTechnology General Ltd, Rehovot 76326, Israel Received 14 May 1998; received in revised form 11 March 1999; accepted 18 March 1999

Abstract Recombinant hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells were stored at various conditions for 12±18 months in their naked form or adsorbed to alum (HBV vaccine). The physical, chemical and immunological parameters during storage at ÿ208C, 48C, room temperature and 378C were investigated. HBsAg particles fully retained the original peptide composition when stored for 6 months, as a dispersion, at ÿ208C and 48C; and as lyophilized powder, at all four temperatures. Ten percent sucrose preserved the size, shape and protein content of the naked particles stored at 48C and ÿ208C for 18 months as a dispersion or lyophilized. Lyophilization in the presence of glucose, sucrose and trehalose, but not mannitol, further improved the 48C stability of size, shape and the protein content during 2 years of storage. Stability of the particle's lipid components was inferior to that of the protein components. Dispersions of naked HBsAg and of particles lyophilized in the presence of sucrose and stored at ÿ208C were the only forms in which the lipid content and composition, including the lipid polyunsaturated acyl chains, were preserved for at least 18 months storage. The level of phospholipid and free cholesterol were most stable in the HBV vaccine which was stored at 48C; they did not change after 1 year of storage. Preservation of immunogenicity was evaluated according to dose-dependent changes in S-speci®c antibody titers in sera obtained from immunized BALB/c mice. The ED50 for achieving seroconversion was 0.07 mg/ml/mouse, indicating that the vaccine is very immunogenic. Freezing or freeze-drying of the HBV vaccine results in the total loss of vaccine immunogenicity (in spite of the good chemical stability), while full immunological potency was retained for at least 2.5 years at 48C. Storing formulated vaccine at 25 and 378C for 4 and 2 weeks, respectively, did not alter the vaccine potency. This study suggests that the vaccine's physical, chemical and immunological characteristics are suciently stable at high temperatures to reduce the need for `cold chain' transportation. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Hepatitis B surface antigen; HBV vaccine; Stability; Lipid and protein composition; Immune response

1. Introduction A new recombinant hepatitis B vaccine was recently evaluated in humans [1]. This vaccine is based on HBsAg particles obtained from genetically engineered CHO cells. CHO-HBsAg has a peptide composition and immunological properties similar to those of previously used plasma-derived hepatitis B vaccines. This

* Corresponding author. Tel.: 972-2-675-8507/9; fax: 972-2-6411663. E-mail address: [email protected] (Y. Barenholz)

vaccine, like other hepatitis B virus (HBV) vaccines, contains alum as an adjuvant. The HBsAg particle is a complex of proteins and lipids assembled into spherical particles with an average diameter of 22 nm (as ascertained by negative staining electron microscopy [2]). It contains three proteins: large (L)>>middle (M)>small (S). These are coded by a single open reading frame. The S component is the major peptide. It is present in two forms: a nonglycosylated form, P24, and a monoglycosylated form, GP27. The M peptide contains, in addition to an S domain, the pre-S2 domain. The M component appears in two forms:

0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 1 4 9 - 8

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D. Diminsky et al. / Vaccine 18 (2000) 3±17

monoglycosylated GP33 and diglycosylated GP36. The L peptide contains, in addition to the M domain, the pre-S1 domain, and it appears in two forms, nonglycosylated P39 and monoglycosylated GP42 [2]. Reduced hepatitis B vaccine stability, which occurs under extreme temperatures (both freezing and high temperatures), reduces its ecacy. The `cold chain' system required during shipment of vaccines [3] is necessary to protect vaccines from heat. However, refrigerated storage and transport of vaccines is very costly and may not always be possible, thus making the use of the vaccine in Third World countries very problematic. Stability assessment of the CHO-HBsAg particles and of the whole vaccine, under various conditions is, therefore, of great importance. This paper focuses on the e€ect of storage temperature and HBsAg lyophilization on the structure, stability and immunogenicity of CHO-HBsAg particles and alum-formulated vaccine. 2. Materials and methods 2.1. HBsAg particles Recombinant HBsAg particles secreted from CHO cells were puri®ed and characterized as described elsewhere [2,4]. The HBsAg particles were kindly supplied by BioTechnology General (Rehovot, Israel). 2.2. HBV vaccine preparation Hepatitis B vaccine was prepared using the above HBsAg particles adsorbed to alum [5], either immediately after particle preparation (to study stability of vaccine) or immediately before vaccination (to test immunopotency of HBsAg naked particles). 2.3. Particle structure and size distribution Particle structure and size distribution were determined by negative staining electron microscopy using 2% phosphotungstic acid as the stain [6]. The size distribution was also measured by dynamic light scattering (DLS), using size distribution processor analysis by the Coulter model N4SD submicron particle analyzer (Coulter Electronics, Luton, UK) [2,6,7]. 2.4. Peptide composition Peptide composition was determined by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) with reducing agent [2], followed by silver staining [8] and Western blot analysis [9]. The ratio between the peptides was determined by computerized image

