(M1530-09-51) Influence of Lyophilization Process Conditions on Crystallization Behavior and Formulation Stability of Doxycycline for Injection

Purpose: The objective of this study is to assess the stability of Doxycycline for Injection formulations prepared with different critical lyophilization process parameters based on the crystalline behaviour determined by X-Ray Diffractometry (XRD) at 2-week time point under accelerated stability condition (40°C/75%RH). The stability samples were analysed for Appearance of cake, pH, Reconstitution time, Loss on drying, Doxycycline Assay, Ascorbic acid assay, and Related substances at 2-month time point to conclude the finding from crystalline behaviour.

Methods: An injectable formulation containing Doxycycline, Mannitol and Ascorbic acid was prepared by Lyophilization process. Various critical process parameters such as Rate of freezing, Annealing temperature, and Primary drying temperature were evaluated (Table 1). All formulations were assessed for stability under accelerated stability conditions for different critical quality attributes. Mainly crystalline behaviour was assessed using X-Ray Diffractometry (XRD), and the Doxycycline assay, Ascorbic assay, Related substances were determined using High Performance Liquid Chromatography (HPLC).

Results: The results of preliminary trials demonstrated that Formulation P1 is stable for 2 weeks and showed cake shrinkage starting after 2 weeks of accelerated storage (Figure. 1). The colour changed to brown colour after 1 month of accelerated storage but the rest of all critical quality attributes were not impacted. Considering these challenges, the design of experiments was executed with different critical lyophilization process parameters to make a variety of formulations as shown in Table 1. All two-week accelerated stability samples showed significant variation in the % crystalline nature of Mannitol and Ascorbic acid except P3 (Figure 2). Formulation P3 showed a comparable percentage of about 20% of stable δ-Mannitol and Ascorbic acid crystalline to T=0 samples, which might indicate a stable formulation under accelerated storage condition. This assumption was confirmed by the 2-month accelerated stability results of other critical quality attributes, including the Appearance of cake, pH, Reconstitution time, Loss on drying, Doxycycline Assay, Ascorbic acid assay, and Related substances, which didn’t show any significant changes after 2 months of storage. In contrast, the impurity profiles of Formulation P2 showed cake shrinkage and higher levels of 4-Epidoxycycline in which higher amounts of unstable β forms of crystalline mannitol and crystalline ascorbic acid (D-Isoascorbic acid) were observed in 2 weeks. Formulation P4 also showed significant change in amorphous form of Ascorbic acid to crystalline form, which impacted stability profile after 2 months. In addition, the placebo formulations did not show any cake shrinkage indicating the possibility of interaction of excipient matrix with Doxycycline. The colour change of the lyophilized cake to brown was also observed in the placebo formulations at higher storage conditions of more than 30º C. This result demonstrates ascorbic acid stored at a temperature of more than 30º C results in brownish cake. However, Ascorbic acid assay was not impacted even there were significant changes in its crystalline behaviour and brownish colour. All these results demonstrate that the development work to finalize a stable Doxycycline for Injection formulation can be completed in a shorter span based on the crystallization behaviours of the formulation. Crystalline behaviour in 2-week stored formulations showed a direct impact on stability profile for 2-month accelerated stored samples.

Conclusion: Assessment of the crystalline behaviour of lyophilized cake using XRD method is an important tool to determine the stability of a lyophilized Doxycycline for Injection product in a short period (approximate 2 weeks). Crystalline behaviour assessment can be used as an important tool in formulation and process optimization helps pharmaceutical companies to shorten product development duration, resources and cost significantly.

Acknowledgements: We acknowledge Northwestern University for assisting assessment of quantification of crystalline nature of the formulations.

Figure 1. Comparison Cake shrinkage between T=0 and 40ºC/75%RH_2 Week stored Samples

Table 1. Screening of Lyophilization Process parameters to Study their Impact on Critical Quality Attributes

Figure 2. Impact of Lyophilization Process Parameters on Crystalline behaviour of product matrix on accelerated stability condition