Protein content estimation of recombinant vaccines at drug product (DP) stage is a crucial lot release and stability indicating assay in biopharmaceutical industries. Regulatory bodies such as US-FDA and WHO necessitates the quantitation of protein content to assess process parameters as well as formulation losses. Estimation of protein content at DP stage in presence of adjuvants (e.g AlOOH, AlPO4, saponin and squalene) is quite challenging, and the challenge intensifies when the target protein is in Virus like particles (VLP) form, owing to its size and structural complexity. Methods available for protein estimation of adjuvanted vaccines mostly suffer from inaccuracy at lower protein concentrations and in most cases require antigen desorption before analysis. Present research work is based on the development of a rapid plate-based method for protein estimation through intrinsic fluorescence by using Malaria vaccine R21 VLP as a model protein. Present method exhibited linearity for protein estimation of R21, in the range of 5-30 µg/mL in Alhydrogel and 4-20 µg/mL for Matrix M adjuvant. The method was validated as per ICH guidelines. The limit of quantification was found to be 0.94 µg/mL for both Alhydrogel and Matrix M adjuvanted R21. The method was found specific, precise and repeatable. This method is superior in terms of less sample quantity requirement, multiple sample analysis, short turnaround time and is non-invasive. This method was found to be stability indicating, works for other proteins containing tryptophan residues and operates well even in presence of host cell proteins. Based on the study, present method can be used in vaccine industries for routine in-process sample analysis (both inline and offline), lot release of VLP based drug products in presence of Alhydrogel and saponin based adjuvant systems.
Keywords: Adjuvanted vaccines; Alhydrogel; Intrinsic fluorescence; Matrix M; Protein quantification; R21 Malaria; Saponin.
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