Inflexal®V a trivalent virosome subunit influenza vaccine: production

Vaccine 20 (2002) B17–B23

Inflexal®V a trivalent virosome subunit influenza vaccine: production Robert Mischler∗ , Ian C. Metcalfe Berna Biotech Ltd., Rehhagstr. 79, CH-3018 Berne, Switzerland

Abstract Inflexal® V, a novel virosome-based trivalent influenza vaccine, has been shown to be highly immunogenic and well tolerated in children, young adults, and the elderly. Here we discuss the techniques for the manufacture of Inflexal® V, highlighting the purity and consistency of the manufacturing process. Key factors to be taken into account in the construction of Inflexal® V are the retention of the natural presentation of antigens, its biodegradability and the presentation of few adverse events. The constituents of the vaccine were also carefully considered based on suitability for human use, adjuvanticity and an innate lack of toxicity. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Inflexal® V; Subunit influenza vaccine; Production

1. Introduction Influenza virus is the cause of costly annually recurrent respiratory tract infections. The socio-economic burden of this disease is reflected in the high incidences of morbidity and mortality, especially in risk groups such as the elderly [1,2]. To date, prophylactic vaccination for influenza has been based on formalin or ␤-propiolactone inactivated whole-virus vaccines, subunit or split-virus vaccines [3]. Through parenteral administration traditional influenza vaccines have been shown to stimulate high levels of serum antibody response [4]. However, the resistance induced is frequently temporal and insufficient, particularly in the elderly [5,6]. In addition, these vaccines tend to be a trade off between immunogenicity and tolerability. For example, inactivated whole viruses show high immunogenicity, but this is accompanied by high reactogenicity, making them unsuitable for use in anyone except healthy adults [7]. Subunit and split-virus vaccines are less reactogenic but are also less immunogenic. As a result several adjuvants have been developed for the administration of these vaccines, including alum based compounds, emulsions (e.g. MF59 [8]), (lipophilic immune stimulating complexes (ISCOMS) containing Quil A adjuvant [9]) and liposomes. A development of the liposomal technique has been the use of immunopotentiating reconstituted influenza virosomes (IRIVs) as antigen delivery systems [10] and has been proven to be highly effective, with two licensed products on the market.

∗

Corresponding author. Tel.: +41-31-980-6600; fax: +41-31-980-6785. E-mail address: [email protected] (R. Mischler).

2. Immunopotentiating reconstituted influenza virosomes The IRIV vaccine delivery system is comprised of spherical unilamellar vesicles with a diameter of approximately 150 nm. Particular attention was paid to the components and processes selected to formulate the IRIVs. Key factors taken into consideration were suitability for human use, adjuvanticity and an innate lack of toxicity [11]. The main constituents of IRIVs consist of naturally occurring phospholipids (PL) and phosphatidylcholine (PC). Previous uses of PC have included numerous pharmaceutical preparations, specifically for malnutrition treatment through oral and intravenous solutions. PC has been shown to be non-immunogenic even when combined with potent adjuvants [11] and forms approximately 70% of the virosomal structure. The remaining 30% of membrane components are composed of envelope phospholipids originating from the influenza virus used to provide neuraminidase (NA) and haemagglutinin (HA) glycoproteins. The NA can readily intercalate into the phospholipid membrane and is a tetramer composed of four equal, spherical subunits hydrophobically embedded in the IRIV membrane by a central stalk. The influenza HA intercalated into the phospholipid bilayer acts to stabilise the liposome base by preventing fusion with other liposomes. It is also the major antigen of influenza virus, containing epitopes on both HA1 and HA2 polypeptides. Furthermore, HA is responsible for the fusion of the virus with the endosomal membrane [12,13]. The NA present on the IRIV’s surface aids its action by the same mechanisms through which it enhances influenza virus pathogenicity. NA catalyses the cleavage of

