University of Groningen
Drying Made Easy
Kanojia, Gaurav
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Publication date: 2018
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Kanojia, G. (2018). Drying Made Easy: Spray drying a promising technology for the production of stable vaccine and therapeutic protein formulations. University of Groningen.
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Maintaining vaccines and therapeutic proteins stable outside the cold chain remains a chal-lenging ordeal. Spray drying has proven to be a promising technique to tackle this problem by providing dry powder formulations, with improved thermostability. In addition, powder particles can be engineered to desired requirements by spray drying, allowing them to be used by various delivery methods and routes.
The aim of this thesis was to investigate the applicability of the spray drying technique to var-ious biopharmaceuticals, including three vaccines and one monoclonal antibody. The inves-tigations focused on aspects such as the composition of the formulation, process conditions and the preclinical efficacy of the produced powders, after applying them via an alternative route of administration (pulmonary administration). Furthermore, spray drying was com-pared with the more traditional freeze-drying process for a monoclonal antibody (infliximab). Chapter 2 provides an extensive literature review on new developments in the field of spray dried vaccines. Key spray drying process stress elements like shear and dehydration stress were identified and potential solutions to overcome these elements were described. Impact of formulation parameters on vaccine quality during drying were outlined, portraying major excipient categories like small sugars and polysaccharides, surfactants, divalent cations, pro-teins and polymers. Further, the concept of Design of Experiments and its implementation to spray dried vaccines is introduced for systematical study of effect of process variables on product characteristics. Finally, several routes of vaccine powder administration were reviewed, and the challenges related to the delivery of powder vaccine using new delivery devices are addressed.
In Chapter 3 a Design of Experiment (DoE) approach was applied to develop a spray dried whole influenza vaccine (WIV). The approach included systematic screening and optimiza-tion of the spray drying process variables like inlet air temperature, nozzle gas flow rate and feed flow rate which may affect essential vaccine product characteristics (including chemi-cal, physical and biological characteristics). The approach gave a descriptive model of parti-cle size, residual moisture content, outlet temperature and powder yield, including a design space in which trehalose based influenza vaccine powder with a product profile of particle size 1-5 µm, powder yield above 70%, residual moisture content below 3% and loss in HA titers less than 10 %, produced on pilot scale, were described. The produced spray dried influenza vaccine retained its antigenicity, and was thermostable for three months upon stor-age at 60 °C. Finally, the descriptive model was suitable to define and subsequently select process settings to produce a vaccine powder with predefined characteristics, as confirmed by experiments.
Due to limited variation in antigenicity between the different formulations in the design space, no regression model could be fitted on the data and influence of process settings on antigenic recovery could not be assessed. For further research, a broader design space with
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higher drying temperatures would be required to investigate the effect of process settings on influenza vaccine antigenicity. Moreover, analytics providing information on bilayer integ-rity and changes in secondary and tertiary structure of proteins may be included in future studies. The DoE approach used here, could also be applied for other vaccine candidates. Such an exercise would of course include adaptation of the design space towards the specific properties of the vaccine candidate and the desired target product profile, characteristics of the new product. Furthermore, the developed vaccine powder was suitable for pulmonary delivery and could be tested in future in vivo experiments to assess immunogenicity. Overall, the approach described in this thesis has shown to be an excellent tool to screen process con-ditions and effects of excipients to obtain the desired vaccine powder, in a controlled manner with minimal formulation efforts.
Chapter 4 describes an investigation into the spray drying of Sabin inactivated polio vaccine (sIPV). It was found that the composition of the formulation that provided maximum stability varied for each of the three serotypes. Which implies that it is preferred to spray dry each serotype separately followed by mixing the three powders to obtain the trivalent vaccine. Moreover, it was more difficult to stabilize serotype 3 than the serotypes 1 and 2. To eluci-date the contribution of each excipient in stabilizing D-antigen during drying, a fractional factorial design was developed around promising formulations. DoE studies demonstrated that trehalose/monosodium glutamate and maltodextrin/arginine combination were crucial for stabilizing serotype 1 and 2 respectively. For sIPV serotype 3, the best formulation con-tained Medium199, glutathione and maltodextrin.