analysis using NIH 1.16 image analysis [2]. For the latter we used mouse monoclonal (#1023) antibodies prepared at the National Institute for Biological Standards and Control (NIBSC), UK, using yeast-derived HBsAg particles as antigen. It recognizes the linear epitope of the S determinant. Following storage for the indicated period, aliquots were lyophilized and stored at ÿ208C until analyzed. 2.5. Lipid composition and total protein Lipid composition, including phospholipids, triglycerides, free cholesterol and phospholipid acyl chains, was analyzed as described elsewhere [2,6]. Total protein content was determined using the method of Lowry et al. after particle solubilization by SDS (5% ®nal concentration) [2]. 2.6. Antigenicity HBsAg concentrations were determined by ELISA, which was validated by BioTechnology General and compared to an international standard. The assay is speci®c for the ®rst loop (epitope 1) of the a linear sequence epitope of the S antigen. Microtiter plates (Maxisorp Nalge, Nunc Int.) were coated with goatanti-HBsAg AD/AY subtypes IgG fraction (purchased from Biodesign International, used at 10 mg/ml) diluted in 0.1 N sodium carbonate pH 9.6. The plates were then blocked with 3% BSA in PBS. After washing with saline containing 0.05% Tween-20, the plates were incubated with the samples containing HBsAg particles for 2 h at 378C and overnight at 48C. After washing, the plates were reacted with biotinylated antiHBs monoclonal antibodies (biotinylated Mab M-6/3IgG2-Ho€man-LaRoche). The detection system utilizes streptavidin-alkaline phosphatase conjugate (Jackson Immuno Research) and p-nitrophenylphosphate as a substrate in 10 vol% diethanolamine bu€er pH 9.8 containing 0.5 mM MgCl2. 2.7. Immunological potency Immunological potency was evaluated by seroconversion in mice and expressed as ED50. All testing was performed using HBV vaccine. Seroconversion test was performed by injecting, i.p., three groups of BALB/c mice (10±15/dose) with either 0.02, 0.1 or 0.5 mg HBsAg formulated as alum-based vaccine. AntiHBs antibodies were determined 30 days after immunization using the AUSAB radioimmunoassay (Abbott). Anti-HBs antibodies are expressed in mIU/ ml using a WHO reference serum.

D. Diminsky et al. / Vaccine 18 (2000) 3±17

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Table 1 HBsAg particle sizea after lyophilization in presence of various sugars and storage for 2 years at 48C Preparation

Size (nm)

Time 0

Size (nm)

After 2 years

Solution

25.82 3 2392 46

99% 1%

32.3 29b 162 238

99% 1%

Lyophilization

17.42 2 7222 160

94% 6%

12302150

100%

Lyophilization+5% glucose

26.42 5 1702 43 19202230

94% 1% 5%

7.12 0.8 37.4 212 387 2120

11% 88% 1%

Lyophilization+10% sucrose

27.52 8 1572 45 12802150

90% 2% 8%

23.9 29 136 259 12402360

89% 2% 9%

Lyophilization+10% trehalose

21.12 6 75.82 43 5292 180

90% 7% 3%

29.5 27 153 254 11402240

83% 3% 140%

Lyophilization+10% mannitol

99.1

100%

40 25 14802410

5% 95%

a b

Determined by dynamic light scattering. Size was determined after 18 months.

3. Results The physical, chemical and immunological stability of HBsAg particles were evaluated following storage up to 18 months at various temperatures. 3.1. E€ect of storage on HBsAg particle size distribution 3.1.1. Storage in suspension at various temperatures HBsAg particles in PBS were stored at four temperatures: 378C, room temperature, 48C and ÿ208C. Determination of the size distribution of HBsAg is somewhat dependent on the method of measurement: Negative staining electron microscopy of fresh HBsAg indicated that the particles are homogeneous and have a mean diameter of 22 nm. Cryotransmission electron microscopy revealed that HBsAg particles are leaky vesicles and less homogeneous in size (unpublished). According to size distribution analysis (DLS), 99% of the particles have a mean diameter of 25.8 2 3 nm (Table 1). The mean size remained constant for 6 months for particles stored at all four temperatures. After 6 months, HBsAg particles stored at 378C lost their particle structure and became aggregated. The aggregation continuously increased during 18 months. At the other three temperatures, the mean size of the particles was almost identical to the size of fresh

HBsAg particles, though some aggregation was observed. Negative staining electron micrographs revealed that at room temperature and at 378C some aggregation occurred already after 4 weeks of storage. 3.1.2. Storage of HBsAg particles at various temperatures after lyophilization with various sugars The particle size was unaltered following 6 months of storage at all of the studied temperatures. Following 18 months of storage at 378C, particle size increased, but to a lesser extent than when stored as an aqueous dispersion. DLS analysis of HBsAg, following lyophilization in the presence of glucose, sucrose, trehalose or mannitol (Table 1) showed that, except for mannitol, the sugars contributed to preserving the HBsAg size. Even after 2 years of storage of these preparations at 48C, more than 83% of the particles in each preparation retained the original size. 3.1.3. E€ect of lyophilization without sugars Lyophilization of HBsAg particles without adding any sugar resulted in a mean particle diameter of 17.4 22 nm (94% of the particles), while the mean diameter of 6% of the particles was 722 nm (Table 1). This may be the result of an artifact associated with the size distribution analysis for resolving broad size distributions to two de®ned distributions.

