The Influence of Arginine and Counter-Ions: Antibody Stability during Freeze-Drying.

Amino acids, for example L-arginine, are used in lyophilisation as crystalline bulking, buffering, viscosity reducing or stabilising excipients. In this study, arginine was formulated with different counter ions (hydrochloride, citrate, lactobionate, phosphate, and succinate). A monoclonal antibody was investigated in sugar-free arginine formulations and mixtures with sucrose regarding cake appearance and protein aggregation and fragmentation. Arginine hydrochloride formulations collapsed during lyophilisation due to its low Tg' and partially crystallised during storage, but provided the best protein stability at low antibody concentration, followed by arginine succinate. Arginine citrate/phosphate/lactobionate formulations resulted in amorphous elegant cakes, but inferior protein stability. Addition of sucrose improved cake appearance and protein stability. Arginine phosphate with sucrose resulted in similar protein stability as the sucrose reference. Mixtures of sucrose with arginine hydrochloride/lactobionate/succinate provided better stability than sucrose alone. While 50 mg/mL antibody improved the cake appearance, only arginine lactobionate provided sufficient protein stability next to sucrose. Overall, sugar-free arginine hydrochloride and lactobionate lyophilisates stabilised the antibody comparably or better than sucrose depending on antibody concentration. The best protein stability was found for mixtures of arginine hydrochloride/lactobionate/succinate with sucrose.

The effect of residual moisture on a monoclonal antibody stability in L-arginine based lyophilisates.

Amino acids are not only used as buffering agents in lyophilisation, but also exhibit cryo- and lyoprotecting characteristics. L-Arginine based lyophilisates were tested regarding their ability to stabilise a monoclonal antibody (mAb) at different residual moisture (RM) levels. Arginine base was formulated with citric, hydrochloric, lactobionic, phosphoric, and succinic acid for pH adjustment. Lyophilisates with less than 0.5% and approx. 2.5%RM were stored for up to 6 months at 40 °C. The mAb aggregation in arginine in combination with hydrochloric acid and succinic acid was similar or even less compared to a sucrose reference formulation. Arginine in combination with citric acid, lactobionic acid, and phosphoric acid resulted in lower protein stability. Overall, arginine formulations with high RM levels resulted in better protein stabilisation despite decreased glass transition temperatures (Tg). Whereas we detected mAb glycation in the sucrose based formulations, this chemical reaction did not occur in arginine based formulations. Arginine hydrochloride and succinate, especially at high RM levels, could be promising alternatives to sucrose for stabilisation of mAb in lyophilisates.

Freeze concentration during freezing: How does the maximally freeze concentrated solution influence protein stability?

During freeze drying of biologics, a highly viscous freeze concentrate (FC) is formed upon the initial freezing due to the crystallisation of ice. Protein stability in this freeze concentrated phase is not yet well understood, but can decide upon the success of the lyophilisation itself. Protein stability may be high below the Tg' as it is typically the case during primary drying but decreases above Tg', e.g. during annealing or during aggressive freeze drying above Tg' in presence of a crystalline bulking agent or, beyond freeze drying, during storage of frozen bulk. Different FCs containing monoclonal antibody, sucrose, histidine or phosphate buffer and sodium chloride were prepared via partial freeze drying and analysed for protein aggregation. No solute crystallisation is visible and the systems are vitrifying during cooling. Increasing sugar or buffer concentration showed positive effects on either melting and aggregation temperature or on protein self-interaction as indicated by A2 values. Protein integrity in the FC was not affected by 1 month storage at temperatures above Tg'. Thus, upconcentration of solutes during freezing does not negatively impact protein stability. Exceeding Tg' during freeze drying e.g. upon annealing or, intentionally or unintentionally, during primary drying does not lead to protein aggregation.

Improvement of arginine hydrochloride based antibody lyophilisates.

Arginine hydrochloride (ArgHCl) can be used as stabiliser of protein drugs in both liquid and freeze dried formulations reducing aggregation. But ArgHCl exhibits a low Tg' value which can lead to cake collapse during lyophilisation. We analysed arginine (Arg) citrate hydrochloride and lactobionate based lyophilisates aiming for elegant cake appearance in combination with minimal protein aggregation using 2 mg/ml monoclonal antibody (mAb) without additional excipients and in combination with sucrose (Suc) as amorphous stabiliser as well as mannitol (Man) and phenylalanine (Phe) as crystalline bulking agents. Furthermore 50 mg/ml mAb was also used. Based on a process which resulted in poor cake appearance for the ArgHCl based cake with 2 mg/ml mAb the combination with sucrose mannitol (Man:Arg 4:1) and phenylalanine (Phe:Arg1:4 1.5:3.5 and 2:3) as well as a high mAb concentration were able to improve cake appearance. Overall the crystalline bulking agent Phe in combination with ArgHCl renders elegant cakes with minimal protein aggregation. The alternative Arg salts did not require cake quality enhancement but mAb stability was inferior to ArgHCl. The combination with mannitol showed reduced stability due to a substantially lower amount of amorphous matrix. Thus the combination of ArgHCl with phenylalanine was superior to any other formulation tested including the sucrose based and may allow for substantial process time reduction due to the crystalline scaffold delivering pharmaceutically elegant cakes.

Method development and analysis of the water content of the maximally freeze concentrated solution suitable for protein lyophilisation.

During freeze-drying of a liquid formulation, a freeze-concentrate is formed in the first phase, the freezing step. Understanding the composition of the maximally freeze concentrated solution can help to judge the process stability of biopharmaceuticals during lyophilisation. Our objective was to develop a suitable method to determine the water content of the maximally freeze concentrated solution using differential scanning calorimetry (DSC). Three different methods were compared: (i) the intercept of the glass transition temperature of the maximally freeze concentrated solution Tg' and the melting temperature Tm for a concentration series, (ii) the linear regression of the melting enthalpy starting from the onset of Tg' until the end of the melting event for a concentration series, and (iii) a one-point determination of the amount of unfrozen water. While Method 1 is accurate but requires the analysis of a high number of samples, Method 3 requires only one single sample, with a loss of accuracy. Method 2 works best taking sample preparation and accuracy into account. Various systems containing sugar (sucrose, trehalose) and other excipients (histidine buffer, phosphate buffer, sodium chloride, arginine hydrochloride, arginine citrate) were evaluated with different antibody concentrations to evaluate the composition of the maximally freeze concentrated solution. The freeze concentrates exhibited a water content of 20-30%, slightly dependent on the excipients, but independent of the antibody concentration. The methodology we developed is broadly applicable for the analysis of the composition of maximally freeze concentrated solutions and can help to elucidate protein stability during lyophilisation.