Challenges with spray drying of serotype 3 were clearly evident as the maximum D-antigen recovery of 56 % was found after spray drying followed by a further loss of 37 % during storage at elevated temperatures (40 °C for a week). Although this is considerably better than fluid serotype 3, which loses all antigenicity at 40 °C for a week, for a feasible process this needs to be improved in future studies, by further identifying and optimizing the components of Medium199 included in the currently identified base formulation. Furthermore, the final product should be optimized for aspects such as particle size and residual moisture content. Finally a the dried powder formulation offers promising opportunities of vaccine delivery via alternative delivery routes [1]. Such powder vaccines have the potential to be used for stockpiling and distribution in developing countries without the need of a cold chain, in cases of future polio outbreaks.Although further improvement is still possible, these findings show the possibility to produce a spray dried vaccine powder based on safer sIPV [with respect to production of virus (no wild- type virus)] [2].
In Chapter 5 the spray drying technology was extended to bacterial vaccines. Developing a spray dried powder for an outer membrane vesicles pertussis vaccine (omvPV) with full re-tention of structural integrity comparable to the liquid formulation. Trehalose (10 % w/v) was used as the stabilizing agent, producing free flowing powders with a mean particle size of ~5
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µm. Drying did reveal some changes in the tertiary structure of proteins, while moderately af-fecting epitope availability of Vag8, yet not for Prn. On storage at 65°C for 4 weeks the anti-body binding epitope of Vag8 antigenicity in powder omvPV was maintained, as determined with a monoclonal antibody, while the liquid omvPV lost its Vag8 epitope within one day of storage. While the vesicle size remained unaffected during storage for both liquid and spray dried products, changes in tertiary structure of the proteins present were more pronounced in the liquid omvPV when compared to the spray dried omvPV. Regardless of changes observed after drying (in vitro), the reconstituted spray dried omvPV when pulmonary administered to mice, induced mucosal IgA and a broad systemic IgG response comparable to liquid omvPV (administered via pulmonary route).
Further work is required to establish the pulmonary delivery of pertussis vaccine as a pow-der, preferably in higher animal models established for pertussis research like baboons [3] or even in humans. Moreover, additional work should be focused on optimizing the formulation for powder delivery in clinical practice with inhaler devices such as Twincer® [4]. Overall, the current work shows promising perspectives for the design of a more stable solid vaccine dosage form prepared by spray drying, for a potential next-generation pertussis vaccine. Finally, the spray drying technology developed so far, was extended to therapeutic proteins. In Chapter 6 the development of a stable spray dried powder of a monoclonal antibody (Infliximab) is described. In prospect of developing an oral dosage form of infliximab, we stabilized it by spray drying and compared it with freeze-drying (vials vs Lyoguard trays). Dextran (6 % w/v) and inulin (6 % w/v) were used in combination with sucrose (5 % w/v) as stabilizing excipients. The drying processes did not affect Infliximab integrity in these formu-lations, i.e. both the physical integrity and biological activity (TNF binding) were retained. Accelerated stability studies (4 weeks at 60°C) showed that the TNF binding ability of In-fliximab was conserved in the dried formulations, whereas the liquid counterpart lost all TNF binding. After thermal treatment, the dried formulations showed some chemical modification of the IgG in the dextran-sucrose formulation, probably due to Maillard reaction products. This observation was limited to storage at elevated temperatures and is not expected to occur at lower temperatures (<25°C).
Recent research [5] have reported the feasibility of infliximab to be formulated into tablets with freeze dried product. However, no attempts were made to investigate its performance in any animal models. Thus, future studies should be directed towards formulating a tablet from the dried powder and evaluating the reduction in TNF locally, when administered via the oral route. For in vivo studies there are several animal models available [6] which can be used for future investigations. To establish the oral route as an alternative for parenteral infliximab administration more clinical studies are required, to prove the safety and efficacy of this route. This study indicated that, with the appropriate formulation, spray-drying may be useful for (bulk) powder production of Infliximab. Finally, the obtained results are a first
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step in the further development of a dosage form of infliximab, stable outside the cold chain for the treatment of patients with Crohn’s disease.
Concluding remarks
The current work addresses the formulation challenges associated with spray drying of in-activated vaccines and therapeutic proteins. Achieving a desired spray dried product was found to be dependent on the biopharmaceutical stabilized by drying. Since it was difficult to predict the behavior of every candidate during drying, any future investigation with new vaccine candidates would require a tailored formulation with optimized drying condition to attain the desired product. Although, one could always skillfully choose the most promising excipients and drying conditions as the starting point for studies with new candidates. How-ever, successful development of a spray dried biological would always require extensive experimentation with formulation and optimization of the process. In addition to formulation and process optimization, analytics for assessing vaccine/antigen quality need to be carefully considered. An extensive set of analytical techniques can be investigated for evaluation of physicochemical and biological activity of antigen/protein of interest. However, this can be challenging when considering diversity of investigated candidates and needs to be select-ed and set as per individual case. Our analytics were fairly specific to proteins of interest (complex omvPV), D-antigenicity (sIPV), HA titers (WIV) and TNF binding and protein aggregation (Infliximab). The extensiveness of such investigations can be expanded or be limited by the scientific question of interest. Considering the viability/replication ability of live attenuated vaccines [7-9], a class of vaccines not studied in this thesis, the formulation challenges associated with their stabilizing by spray drying, could be very different from the one encountered in the current work and would require additional consideration.