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D. Diminsky et al. / Vaccine 18 (2000) 3±17

Table 2 Quantitative analyses of soluble HBsAg peptides (batch 4-38P-151) in SDS-gel electrophoresis (wt%) Storage temperature Storage time S M L M.W. M.W. (months) (under 20 kDa) (above 50 kDa) 24 kDa 27 kDa Total 33 kDa 36 kDa Total 39 kDa 42 kDa Total Untreated 48C (ÿ208C) 378C Room temperature 48C Room temperature 378C (ÿ208C) 48C (ÿ208C) Room temperature 378C a

0 3 3 3 3 6 6 6 6 18 18 18 18

29.7 46.9 47.4 25.2 33.3 33.2 33.7 42.8 31.9 54 54.3 58.8 52.9

24.2 29.7 26 12.4 16.3 26.3 23 22.3 27.8 22.6 22.4 22.9 16.8

53.9 76.6 73.1 37.6 49.6 59.5 56.7 65.1 59.7 76.6 76.7 81.6 69.7

19.8 16.2 19.8 11.2 16.5 4.7 1.7 20.1

10.2 1.8 3.8 B.D. 6.9 0.2 0.3 7.6

30 1.3 18 23.8 8.2a B.D. 11.2 B.D. 23.4 5.5 4.9 2 27.7 4.5 7.2a 5.8a 0.3a 6.3a

4.9 B.D. B.D. 1 0.7

6.2 2.2a 2.9a B.D. B.D. 6.5 13.9a 12.8a 5.3 2.9a 1.5a 0.9a 2.3a

9.9 B.D. B.D. 12.7 11.4 10.6 14.3 15.1 5.2 7.2 16 17.3 21.7

B.D. B.D. B.D. 41.5 27.8 B.D. 3.1 5 2.1 6.1 B.D. B.D. B.D.

The bands could not be analyzed separately. The location of 42 kDa peptide is similar to the HBsAg dimer. B.D. means below detection.

3.2. Protein content After 18 months there was no change in the total protein content (see Section 2) in all HBsAg particle preparations stored at 48C, either as lyophilized forms or as aqueous dispersions. However, there was an average decrease in protein content of 35% in preparations which were stored at room temperature or higher temperatures (data not shown). 3.3. Peptide composition 3.3.1. Storage of HBsAg particles as aqueous dispersions HBsAg particles stored as aqueous dispersions at 48C or ÿ208C for 6 months fully retained their original peptide composition as determined by SDS-PAGE (Table 2), while the composition of such particles stored at room temperature and 378C was markedly changed. The amount of M peptides was reduced and only 5±10% of the original amount of M peptides was still present after 6 months. In addition to the persistence of the six bands of the HBsAg, additional components with MW below 20 kDa and above 42 kDa became more prominent after 18 months. The high molecular weight bands are probably a result of aggregates formed. These aggregates interfere with the analysis of L peptides. The low molecular weight peptides (under 20 kDa) may represent protein degradation and oxidation products. HBsAg dispersions stored at 48C for 1 year were subjected to Western blot analysis using monoclonal anti-S antibodies, following SDS-PAGE. In support of the results obtained by silver staining, Western blot analysis of the gels also indicated that all of the six HBsAg peptides persist for the duration of the study.

Intensity measurements of the various bands have shown that the relative amounts of the S, M and L peptides do not change signi®cantly during storage at 48C. Western blot analysis using pre-S1 and pre-S2 domain-speci®c antibodies was done for comparative evaluation of the stability of these peptides. Storage at 48C, for as long as 9 months does not appear to lead to any degradation of these peptides. 3.3.2. E€ect of storage on HBsAg particles lyophilized in 10% sucrose Lyophilization of HBsAg in the presence of 10% sucrose stabilizes the protein composition, especially by partially protecting M peptides, as found after 6 months storage at all four temperatures (Table 3). At room temperature the amount of M peptides in the preparation was reduced. After 18 months at all four temperatures there was a sharp decrease in the amount of M peptides, and increase in the amount of 24 kDa S peptide. Low molecular weight peptides (under 20 kDa) were observed in all preparations (except following storage at 48C (Table 3)). 3.3.3. E€ect of storage on HBsAg particles lyophilized with various sugars Lyophilization of HBsAg in PBS or in the presence of trehalose, glucose or mannitol (Table 4), and storage for 1 month at 48C, resulted in a decrease in the M peptide content with a concomitant increase in the S peptides, especially in the 24 kDa species. 3.3.4. Storage of HBV vaccine composition Alum-formulated HBsAg vaccine was stored in upright or inverted vials at 48C, 258C and 378C for up to 30 days, and at 508C for at least 24 h. The antigen

B.D. 2.3 2.5 2.7 1.4 B.D. B.D. B.D. B.D. .9 4.4 7.1 3.3 5.4 B.D. 19 14.4 11.9 0.3 2.9

6.2 3.1a 3.2 7.6a 2.8a 6.4a 2.3a 2.1a 1.4a 4.9 1.3

30 16.5 25.4 23.9 10.3 6.6a 9.9a 7.6a 4.6a

3.4. Lipid composition 3.4.1. Phospholipid content of HBsAg particles Total phospholipid content was calculated from total phospholipid phosphorus extracted from HBsAg particles. The phospholipid phosphorus content of aqueous HBsAg particle dispersions continuously decreased during 18 months at the four storage temperatures. According to this analysis, the smallest decrease in phospholipid content occurred in the preparations stored at ÿ208C and 48C (Fig. 1).

