Lyophilisation of high concentration protein formulations

Antibodies form the main class of commercial therapeutic proteins and are intended for the treatment of several chronic diseases. The current trend is to formulate antibodies at high concentrations in order to deliver a large therapeutic dose in small volumes (1-2 mL) subcutaneously (SC). Thus, enabling patients to self-administer these medications at home rather than in a hospital setting through intravenous (IV) infusions. However, several challenges can be encountered when formulating proteins at high concentrations. In the liquid state, high concentration protein formulations are prone to aggregation and exhibit high viscosities. In the lyophilised state, they show high total solute concentrations and product dry layer resistance, that can prolong primary drying, increase overall process time, costs of manufacturing and extend reconstitution time. The overall aim of this thesis was to obtain a stable, lyophilised, high concentration antibody (immunoglobulin 1, IgG1) formulation via rational selection of the excipients (type and ratio) and optimisation of the lyophilisation process with the aid of Quality by Design (QbD) approaches. Formulations selected include BSA as model protein and an IgG1 as the target protein. Arginine/arginine-HCl (arg/arg-HCl) and sucrose were selected as excipients as a result of an overview of the composition of liquid and lyophilised protein products approved in the European Union since 1995. Polysorbate 80 was also employed as a surfactant at a fixed concentration. The ratio of protein:excipients relative to the total solute concentration was determined with the aid of a mixture Design of Experiment (DOE) tool. The DOE was used to generate empirical models for critical temperatures optimisation and prediction. The lyophilisation cycle optimisation was conducted using a lyomodelling tool for primary drying prediction and the process was monitored using a range of temperature monitoring systems. CQAs of the optimised lyophilised formulations (glass transition temperature (Tg), residual moisture, product appearance, reconstitution time and biophysical stability) were assessed and stability was monitored over a six month period in the presence and absence of cold chain storage. The empirical model generated from the DOE provided formulations containing 100 mg/mL of protein with maximised critical temperatures. The empirical model was also capable of accurately predicting both critical temperatures (glass transition of the maximally freeze concentrated solution, Tg’ and collapse temperature, Tc) within the design space. The lyophilisation process was successfully optimised. A significant reduction in primary drying time (45%, -11h) was obtained when shelf temperature (Tshelf) was increased from -25°C to -15 °C. Additionally, an aggressive lyo-cycle conducted at a Tshelf of 35 °C provided an extremely short primary drying time (4.75h). The lyophilised products obtained by this lyo-cycle did not show any collapse and had similar Tg and residual moisture to the products lyophilised at conservative conditions (95-100 °C and 0.1-0.3% respectively). The high Tg renders these products good candidates for the elimination of cold chain during storage. Formulations selected were amorphous and maintained their physical solid state over six months storage and exposure to high relative humidity (up to 70%) conditions. The high concentration of the protein was capable of inhibiting excipient recrystallisation, providing a stable amorphous product. A non-destructive technique was used to determine the vial headspace residual moisture which correlated with product moisture and Tg. A correlation model was developed to estimate product moisture and Tg by performing a non-destructive technique and retaining the samples at each time point of the stability programme. The lyophilised formulations were biophysically stable over six months in the presence and absence of cold chain. Aggregation was not detected. The challenge of prolonged reconstitution times for high concentration protein formulations was overcome by lyophilising the product at a low fill volume (1.1 mL). Finally, the addition of arg/arg-HCl to the lyophilised formulations showed some advantages; reducing the specific surface area and improving the product visual appearance of lyophilised formulations. Furthermore, arg/arg-HCl provided a stabilising effect, reducing protein aggregation in formulations exposed to high intensity light. This study provides a rational approach, insights and strategies that can be applied to overcome some of the challenges encountered during formulation and manufacturing of lyophilised high concentration protein formulations. The use of QbD approaches aids the development of stable, lyophilised, high concentration antibody formulations. The formulation strategy identified allows reduction of the manufacturing time and enables storage of these formulations at ambient conditions rending the development of lyophilised high concentration protein formulations more cost effective