The potential of spray dried vaccines to be stored and transported outside the cold chain would simplify vaccine delivery to remote areas and reduce the economic burden of vacci-nation programs [10]. Moreover, production of controlled engineered particles of desirable range gives spray drying the flexibility to produce antigen that can be administered via di-verse routes of administration [11]. This should motivate researchers to include spray drying technologies in their development plans. The best approach for such development could be either an approach in which many excipients are screened in a DoE setup or an approach in which first the degradation mechanism of a vaccine is extensively characterized, followed by a more focused choice of excipients able to inhibit the known degradation pathway(s)[e.g. protein denaturation, membrane disruption, RNA damage etc.]. In addition, the process can also be optimized using a DoE setup. Moreover, the growing scientific interest in the field of particle engineering and new nano and micro technologies could add to the advancement of spray drying and needle free approaches for vaccination.
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To date, there are no marketed spray dried vaccines. However, several spray dried vaccines are in the pipeline and some powder vaccines are in early clinical trials [12]. Recent advances with asceptic spray drying of biopharmaceuticals [13] has attracted interest from the industry to apply spray drying for stabilization of vaccines. An important aspect that needs to be ad-dressed is technology to accurately fill vaccine powder doses in final containers. Technology used for filling spray dried inhalable insulin product into 1 and 3 mg dose blisters exists on a manufacturing scale, but application of this technology for vaccines including validation is still lacking (at least in literature). Any future use of spray drying for drying vaccines and therapeutic proteins would require addressing the concerns of upscaling, biosafety, regulato-ry, fill and finishing aspects.
In conclusion, the research in this thesis presents the strategies to address the challenges associated with formulation of vaccines and therapeutic proteins via spray drying. With new opportunities and challenges arising at every step of formulation development with spray drying, it can be said that there is still a lot to learn and improve in the near future.
1. Kraan, H., et al., Intranasal and sublingual deliv-ery of inactivated polio vaccine. Vaccine, 2017. 35(20): p. 2647-2653.
2. Verdijk, P., et al., Safety and immunogenicity of a primary series of Sabin-IPV with and without alu-minum hydroxide in infants. Vaccine, 2014. 32(39): p. 4938-44.
3. Warfel, J.M., et al., Nonhuman primate model of pertussis. Infect Immun, 2012. 80(4): p. 1530-6. 4. Hoppentocht, M., et al., Tolerability and
Phar-macokinetic Evaluation of Inhaled Dry Powder Tobramycin Free Base in Non-Cystic Fibrosis Bronchiectasis Patients. PLoS One, 2016. 11(3): p. e0149768.
5. Maurer, J.M., et al., Development and potential application of an oral ColoPulse infliximab tablet with colon specific release: A feasibility study. Int J Pharm, 2016. 505(1-2): p. 175-86.
6. Pizarro, T.T., et al., Mouse models for the study of Crohn’s disease. Trends Mol Med, 2003. 9(5): p. 218-22.
7. Wong, Y.L., et al., Drying a tuberculosis vaccine without freezing. Proc Natl Acad Sci U S A, 2007. 104(8): p. 2591-5.
8. Ohtake, S., et al., Heat-stable measles vaccine produced by spray drying. Vaccine, 2010. 28(5): p. 1275-84.
9. Lovalenti, P.M., et al., Stabilization of Live Atten-uated Influenza Vaccines by Freeze Drying, Spray Drying, and Foam Drying. Pharm Res, 2016. 10. PATH;, W., Some vaccine costs are hidden below
the surface. Project Optimize - Ideal supply sys-tems for the future. 2012.
11. Kanojia, G., et al., Developments in the formulation and delivery of spray dried vaccines. Hum Vaccin Immunother, 2017. 13(10): p. 2364-2378. 12. group, M.a., et al., Safety and immunogenicity of
dry powder measles vaccine administered by in-halation: a randomized controlled Phase I clinical trial. Vaccine, 2014. 32(50): p. 6791-7.
13. Siew, A. Exploring the Use of Aseptic Spray Drying in the Manufacture of Biopharmaceutical Inject-ables. 2016; Volume 40, Issue 7, pg 24–27.