53.9 73.6 62.1 62.5 80.1 84.3 68.8 75.9 82.1 a

The bands could not be analyzed separately. B.D. means below detection.

24.2 31.9 29 27.5 30.4 32.4 27.6 20.8 18.8 29.7 41.7 33.1 35 49.7 51.9 41.2 55.1 63.3 Untreated Lyophilization+10% Lyophilization+10% Lyophilization+10% Lyophilization+10% Lyophilization+10% Lyophilization+10% Lyophilization+10% Lyophilization+10%

sucrose, 48C sucrose (ÿ208C) sucrose, 378C sucrose, room temperature sucrose, 48C sucrose (ÿ208C) sucrose, 378C sucrose, room temperature

0 6 6 6 6 18 18 18 18

27 kDa

7

was desorbed from alum prior to electrophoresis under reducing conditions. The peptide composition was determined by polyacrylamide-gel electrophoresis in the presence of SDS and mercaptoethanol. The gels were either stained with silver reagents or evaluated by Western blot analysis after transblotting and probing with S-speci®c Mab antibody. All of the samples stored at the di€erent temperatures retained all six HBsAg peptides. The intensities of additional high and low molecular weight bands did not appear to change in a time- or temperature-dependent fashion.

19.8 12.5 18.9 18.3 8.5

10.2 4 6.5 5.6 1.8

39 kDa Total 36 kDa 33 kDa 24 kDa

Total

M S

Storage time (months) Treatment

Table 3 Quantitative analyses of lyophilized HBsAg peptides (batch 4-38P-151) in SDS-Gel (wt%)

L

42 kDa

Total

M.W. (under 20 kDa)

M.W. (above 50 kDa)

D. Diminsky et al. / Vaccine 18 (2000) 3±17

3.4.2. Phospholipid content of HBsAg stored in lyophilized form The phospholipid phosphorus content of all lyophilized preparations which contained sucrose was better preserved than that of the aqueous dispersions (Fig. 1). In these preparations, storage at 48C or ÿ208C stabilized the total phospholipid content to a greater extent than storage at higher temperatures (Fig. 1). Formulation of HBsAg with alum preserved the phospholipid phosphorus content of the adsorbed particles for at least 1 year when such preparations were stored at 48C. The stability of the HBsAg phospholipid composition was also evaluated. The relative amounts of the primary phospholipids in fresh particles are: phosphatidylcholine (PC)>sphingomyelin (SPM)>phosphatidylethanolamine (PE). After storage of HBsAg for 18 months, under di€erent conditions, the PC and PE content signi®cantly decreased, especially when the HBsAg was stored as an aqueous dispersion or as a lyophilized powder at 378C. Storage of HBsAg lyophilized with 10% sucrose at ÿ208C preserved the content of all of the various phospholipid species (data not shown). 3.5. Lipid acyl chain composition 3.5.1. Fresh HBsAg particles Oleic acid (C18:1) is the main acyl chain of HBsAgassociated lipids, comprising 52.7 2 5% of the fatty acid chain composition. The percent composition of other acyl chains is: C16:0 (19.62 1.5%)>C16:1

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D. Diminsky et al. / Vaccine 18 (2000) 3±17

Table 4 Peptide composition of lyophilized HBsAg with di€erent sugars following 1 month of storage at 48C (wt%) Treatment

S

M

L

M.W. (under 20 kDa) M.W. (above 50 kDa)

24 kDa 27 kDa Total 33 kDa 36 kDa Total 39 kDa 42 kDa Total Solution, 48C Lyophilization Lyophilization+10% Lyophilization+10% Lyophilization+10% Lyophilization+10% a

33.3 37.2 sucrose 38 trehalose 44 glucose 39.2 mannitol 41.5

24.1 28.9 28 30.3 26.4 25.8

57.4 66.2 66 74.3 65.6 67.3

19.3 16.2 16.7 13 18.4 16.8

10.4 6.6 4 1.9 4.4 4.8

29.7 22.8 20.7 14.9 22.8 21.6

1

5.4

3.7 1.5 1.64

B.D. 3.3 2.5

6.4 4.7a 3.3a 3.7 4.8 4.1

6.4 4.8 9.3 5.1 6 6.3

B.D. 1.4 1 1.9 0.6 0.7

The bands could not be analyzed separately. B.D. means below detection.

(9.4 2 2%) [3] C18:0 (8.8 22%). Small amounts of other acyl chains, including C20:4 (3.5 20.7%), C22:6 (1.1 2 0.6%) and C18:2 (0.4 2 0.1%) are also present. Monitoring of the latter three has been used as an index for quantifying oxidative damage [10,11]. 3.5.2. E€ect of lyophilization and storage temperature on the acyl chain pro®le The amount of the saturated acyl chains and the amount of C18:1 did not change even after 18 months of storage at all conditions. After 3 months there was almost no decrease in all the acyl chains composition

of HBsAg particles. A sharp decrease (close to complete) in the amount of C16:1 was observed in HBsAg particle aqueous dispersions at 48C and at room temperature. Similar decrease was observed in samples lyophilized in the presence of sucrose stored at room temperature and at ÿ208C. The suspended and the lyophilized HBsAg particles fully preserved the C18:2 and C20:4 acyl chains at ÿ208C, frozen or lyophilized, while these acyl chains are below detection limit after storage at other temperatures for 18 months, suggesting temperature-dependent oxidative damage to polyunsaturated acyl chains. The very low level of

Fig. 1. Stability of phospholipids of HBsAg, stored in di€erent conditions for 18 months. Total phospholipid of HBsAg particles was analyzed as described by Barenholz and Amselem [7]. Lyophilization in presence of sucrose at ÿ208C and 48C preserved the amount of phosphorus for 18 months. The same result was observed when HBsAg particles adsorbed to alum were stored at 48C for 1 year.

