Worldwide vaccination programmes have seen considerable progress in fighting diseases but instability and degradation of the active ingredient can be a major hurdle to their use in developing countries. Martin Scholz (CSO) and Wolfram Altenhofen (VP Business Development), Leukocare, describe a novel approach to stabilising vaccines.
Instability and degradation of the ‘active ingredient’ continue to be major drawbacks in the production, transport and storage of vaccines. A broad range of biophysical methods to characterise vaccine formulations are currently being investigated and it is obvious that there is no single answer to the existing challenges on the formulation side. Finding an ‘optimal’ formulation is still largely empirical.
Researchers at Leukocare, however, have developed a novel approach to stabilising vaccines that has its roots in stabilising biologics in medical device combination products and even allows for terminal sterilisation of biologics by irradiation or ethylene oxide (EtO). The results from the initial investigation into this Stabilising and Protecting Solutions (SPS) platform show that it also provides a powerful way of stabilising vaccines.
Vaccination is undoubtedly the most important disease-preventing tool in global healthcare. There is no other class of pharmaceuticals, however, that is more diverse and more complex in terms of formulation requirements. Active ingredients can be live attenuated, genetically modified live, inactivated, synthetic peptide-based, nucleic acid or subunit vaccines1-3 and each class defines a chemical universe in itself. Also, access to vaccines is still largely constrained by the need to have a cold chain in place.
Finding formulations that are tolerant to moderate stress conditions has obvious merits in terms of coverage, especially in second and third world countries.4 Increasing effort is, therefore, also put into the development of solid dosage forms that tend to be less sensitive to thermal stress.4
Many of the currently used excipients for solid dosage forms contain sugars or polyols that can have limited stabilising properties.4 It is important to note, therefore, that the SPS platform of excipients is free of sugar and polyols. This, among other specifics, allows biologics stabilised by SPS to be submitted to terminal sterilisation by irradiation. Besides increasing product safety and decreasing production costs, terminal sterilisation by irradiation may also be used to inactivate vaccines while preserving the three-dimensional structure of the specific antigen.
Figure 1: Schematic depiction of protecting mechanisms elicited by the Leukocare technology. As an example, an IgG antibody is embedded in the stabilising and protecting solution (SPS) (A). After drying, the small molecules form a protective layer around the target molecule that prevents irradiation and stress mediated damage (B)
developed for medical devices
Leukocare, a clinical stage biotechnology company based in Germany, developed the SPS technology in the context of surface modifications of medical devices using biologics such as antibodies or growth hormones.
Antibody-coated catheters can be used to collect circulating tumour or foetal cells for diagnostic purposes; and growth hormones coupled to the surfaces of dental or orthopaedic implants have been shown to significantly improve the bio integration. Major challenges in the production of biologically functionalised devices are to maintain the functionality of the pharmacologically active biologic – typically in absence of water – to ensure reasonable shelf-life for successful commercialisation, and to enable terminal sterilisation of the combination product.
The specific composition developed by Leukocare consists of five to seven different small molecule type excipients, including a rigid amphiphilic molecule, is free of proteins, sugars, and salts that are known to have limited stabilising properties,4 and can be adjusted to the specific requirements of the biologic.
The constituents are 100% GRAS, available in pharmaceutical grade and do not exhibit pharmacological or toxicological effects; a drug master file is currently being prepared.
Compared with standard lyophilisation buffers containing sugars and polyols, the cake properties, reconstitution, glass transition temperatures and the amorphous characteristics of the excipient were found to be at least similar, if not superior. Protein integrity after sterilisation was far superior to standard formulations and the functional activity proved to be significantly better.
Mechanistically, SPS works by replacing stabilising interactions between the protein and water with similar interactions of less reactive small molecules (Figure 1A). On removal of water these molecules form an amorphous coating (Figure 1B) that protects the substrate against physical destabilisation during storage (Figure 2) and against the destructive impact of irradiation or EtO (Figure 3). This technology allows embedding of biologics, including antigens for vaccine production, in an amorphous layer that substitutes for the natural hydration shell. Even though primarily developed for dry formulations, SPS also stabilises biologics in liquid storage.
Figure 2: SPS-mediated prolonged stability of a highly instable IgM antibody during storage. Only a minor loss in functionality occurs when the protein is embedded in SPS versus a standard formulation
vaccine applications
Several studies provided significant evidence that SPS is applicable to:
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a) the safe generation of antigens for vaccine production
b) the stabilisation of dry vaccines during thermal and irradiation stress
c) the stabilisation of vaccines in liquid formulation
Safe generation of antigens for vaccine production: As outlined above, the SPS-mediated protection of the molecular integrity and functionality of a broad spectrum of therapeutically relevant biologics under various stress conditions had been previously shown. Given the potency of toxins to be masked by vaccination, the requirement to maximise safety and efficacy in vaccines is even more important.
