High-throughput screening of excipients for the formulation of protein-based vaccines
(2012)
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- (en) From manufacturing to patient administration, vaccines undergo various stresses such as exposure to light, temperature and shaking. Under these conditions, antigen integrity and therefore vaccine efficacy may be impacted. Among these stresses, hydrophobic air-liquid interfaces which are present at different steps of the production process as well as in the final container are a common cause of protein aggregation. Ensuring antigen integrity is a critical element in the development of new vaccine candidates. Traditional formulation methods allow the testing of a limited number of excipients. Recent advances in sample miniaturization, microplate-based analytical techniques, robotics and information technologies have promoted the emergence of high-throughput screening (HTS) techniques. Our objective was to develop new HTS techniques for vaccine formulation, in order to evaluate in parallel the effect of a large number of excipients on antigen stability. Vaccine antigens belong to a large variety of biological families, such as bacterial and viral inactivated units, proteins, virus-like particles, and polysaccharide conjugates. The effect of excipients on the stability of these antigen groups was first reviewed. A stress test in microplate was developed for studying protein sensitivity to aggregation at air-liquid interface. Briefly, air bubbles were blown in microplate wells, while avoiding extensive foaming. Analysis of the aggregates by size-exclusion chromatography correlated with changes in tryptophan fluorescence emission (TF). This label-free method was used to follow prevention of aggregation of a protein antigen at air-water interface by a set of 44 excipients (amino acids, sugars, polyols, polymers and surfactants), in HTS mode. Preservation of antigen integrity by 5 surfactants and 2 cyclodextrins was demonstrated. HTS assays rely upon simple and rapid analytical methods. Despite the widespread use of ultraviolet absorption spectroscopy (UA), the ability of this technique to follow protein conformational stability is currently not used in HTS. The conformational stability of two protein antigens was assessed in the presence of the set of 44 excipients. UA was compared to TF and differential scanning fluorimetry, both common HTS methods. The same protecting excipients were identified by all three methods. Results show that protein scientists can extract information on protein conformational stability from UA spectra recorded in microplate. This approach allows for a rapid identification of protein-stabilizing excipients in HTS studies. The high throughput platform based on limited material per testing condition and rapid analytical read-outs, allows screening of a larger area of the formulation space in a limited time period and enables rapid identification of candidate excipients for further formulation development.
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Dasnoy, S. (2012). High-throughput screening of excipients for the formulation of protein-based vaccines. https://hdl.handle.net/2078.5/158504