D. Diminsky et al. / Vaccine 18 (2000) 3±17

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Fig. 2. Cholesterol determination of HBsAg stored at di€erent conditions for 18 months.

polyunsaturated acyl chains in the CHO-HBsAg particles compared with yeast-derived particles may result in reduced sensitivity to oxidative damage. 3.6. Cholesterol

3.6.3. Storage of HBV vaccine The cholesterol content was unchanged for at least 1 year when stored at 48C. 3.7. Structural HBsAg changes

3.6.1. Storage of HBsAg particle dispersions The level of nonesteri®ed cholesterol was determined in HBsAg particles stored for as long as 18 months. Fig. 2 shows that the cholesterol content was signi®cantly a€ected by the storage temperature. Only storage of dispersions at ÿ208C preserved more than 80% of the cholesterol content. Following storage at all other storage conditions, the cholesterol decrease was larger. The stability of dispersions was worse than lyophilized material. The decrease was most prominent when samples were stored at 378C.

3.7.1. E€ect of temperature The spherical structure of HBsAg particles was maintained following 18 month storage at 48C and ÿ208C, for both HBsAg aqueous dispersions and following lyophilization in the presence of sucrose (Fig. 3(a)). Storage of HBsAg as dispersions or lyophilized material at 378C or room temperature caused signi®cant aggregation, or loss of vesicular structure compared to the fresh HBsAg particles' spherical structure (Fig. 3(b) and (c)).

3.6.2. Storage of lyophilized HBsAg particles CHO-HBsAg particles lyophilized in the presence of sucrose maintained the original cholesterol content when stored for 3 months at any of the four temperatures. After 18 months, the cholesterol content was reduced regardless of storage temperature, although the degree of cholesterol reduction was lower in the lyophilized preparations than in the dispersions. The cholesterol content was most stable in particles lyophilized in the presence of sucrose and stored for 18 months at ÿ208C.

3.7.2. Storage of HBsAg particles lyophilized in the presence of various sugars The HBsAg particle structure stability was evaluated using negative stain electron microscopy. Lyophilization of HBsAg particles in the presence of sucrose, glucose, trehalose or with no added sugars appears to preserve HBsAg particulate structure. Lyophilization in the presence of 10% mannitol caused aggregation, fusion and signi®cant changes in the HBsAg particle shape (Fig. 3(d)).

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D. Diminsky et al. / Vaccine 18 (2000) 3±17

Fig. 3. Electron micrographs of CHO-HBsAg particles stored at di€erent conditions. The particles were visualized by negative staining using 2% phosphotungstic acid as the stain. (a) HBsAg after lyophilization in presence of 10% sucrose, stored for 18 months at ÿ208C. The HBsAg particles preserved their size and structure. (b) HBsAg after lyophilization in presence of 10% sucrose, stored for 18 months at 378C. Most of the particles were aggregated or lost their vesicular structure. (c) HBsAg stored as a liquid dispersion at room temperature for 18 months. Some aggregation of HBsAg particles can be seen. (d) HBsAg after lyophilization in presence of 10% mannitol. Most of the preparation contains aggregates of di€erent sizes.

3.8. Immunologic parameters 3.8.1. E€ect of temperature on antigenic activity of HBsAg Stability of HBsAg antigenicity was determined using ELISA speci®c for the S peptide. Samples of HBsAg particles were incubated at 48C, 258C and 378C for up to 1 year. At each time interval, samples were withdrawn and evaluated using S-speci®c ELISA to determine the concentrations of speci®c S peptides compared to a reference sample. As can be seen in Fig. 4, storage of various HBsAg batches at 48C for

up to 6±7 months was accompanied by a small decrease in activity followed by a gradual increase in the availability of S peptides. Six months storage of HBsAg batches at 258C caused a >10% reduction in antigenicity, and storage at 378C caused an 80% loss in antigenicity after 3 months for all of the batches. 3.8.2. Potency of the HBV vaccine as evaluated by seroconversion in BALB/c mice The HBV vaccine was prepared by adsorption of CHO-HBsAg particles to alum, and it was subsequently stored at 48C. Ten lots of HBV vaccine were

D. Diminsky et al. / Vaccine 18 (2000) 3±17

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Fig. 4. E€ect of storage of HBsAg on antigenic activity: ®ve unformulated HBsAg batches were stored at 48C, 258C and 378C for 1 year. The antigenicity was determined by ELISA.