Current production protocols, however, allow for a compromise: for example, the safe generation of influenza A antigens for vaccine development is achieved by treatment of the virus with formaldehyde. Formaldehyde has the disadvantage that it chemically cross-links antigenic structures and aggregates protein structures, as was observed with diphtheria, pertussis, and tetanus toxoids.4-6
On the other hand, irradiation of antigens normally results in destruction of protein structures due to the high energy input.7 By using SPS in conjunction with larger doses of irradiation (40kGy in this case) the company could show that proteins were stabilised and that virus titers were efficiently reduced. Titers of human adenovirus and porcine parvovirus that are used as standards in disinfection studies were reduced by 5-6 log units. Thus, the method is of particular interest for the preparation of stable and safe antigens for vaccine production.
Stabilisation of vaccines during thermal and irradiation stress in dry formulations: Another major issue is the loss of vaccine stability during storage and transport, especially in countries where the cold chain cannot be fully maintained.4 The company investigated the protective effects of its stabilising technology on dried preparations of inactivated influenza A under thermal stress, both with and without previous irradiation and with subsequent storage.
Figure 3: Example for the protection of biologics by SPS during and after beta-, gamma-irradiation, and after EtO sterilisation
Suitable methods for drying a viral or bacterial preparation include, for example, lyophilisation (freeze-drying), spray-drying, freeze-spray drying, air drying or foam drying.8
Virus activity, as shown by the hemagglutination properties of influenza A, was significantly reduced after drying and reconstitution, during irradiation, and storage at 40°C when the standard formulation was used. In contrast, virus that had been previously rebuffered in the SPS protecting solution remained active under the stress conditions mentioned above. The effect of SPS on the stability of adjuvants9 is currently being investigated.
Stabilisation of vaccines in liquid formulation: SPS technology was also applied successfully to enhance the stability of liquid live virus formulations: highly unstable enveloped DNA viruses such as Herpes Simplex Virus-1 (HSV-1) were studied under different thermal stress conditions. Given that pH, ionic strength, osmolarity and the type of excipients are considered carefully,4 repeated freeze (-20°C) thaw experiments, storage at 4°C, at room temperature and at 40°C resulted in a significantly higher virus titer over prolonged storage time compared with standard formulations.
| Table 1: Various applications for SPS technology | |
| Field | Applications |
| Medical devices | Orthopaedic and dental implants |
| Stents | |
| Wound dressings | |
| Catheters | |
| Extracorporeal blood treatment devices | |
| In vitro and in vivo diagnostics and biochemical research products | Biofunctionalised micro- and nanoparticles |
| Diagnostic devices | |
| Cell culture products | |
| Protein arrays and microtiter plates | |
| Biopharmaceuticals | Therapeutic antibodies, vaccines and other proteins (lyophilised, spray-dried, micro-crystallised or other finished or bulk dry formulations) |
In summary, the SPS technology has been proven to protect and stabilise a broad range of biologics including vaccine relevant antigens as dry and liquid formulations (Table 1). Therefore SPS has a high potential to enable the production of more stable vaccines and other therapeutics in the future. Experiments underlining the stabilising and protecting effects of SPS technology in the field of genetically modified organisms (GMOs) are currently ongoing. GMOs may be used for vaccination against infectious diseases but also against other diseases such as cancer.
references
1. Abdul-Fattah A.M. et al. Pharm Res. 2007, 24:715-27.
2. White D.O. and Fenner F.J. Medical Virology. 3rd ed., Academics, Orlando Fl,1987.
3. Crommelin D.J.A. and Sindelar R.D. Pharmaceutical Biotechnology: An introduction for pharmaceuticals and pharmaceutic scientists, 2nd ed. Taylor and Francis, New York, 2002.
4. Brandau D.T. et al. J Pharm Sci. 2003, 92:218-31.
5. Schwendeman S.P. et al. Proc. Natl. Acad. Sci. USA. 1995, 92:11234-38
6. Bolgiano B. et al. Dev- Biol. 2000, 103:51-9.
7. Garrison W.M. et al. Radiat. Res. 1962, 16:483-502.
8. Chang L. and Pikal M. J. Pharm Sci. 2009, 98:2886-908.
9. Clausi A.L. et al. J Pharm Sci. 2008, 97:2049-61.