0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 2 ) 0 0 5 1 2 - 1

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N-acetylneuraminic acid (sialic acid) from bound sugar residues [14] resulting in a decreased viscosity of the host’s mucus and allowing the influenza virus easier access to epithelial cells. The same process leads to destruction of the HA receptors within the cell membrane to which viruses and IRIVs bind (see below). This allows the virus particles to avoid aggregation, as newly formed virus particles do not adhere to the infected host cell membrane after budding, allowing the influenza virus to retain its mobility. The actions of NA greatly enhance the infectivity of the virus and therefore the action of virosomes. With IRIVs the actions

of NA may be useful, as, after coupling with HA, IRIVs not absorbed into the cell by endocytosis can be cleaved off to potentially react with alternative cells. As an additional benefit the reduction of viscosity in the host’s mucus may prove useful in the development of an intranasal vaccine. The mode of action of IRIVs is dependent on the influenza HA which is intercalated into the liposomal bilayer. The HA is formed from two polypeptides, HA1 and HA2, that are responsible, upon conformational change, for the fusion of the virus with the endosomal membrane. The HA1 globular head contains a receptor site that has a high affinity for sialic

Fig. 1. Inflexal® V production procedures. The constituents, shown on the left, were selected for suitability for use in vaccines for humans and an innate lack of toxicity. The process validation for Inflexal® V is based on the complete inactivation of the influenza viral strains (assessed by bioburden), as well as the HA:PL ratio, the purity of the influenza active ingredients and the consistency of particle size; the test procedures for validation are displayed on the right.

R. Mischler, I.C. Metcalfe / Vaccine 20 (2002) B17–B23

acid, present in relatively high concentrations on the surface of antigen presenting cells (APCs), such as macrophages and lymphocytes, and therefore facilitates the binding of the IRIV. The actual fusion of the IRIVs with the endosomal membranes is mediated by the HA2 polypeptide. The low pH of the host cell endosome (approximately pH 5.0) produces a conformational change in the HA2, exposing its fusogenic peptide, that is a prerequisite for fusion to occur. The fusion mechanism of IRIVs enables stimulation of the MHC I or II class pathway, depending upon how the antigens are presented to the APCs. Antigens linked to the surface of virosomes are degraded upon endosomal fusion within the endosome and are presented to the immune system by MHC

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II class receptors. Antigens encapsulated in the virosomes are delivered to the cytosol during the fusion event, thus entering the MHC class I pathway. Therefore, virosomes are able to induce either a B- or T-cell response. IRIVs can be produced on a large scale, with lot sizes of up to 500,000 doses as standard, the procedure for which is shown in Fig. 1 [15]. Initially the monovalent whole-virus pools are separately diluted with a phosphate buffer saline (PBS) solution and ultracentrifuged. The pellet of the whole virus is then solubilised with the detergent octaethyleneglycol (OEG). The solutions are then ultracentrifuged for a second time and the supernatants are mixed with lecithin and solubilised. The preparations are then put through a

Table 1 Virosomal pool production for the three monovalent virosomes Lot no.

HA (g/ml)

PL (g/ml)

Ratio HA:PL (1:X)

Bioburden (microorganisms/ml)

A strain (H1N1) A/New Caledonia/20/99-like (IVR-116) VP 1 524.5 Negative VP 2 587.7 Negative VP 3 597.0 Negative VP 4 545.8 Negative VP 5 521.4 Negative VP 6 494.4 Negative VP 7 507.4 Negative VP 8 487.0 Negative VP 9 556.0 Negative VP 10 641.0 Negative

1446 1439 1499 1328 1217 1295 1367 1563 1472 1549

2.8 2.4 2.5 2.4 2.3 2.6 2.7 3.2 2.6 2.4

0 0 0 0 0 0 0 0 0 0

Mean S.D.

1418 107

2.6 0.2

0 0

A strain (H3N2) A/Moscow/10/99-like (A/Panama/2007/99) VP 1 548.0 Negative VP 2 594.0 Negative VP 3 524.8 Negative VP 4 616.0 Negative VP 5 637.0 Negative VP 6 542.0 Negative VP 7 538.0 Negative VP 8 546.0 Negative VP 9 570.0 Negative VP 10 594.0 Negative

1407 1495 1405 1420 1576 1440 1485 1506 1470 1521

2.6 2.5 2.7 2.3 2.5 2.7 2.8 2.8 2.6 2.6

0 0 0 0 0 0 0 0 0 0

Mean S.D.