tested for the stability of their ecacy. Vaccine potency was evaluated by measuring anti-S titers in immunized mice as described in Section 2. Storage of nine di€erent batches of HBV vaccine revealed that there was no signi®cant change in potency (ED50 of 0.07) during the 30-month storage period (data not shown). This indicates that there was no loss in the immunizing potential of the vaccine during this period. 3.8.3. HBsAg vaccine potency after storage at 258C or 378C Samples were stored at 378C or 258C for 14 or 30

days, respectively. A summary of the potency of these samples is presented in Table 5. These results indicate that exposure to these conditions does not signi®cantly alter vaccine potency. 3.8.4. Immunopotency of HBsAg (preformulated) and of HBV vaccine, stored as lyophilized powder Alum-formulated HBsAg vaccine samples as well as HBsAg particles (210% sucrose) were lyophilized and stored at 48C. After 2 years of storage at 48C, the alum-formulated HBsAg preparations were suspended in bidistilled water, and BALB/c mice received a single

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D. Diminsky et al. / Vaccine 18 (2000) 3±17

Table 5 The immunopotency of HBV vaccine Time

Temperature

ED50 mg

6 months 30 daysa 14 daysa

48C 258C 378C

0.09 (0.05±0.15) 0.26 (0.19±0.36) 0.19 (0.09±0.4)

a

Following 10 months at 48C.

intraperitoneal injection. The lyophilized HBsAg particles were dispersed in PBS and adsorbed to alum in PBS prior to injection (see Section 2). In all cases, 0.5

mg/ml (in 1 ml) HBsAg was injected. The anti-S antibody titers were measured after 1, 2 and 3 weeks and compared to those in mice vaccinated with fresh vaccine (Fig. 5). The immunopotency of preformulated HBsAg vaccine was completely lost following lyophilization and storage, while lyophilization of HBsAg particles with or without sucrose, followed by adsorption to alum after hydration immediately before injection, preserved the immunogenicity of the HBsAg particles. The magnitude of the antibody titer generated by lyophilized (unformulated) HBsAg was similar to that observed after injection of the fresh vaccine for all time periods measured.

Fig. 5. The immunogenic potentcy of formulated HBsAg stored as lyophilized powder for 2 years at 48C. The powder was dispersed in PBS and the HBsAg adsorbed to alum prior to injection. The potency was evaluated by measuring anti-S titers in mice after 1, 2 and 3 weeks of injection.

D. Diminsky et al. / Vaccine 18 (2000) 3±17

4. Discussion Hepatitis B surface antigen particles serve as the antigen for most current vaccines against hepatitis B virus. Alum is the sole adjuvant approved for human use [12,13]. In order to ensure reproducible ecacy of the vaccination it is crucial to know: (a) the stability pro®le of the antigen, (b) the preferred conditions for its storage and handling, (c) the optimal storage conditions for the HBsAg particles and the whole vaccine (antigen+adjuvant) and (d) that the manufacturing process consistently yields a product with particular chemical, physical and immunological characteristics. These criteria are especially important for antigens which are not de®ned molecules (proteins or peptides), but rather complex assemblies like the HBsAg particle, which is a natural proteoliposome composed of lipids and proteins [2] and for which the presence of lipids is essential for optimal immune response (unpublished). There are many indications [2,14] that the particulate nature of the HBsAg proteoliposome is more important than the exact lipid composition. The particulate nature of the HBsAg and the presence of lipids in the particle impose new stability issues for preparation, storage and handling of the antigen, in addition to the current problems of protein stability of all multiunit vaccines. Numerous chemical, physical and immunological parameters possibly relevant to immunogenic potency were evaluated in this study. 4.1. Physical stability The e€ect of 18 months of storage on stability and antigenic potency of CHO-HBsAg particles, in their naked form and adsorbed on alum, was studied. HBsAg aqueous dispersions (liquid or frozen) or lyophilized powders (with and without various sugars) were stored for 18 months at ÿ208C, 48C, room temperature and 378C. The particle size of HBsAg dispersions or powders lyophilized with sugars (other than mannitol) remained fairly constant when stored at 48C and ÿ208C for 18 months. At room temperature a fraction of aggregate appeared, which became larger at 378C. Lyophilization of HBsAg in the presence of glucose, sucrose and trehalose, but not mannitol, contributed to preserve HBsAg size and structure (even after 2 years of storage at 48C). These sugars may replace the water around polar regions in membrane phospholipids and proteins, maintaining their integrity in the absence of water [14]. Vega et al. [15] reported no size or structural changes in yeast-derived HBsAg particles stored at 48C and 288C for 10 months. However, in those studies, freeze-drying of the particles caused aggregate for-

