Quality by Design applied to the development of a recombinant veterinary product liquid formulation

RODRÍGUEZ, MARÍA CELESTE; VILLARRAZA, CARLOS JAVIER; TARDIVO, MARÍA BELÉN; ANTUÑA, SEBASTIÁN; FONTANA, DIEGO; CEAGLIO, NATALIA; PRIETO, CLAUDIO

Viena

Encuentro; ESACT FRONTIERS RETREAT; 2022

European Society of Animal Cell Technology

BACKGROUND AND NOVELTYProtein formulation and drug characterization are one of the most difficult and time-consuming tasks because the complexity of biotherapheutic proteins. Hence, maintaining a protein drug in its active state typically requires preventing changes in its physical and chemical properties for maintaining its shelf-life.Quality by Design (QbD) is a systematic approach that emphasizes product and process understanding and process control. Design of Experiments (DoE) is one of the most important QbD tool, allowing the possibility to modify the formulation attributes within a defined design space. This is particularly important for complex dosage forms such as recombinant protein products in liquid state. The aim of this work was to apply QbD concepts to obtain an optimized liquid formulation of a bioactive recombinant form of equine chorionic gonadotropin (reCG) (Villarraza et al., 2021) with a predefined quality product profile.EXPERIMENTAL APPROACHThe critical quality attribute (CQA) considered in this analysis was reCG stability, evaluated as the intact reCG (%) amount obtained by a previously validated RP-HPLC method. The design space was defined throughout a DoE approach. First, we applied a screening Placket Burman design (PBD) to determine the effect of several factors on the reCG stability, under accelerate conditions (25°C, 60% RH, for seven days). Several formulation components as stabilizers (sucrose, mannitol, arginine, methionine), surfactant (Pluronic F68), molarity and pH of the buffer, and concentration of the API were evaluated. Thereafter, an optimization procedure through the response surface methodology (RSM) approach was applied to define a design space region in which CQAs are attended. A Central Composite design (CCD) was applied, consisting in 27 experiments with a triplicated in the central point, under accelerate and stress conditions (40°C, 75% RH). Finally, one of the optimum liquid formulation condition predicted by a polynomial model was validated. This formulation was prepared as three individual formulation batches and evaluated after zero, 15, 45, 90, 120, 150 and 180 days of store at real (4°C), accelerate and stress conditions.RESULTS AND DISCUSSIONSelection of appropriate excipients for a formulation is crucial since many of the excipients may interact with each other or with the protein of interest. Hence, a DoE and RMS approach simplify these studies, improving the quality of the obtained results in comparison with traditional univariate strategies. Here, a robust design space was obtained, which should facilitate the scaling-up stage and allow possible modifications of the obtained liquid formulation within the design space. After 180 days under accelerated conditions 96 ± 10% of reCG remained intact, whereas at real conditions (4°C) the reCG persisted unaltered, demonstrating the high versatility and efficiency of QbD tools as DoE during biotherapeutics' formulation stage.It should be highlight that, this is the first time that a liquid formulation was reported for an eCG molecule, since pregnant mare serum gonadotropin (PMSG) commercial preparations are presented in solid state as a lyophilized product, reducing the cost and time of the reCG manufacturing process.