1473 52

2.6 0.1

0 0

B strain B/Beijing/184/93-like (B/Yamanashi/166/98) VP 16 482.0 Negative VP 17 480.0 Negative VP 18 525.3 Negative VP 19 551.1 Negative VP 20 569.0 Negative VP 21 549.0 Negative VP 22 524.0 Negative VP 23 483.0 Negative VP 24 557.0 Negative VP 25 594.0 Negative

1404 1534 1453 1565 1605 1544 1499 1502 1553 1558

2.9 3.2 2.8 2.8 2.8 2.8 2.9 3.1 2.8 2.6

0 0 0 0 0 0 0 0 0 0

Mean S.D.

1522 56

2.9 0.2

0 0

546.2 47.1

571.0 35.6

531.4 37.7

OEG

– –

– –

– –

Bioburden (microorganisms/ml) is assessed after the second ultracentrifugation and subsequent solubilisation with lecithin (Fig. 1). The haemagglutinin:phospholipid (HA:PL) ratio and presence of PL (g/ml), OEG and HA (g/ml) are quantified from the virosomal pools formed after sterile filtration, batch-chromatography and dilution with PBS.

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sterilising filtration process followed by batch-chromatography and dilution with PBS, the result of which is a virosomal pool (VP). The VPs are then analysed for phospholipid content, viral neuraminidase (NA) and haemagglutinin (HA) content, purity, OEG and endotoxin presence, sterility, virosome size and ratio of LAL:15 g HA and HA:PL. The virosomal pools from the WHO recommended influenza virus strains are then pooled to form a final bulk.

3.

Inflexal® V

Berna

The use of virosomes to deliver influenza antigens to the endosome and stimulate a strong immune response of immunocompetent cells forms the basis of Inflexal® V Berna. This parenterally administered trivalent virosome influenza vaccine has been licensed in Switzerland since 1997 and is currently licensed in 25 countries worldwide. The actual composition of Inflexal® V consists of a mixture of three monovalent virosome pools, each formed with one influenza strain’s specific HA and NA glycoproteins. The influenza strains chosen are dependent on the yearly recommendations of the WHO. Inflexal® V has an exemplary safety and efficacy report with more than 2500 healthy volunteers vaccinated in 18 clinical trials. This vaccine possesses excellent tolerability in children and more than 10 million vaccine doses have been administered. Following the production techniques used for individual monovalent concentrates of the IRIVs, each of the three monovalent influenza virus strain bulks, currently H1N1, H3N2 and B, are used as the starting material for the manufacture of Inflexal® V. Due to the nature of the vaccine’s production, the VPs can be used for two successive seasons, so long as the corresponding influenza strain is again recommended by the WHO, and the stability has been demonstrated. The manufacturing process for Inflexal® V is comprised of two production blocks: purified and inactivated influenza whole-virus antigen, and the formulation of the influenza glycoprotein subunits. Particular attention is paid to confirm the complete inactivation of each seasons’ new influenza strains. From each new strain an inactivation kinetic is performed to guarantee the inactivation of the bulk influenza virus. The process validation for Inflexal® V is also based on the haemagglutinin to phospholipid ratio, the purity of the influenza active ingredient and the consistency of particle size. The lot consistency and purity of each of the virosomal products is represented in Table 1. It can be seen that, throughout 10 consecutive lots for each of the 3 influenza strains chosen, there is very little variation in the ratio between HA and phospholipids which form the virosomes (mean ratio HA:PL; 1:2.6 and 1:2.9 for the ‘A’ and ‘B’ influenza strains, respectively). In addition, the data in Table 1 also show the virosome pools to be free from detergents. Detergent (OEG) presence is based on <50 mg per human dose, this is compared to some products claiming to be de-

tergent free at <500 mg per human dose. In addition the sensitivity of the tests used for detergent presence can detect as little as 54 ␮g per human dose. The high purity of the influenza surface glycoproteins present in Inflexal® V has Table 2 Size distribution of virosomal pools Lot no.

Mean size (nm)

A strain (H1N1) A/Beijing/262/95-like (X-127) VP 1 232.6 VP 2 189.5 VP 3 170.2 VP 10 179.9 VP 11 200.5 VP 12 172.6 Mean S.D.

190.9 21.28

A strain (H1N1) A/New Caledonia/20/99-like (IVR-116) VP 1 165.5 VP 2 171.9 VP 3 163.5 VP 4 163.6 VP 5 181.3 VP 6 150.5 Mean S.D.