13

mation Ð a phenomenon not observed in the present study of CHO-HBsAg. The stability of particle size correlates, to some extent, with the stability of immunological potency, thereby supporting the conclusion reached in the Hansenula-HBsAg/CHO-HBsAg study [2] regarding the requirement for particulate structure. 4.2. Chemical stability 4.2.1. Total protein and peptide composition The total protein content of aqueous HBsAg particle dispersions was unchanged after 18 months of storage at ÿ208C and 48C, but was reduced after storage at the higher temperatures. The protein content of samples lyophilized in the presence of various sugars and stored at 48C for 2 years did not change. After 6 months storage, HBsAg stored as aqueous dispersion at ÿ208C and 48C and as a lyophilized powder at all four temperatures, fully retained the original peptide composition, while after storage at room temperature and 378C the amount of M protein was reduced sharply, and new low- and high-molecular weight bands appeared in the aqueous dispersions. Lyophilization of HBsAg in the presence of 10% sucrose improved the stability of the particle peptides, since only after 6 months at all four temperatures did the M protein content decrease sharply and the S protein content increase. The M peptide of the HBsAg peptides is the most sensitive to enzymatic cleavage (data not shown), perhaps due to its location on the surface of the particles [16,17]. The M protein can be cleaved to yield S peptides and another low molecular weight peptide, thus explaining the increase in the amount of S protein. Addition of desferal or diethylenetriaminepentaacetic acid (DTPA) may prevent the protein degradation and oxidation. The appearance of high molecular weight species, especially after storage at room temperature or 378C for 18 months, are a result of covalent (but not disul®de) bonds between the peptides. It is not yet clear if this covalent oligomerization of the HBsAg peptides is only intraparticle, or if interparticle oligomerization occurs as well (especially in aggregated particles). 4.2.2. Total phospholipids The amount of HBsAg lipid was signi®cantly in¯uenced by the storage conditions. The phospholipid phosphorus content of HBsAg particle dispersions sharply decreased after 18 months of storage at the four temperatures studied. The phospholipid phosphorus content of HBsAg lyophilized with 10% sucrose and stored at ÿ208C and 48C did not change; likewise, that of alum-formulated HBsAg remained stable after 1 year of storage at 48C.

b

Except for disappearance of C18:2 and C20:4. After 2 years. c After 1 month. d After 1 year. e After 30 months.

a

HBV vaccine Lyophilized HBV vaccine

Lyophilization

Lyophilization+10% sucrose

Q Q Q Qb -b -b Qb -b N.D.

Temperature

48C ÿ208C Room temperature 378C 48C ÿ208C Room temperature 378C No sugar 5% glucose 10% trehalose 10% mannitol 10% sucrose 48C ÿ208C

Size and shape

Parameter

q q -b -b -b -b -b N.D.

Total protein Q Q Q Q Q Q Q Q -c -c -c -c -c -c N.D.

S q q q q q q q q -c -c -c -c -c -c N.D.

M q q q q q q q q -c -c -c -c -c -c N.D.

L

Peptide composition

Q Q Q Q Q Q Q Q N.D. N.D. N.D. N.D. N.D. N.D. N.D.

New peptides q q q q q q N.D. N.D. N.D. N.D. N.D. -d N.D.

Total phosphorus

Phospholipid composition + N.D. N.D. N.D. N.D. N.D. N.D. N.D.

Lipid acyl chain composition -a -a -a -a -a -a N.D. N.D. N.D. N.D. N.D. N.D. N.D.

Table 6 The e€ect of 18 months storage of HBsAg particles and HBV vaccine. - no changes; + change; Q increase; q reduction; N.D. - not determined

q q q q q q q q N.D. N.D. N.D. N.D. N.D. N.D. N.D.

Free cholesterol

N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. +b N.D. N.D. N.D. +b -e qb

Immunogenicity

14 D. Diminsky et al. / Vaccine 18 (2000) 3±17

D. Diminsky et al. / Vaccine 18 (2000) 3±17

4.2.3. Phospholipid composition The phospholipid composition of HBsAg lyophilized with 10% sucrose and stored at ÿ208C was stable. The PC and PE content of all liquid dispersions decreased to a minor extent during the 18-month storage period. 4.2.4. Lipid acyl chains The pro®le of the lipid acyl chain composition did not change signi®cantly during storage at di€erent temperatures, except for the loss of oxidation-sensitive C18:2 and C20:4 polyunsaturated acyl chains. The content of these two acyl chains was unaltered only when aqueous dispersion or lyophilized powders were stored at ÿ208C. 4.2.5. Free cholesterol The cholesterol content of the particles remained constant for 18 months when aqueous dispersions or freeze-dried powder were stored at ÿ208C. All other storage conditions decreased the cholesterol content, especially in the aqueous dispersion preparations. The amount of free cholesterol in alum-formulated HBsAg vaccine was completely preserved during at least 1 year of storage at 48C. 4.2.6. Chemical stability-related conclusions All variables tested were a€ected by storage conditions to various degrees. In general, storage at ÿ208C retained the gross and the ®ne chemical composition of the HBsAg particle peptides and the lipids. The stability follow-up suggests that the HBsAg particles are best stored as a powder at ÿ208C (after lyophilization in the presence of 10% sucrose). The relative order of storage conditions conducive to HBsAg particle stabilization is: lyophilized, stored at ÿ208C>lyophilized, stored at 48C>lyophilized, stored at room temperature=lyophilized, stored at 378C>frozen aqueous dispersion, stored at ÿ208C> aqueous dispersion, stored at 48C>aqueous dispersion, stored at room temperature>aqueous dispersion, stored at 378C. This is summarized in Table 6. The relevance of the various aspects of HBsAg particle chemical instability to the immune potency will be discussed below. 4.3. Stability of immune potency The central characteristic of any study concerned with the development of a product is performance stability. In the present study, this concerns immunological potency as measured by antibody titers following injection. 4.3.1. CHO-HBsAg particles The antigenic activity of HBsAg particles was determined by ELISA measurement of the S peptide ®rst