166.1 9.32

A strain (H3N2) A/Sydney/5/97-like (Resvir-13) VP 1 235.9 VP 2 183.6 VP 3 188.4 VP 10 269.9 VP 11 258.8 VP 12 182.7 Mean S.D.

219.9 36.43

A strain (H3N2) A/Moscow/10/99-like (A/Panama/2007/99) VP 1 178.5 VP 2 129.0 VP 3 151.6 VP 15 183.9 VP 16 142.2 VP 17 155.1 Mean S.D.

156.7 19.24

B strain B/Beijing/184/93-like (B/Harbin/7/94) VP 1 211.6 VP 2 178.4 VP 3 198.8 Mean S.D.

196.3 13.67

B strain B/Sichuan/379/99-like (B/Victoria/504/2000) VP 1 165.5 VP 2 157.7 VP 3 154.8 Mean S.D.

159.3 4.52

Although variation occurs between strains, the data show that virosomes of the same stem have minimal variation in particle size. Particle size is assessed through light scattering techniques before the VMVPs are mixed to form the Inflexal® V final bulk.

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Table 3 Production control of Inflexal® V final product Lot no.

(H1N1) HA (g/ml)

(H3N2) HA (g/ml)

HA (g/ml)

Protein (g/ml)

Ratio HA:protein (1:X)

OvA (ng/ml)

Ratio (ppm OvA/1 g HA)

Endotoxin (IU/ml)

Thiosermal (g/ml)

00IVFB28 00IVFB29 00IVFB30 00IVFB31 00IVFB32 01IVFB38 01IVFB49 01IVFB40 01IVFB41 01IVFB42

32.7 36.7 35.7 34.6 32.4 35.0 32.1 32.8 33.0 35.7

32.6 33.1 32.1 33.8 33.9 34.4 35.0 32.9 34.2 32.1

32.8 31.8 36.9 32.6 31.8 33.7 31.1 32.1 31.8 35.1

190 140 140 150 170 130 120 150 150 150

1.9 1.4 1.3 1.5 1.7 1.3 1.2 1.5 1.5 1.5

4 4 5 5 4 4 4 3 3 3

40.8 39.4 47.8 49.5 40.8 38.8 40.7 30.7 30.3 29.2

1 1 1 2 1 1 1 0 1 2

Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

Mean S.D.

34.07 7.57102

33.41 0.94705

32.97 1.70414

149 18.68154

1.5 0.2

3.9 0.7

38.8 6.6

1.1 0.56765

– –

After combination of the VMVPs, tests for each strains’ HA content (g/ml), protein content, ratio of ppm ovalbumin (OvA):1 g HA, and presence of ovalbumin (ng/ml), endotoxin (IU/ml) and thiosermal (g/ml) are quantified.

been ascertained by polyacryl gel electrophoresis and total protein determination. The results of the gel electrophoresis have fulfilled all the requirements outlined by the WHO and European Pharmacopoeia. Particle size may be used as a determinant of a consistent production process. Table 2 represents the data from various lot numbers of each of the three influenza strains that presently constitute Inflexal® V. The size variation between viral strains is not reflected within the specific strain pools, as can be seen from the data in Table 2, within strains the particle size remains in a well defined range. However, due to annual changes of influenza viruses, as recommended by the WHO for influenza vaccines, the amino acid composition of the HA and NA will change resulting in variability in the mean diameter of virosome particle size. This variability means it is impossible to rely on a fixed particle size and therefore, lot release has not been made dependent on the results of light scattering measurements even though it is measured in each lot as part of the summary protocols. The analysis of the trivalent final bulk vaccine is shown in Table 3. Here it can be seen that the presence of ovalbumin (OvA) (detected to levels of 0.313 ng per dose) and endotoxin fall well within the safety limits imposed by the authorities. For Inflexal® V, OvA presence is more than 500 times less than the maximum OvA allowed (2 ␮g/ml) with

a mean of 3.9 ng/ml. In addition, up to 200 IU/ml of endotoxin is allowed in the final vaccine and Inflexal® V consistently shows 200 times less than this limit with only 1 IU/ml. Table 3 also shows the consistency of the HA presence for each of the viral strains included in the final product for each of the lots detailed. For the 10 lots shown the HA (g/ml) has a standard deviation of not more than 1.7 g/ml for any of the three viral strains. This is in addition to a mean HA to protein ratio of 1:1.5, displaying the high purity of the final vaccine bulk (the minimum HA:total protein ratio is 1:3.6). When compared to other influenza vaccines (Table 4) it is important to note that the levels of both detergent and OvA in the final vaccine are lower in Inflexal® V than the comparable split and subunit vaccines available. Table 4 shows Inflexal® V to have adequate presentation of HA for each of the virus strains indicated (HA A/NC 34.3 g/ml; HA A/Pa 30.9 g/ml; HA B/Ya 32.1 g/ml). Negative results for detergent and less ppm OvA/1 g HA than the alternative vaccines also place Inflexal® V in a very favourable light. An important consideration in the production of a vaccine is its stability under varying storage conditions. The monovalent virosome pools for each viral strain forming Inflexal® V have been shown to maintain high levels of HA content over a period of 24 months (Fig. 2). Additional stability reports, required for the final bulk vaccine, are based on the

Table 4 Comparison of different influenza vaccines compositions (influenza season 2000–2001) Manufacturer supplying vaccine

Vaccine type

Lot no.

HA A/NC (g/ml)

HA A/Pa (g/ml)

HA B/Ya (g/ml)

Detergent

HA (ppm OvA/1 g)

Virosome size (nm)

1a 1b 2a 2b 3 4

Split Split Split Subunit (adjuvanted) Subunit Inflexal® V

18514C9 18531A9 030021 1402 R-0805 00IVFB32

32.8 31.8 10.1 34.2 34.3 34.3

35.1 33.1 14.5 31.9 28.9 30.9

30.2 30.3 10.0 32.7 32.6 32.1

Positive Positive Negative Negative Negative Negative

100 63 404 1190 750 51

122.1 12.7 252.8 178.6 93.4 162.9

The results show a minimal presence of ovalbumin per 1 g HA, negative detection of detergent (<50 mg per human dose) and comparable presence of HA for each strain in Inflexal® V versus alternative vaccines.

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Fig. 2. Stability of virosome pools for Inflexal® V identified by influenza virus strain. The loss of HA content from the virosome pools is below 10% for all influenza virus strains over a 24-month period conforming to the requirements for stability.

Table 5 Stability report results for Inflexal® V final bulk product (2000–2001) Time

Baseline 18 months at 5 ◦ C 3 months at 25 ◦ C 28 days at 37 ◦ C

LAL (IU/ml)

pH

1 <0.5 – –

7.4 7.4 7.4 7.4

Phospholipid content (mg/ml)

Viral HA content A/New Caledonia (g/ml)

A/Panama (g/ml)

B/Yamanashi (g/ml)

0.283 0.268 0.295 0.260

36.7 32.6 30.6 26.8

33.1 30.5 33.1 27.8

31.8 34.5 30.2 29.1

Sterility

Virosome size (nm)

Sterile Sterile Sterile Sterile

171.5 178.5 174.4 172.9

The results show the long-term stability of Inflexal® V when stored at 2–8 ◦ C over 18 months. In addition, when stored under stress conditions Inflexal® V has also been shown to remain stable for 3 months at 25 ◦ C and 28 days at 37 ◦ C.

following seven criteria: endotoxin levels (200 IU/ml), pH (6.5–7.8), phospholipid content (0.08–0.40 mg/ml), potency (LCL 95% ≥ 24 g HA/ml), sterility, virosome size (nm). The long-term stability of Inflexal® V packaged in syringes has been monitored over 18 months at 5 ◦ C, 3 months at 25 ◦ C and 28 days at 37 ◦ C. The results showed that at conditions of 2–8 ◦ C Inflexal® V is stable for a period of at least 18 months. In addition, when tested under accelerated and stress conditions Inflexal® V has been shown to be stable for at least 3 months when stored at 25 ◦ C and 28 days at 37 ◦ C (Table 5).

4. Conclusions In summary the improved immunological action of the trivalent virosomal influenza vaccine, Inflexal® V originates from three factors: • high purity of the influenza glycoproteins intercalated into the phospholipid bilayer; • preserved biological activity of the HA molecule and • the natural presentation of antigens. The analysis of the data represented here describes a consistent and stable procedure for the production of an efficacious and well tolerated trivalent virosomal influenza vaccine, Inflexal® V.

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