15

loop (epitope 1) using the Ho€man-La Roche biotinylated Mab M6/3-IgG2 antibody (see Section 2). This antibody recognizes a sequence epitope as it binds to both the intact HBsAg particles in the ELISA and also to the `soluble' S peptide (after solubilization of the particle by SDS) in the Western blot. Storing of the HBsAg particles at 48C did not decrease for 12 months. The actual shelf life at this temperature is longer since signi®cant changes have not yet occurred (study still in progress). This parameter was signi®cantly reduced after storage at 258C, and even further reduced after storage at 378C. Vega et al. [15] showed no decrease in antigenic activity or purity of yeast-HBsAg (no aggregates or low molecular weight bands of S peptide) following storage for 16 months at 48C. All six HBsAg determinants persist after 3 months of storage regardless of the storage temperature, but the intensities of the 27 kDa (S) band and the 33 kDa (pre-S2) band were reduced following storage at 378C. GoÂmez-GutieÂrrez et al. [18] found that the structure and antigenicity of HBsAg particles puri®ed from plasma remained unchanged after they were stored for 19 h at 378C. Above this temperature, the decrease in antigenic activity paralleled the conformational changes induced by temperature. Circular dichroism showed a cooperative transition with a midpoint at 498C. The conformational changes reduced the helical content of HBsAg S proteins from 49% at 238C to 26% at 608C and abolished the antigenic activity. It seems that antigenic determinants are dependent on the structure of HBsAg and on the dynamics and organization of the lipid. An examination of the e€ects of temperature and time on discrete antigenic determinants showed that storage at 48C for 9 months does not lead to any degradation of the six determinants. GoÂmez-GutieÂrrez et al. [18] showed that the transition temperature of conformational changes, which alters the secondary structure of the antigenic determinants and abolishes binding of the monoclonal antibodies, is between 48±528C. Lelie et al. [19] observed enhanced HBsAg immunogenicity after heat treatment. This phenomenon may have been due to enhanced exposure of particular antigenic determinants resulting from higher lipid ¯uidity caused by elevated temperature. 4.3.2. HBV vaccine Evaluation of seroconversion in mice after injection of alum-formulated CHO-HBsAg vaccine indicated that the vaccine is very immunogenic, with ED50 of 0.07. No loss in vaccine potency was observed during 30 months of storage at 48C. Studies with yeast-derived HBsAg vaccines demonstrated that they were as potent as human plasma-derived vaccine in stimulating antibodies in mice for at least a year. However, the ED50 of these vaccines is much higher (ED50=0.6)

16

D. Diminsky et al. / Vaccine 18 (2000) 3±17

[20,21]. Exposure of CHO-HBsAg vaccine to elevated temperatures (258C and 378C) during storage for 2 weeks did not alter the vaccine's potency. In conclusion, this study describes the physical, chemical and immunological limits of the stability of CHO-HBsAg particles and of the alum-vaccine derived from these particles. The naked particles can be stored either as an aqueous dispersion for 30 months or in a freeze-dried form in the absence or presence of sugars (except mannitol). It seems that in spite of the changes in the peptide composition and lipid composition of HBsAg particles caused by storage under di€erent conditions, the immunopotency was not reduced. Exposure of HBsAg naked particles to phospholipase-C treatment (which hydrolyzes most phospholipid headgroups) or to termolizin treatment (to cleave the proteins) did not a€ect the spherical structure of the HBsAg particles; while the immunopotency of these HBsAg-treated particles was even enhanced (unpublished data). Together, these ®ndings suggest that the particulate structure of HBsAg is required and that it has a strong in¯uence on immunogenicity. This claim is supported by recent experiments of Schirmbeck et al. [22,23] who showed that solubilization of HBsAg particles and injection of the S-monomer into mice did not stimulate an antibody response against the solubilized S-peptide. The relationships between HBsAg particle aggregation, peptide covalent (non-S±S-) oligomerization and the particle immunogenicity are still unclear. The chemical stability of the HBV vaccine (alumadsorbed HBsAg particles) is better than, and the immunological stability is similar to, those of naked HBsAg particles stored in their naked form and formulated immediately before the vaccination. However, the HBV vaccine (but not naked HBsAg particles) completely loses its immunological potency upon freezing or freeze-drying. This study suggests that the vaccine is stable enough to facilitate its application and distribution even under suboptimal environmental conditions.

[2]

[3] [4]

[5] [6] [7]

[8]

[9] [10]

[11] [12]

[13]

[14] [15]

Acknowledgements This study was supported in part by EC Biotech Contract No. PL 97-0002 grant to Y.B. Dr. Y. Guy from BioTechnology General, Rehovot and Mr. S. Geller are acknowledged with pleasure for editing, and Mrs. B. Levene for typing the manuscript.

[16] [17] [18]

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[22] Schirmbeck R, Melber K, Mertens T, Reimann J. Antibody and cytotoxic T-cell responses to soluble hepatitis B virus. J Virol 1994;1418±25. [23] Schirmbeck R, Melber K, Mertens T, Reimann J. Selective stimulation of murine cytotoxic T-cell and antibody responses by particulate or monomeric hepatitis B virus surface (S) antigen. Eur J Immunol 1994;24:1088±96.