(M1130-02-14) Enabling Inline Concentration Control: Refractometry as a PAT Tool in Continuous Manufacturing of Lipid-Based Therapeutics

Purpose: While offline refractometry is established for single component analysis, its application as a real-time process analytical technology (PAT) in continuous manufacturing (CM) of lipid-based therapeutics is not well established. This work quantifies how formulation and process variables, including cryoprotectants, solvents, and lipids influence refractive index (nD) inline. It also investigates non-linear and synergistic effects in binary and ternary mixtures, providing a predictive framework for refractometry-based PAT. These insights address a critical gap in real-time process monitoring, enabling more robust, efficient, and scalable manufacturing of advanced biopharmaceuticals.

Methods: Inline and static refractive index measurements were performed using a Vaisala PR-23 refractometer (589 nm) integrated with a continuous manufacturing (CM) platform.^1,2 Concentration calibration curves were obtained from Vaisala. Monodisperse liposome formulations (50–200 d.nm) with varied charged and PEG-lipid content were prepared. Single-component systems were first characterized for cryoprotectants (Fig 1), solvents (Fig 2), lipids, liposome size, temperature (25-50°C), flow rate (0-1600 mL), buffer type, and pH (4-10). Various 3-factor design of experiments (DoEs) were employed to evaluate different combinations with a focus on formulation components with greater refractive index effects or relevance to liposome manufacturing. Multiple non-linear regression and Pearson correlation analyses validated predictive models for both single and multi-component systems.

Results: The process refractometer was able to accurately measure the concentration of a variety of formulation components in-flow. Among cryoprotectants sucrose produced the largest nD shift (0.021 nD per 10% w/v) followed by maltodextrin, trehalose, glycerol, lactose, and mannitol (0.016 nD per 10% w/v). The solvents ranked greatest to least influence were DMSO, TCM, DCM, IPA, EtOH, ACN, and MeOH. However, solvents showed non-linear nD response when mixed with aqueous phases, attributed to competing effects of hydrogen-bond driven solvent restructuring and density/polarizability changes. This also applied to moderately immiscible solvents, such as TCM and DCM, with aqueous and organic rich phases displaying different nD reactions to changing mixture concentrations. These interactions are system-specific, requiring full characterization for accurate PAT implementation. The largest amount of each factor that could individually influence nD (Fig 3) was from cryoprotectants (0.14 nD), then alcohols (0.04 nD), followed by buffer molarity (0.03 nD), temperature, lipid concentration, flow rate, pH, lipid composition, and liposome size. Three-factor design of experiments (DoEs) were employed to investigate formulation component interactions and test if predictions based on single components were accurate for multicomponent systems. The resulting predicted and actual refractive index values showed excellent agreement (Pearson’s r=0.999) if individual components were diluted with the same solvent. Additionally, inline refractometry was demonstrated to track concentration changes at multiple CM stages, including after particle formation, TFF, and post-loading.

Conclusion: This work establishes refractometry as a PAT tool for continuous manufacturing of lipid-based therapeutics capable of real-time, multi-point concentration monitoring even in complex, multi-component systems. Field calibration under actual process conditions is recommended to achieve optimal accuracy. These findings lay the groundwork for broader adoption of refractometry-based PAT in GMP environments supporting quality focused biopharmaceutical manufacturing.

References: 1. Costa, A.P., Xu, X., Khan, M.A. et al. Liposome Formation Using a Coaxial Turbulent Jet in Co-Flow. Pharm Res 33, 404–416 (2016). https://doi.org/10.1007/s11095-015-1798-8
2. Yenduri, G., Costa, A.P., Xu, X., Burgess, D. Impact of critical process parameters and critical material attributes on the critical quality attributes of liposomal formulations prepared using continuous processing, International Journal of Pharmaceutics, Volume 619, 2022, 121700, ISSN 0378-5173.

Acknowledgements: The authors acknowledge the University of Connecticut Pfizer Distinguished Chair Funds as well as the Department of Pharmaceutical Sciences, University of Connecticut Singiser Fellowship award to LDB for supporting this research.


Figure 1: Cryoprotectant Refractive Index by Concentration


Figure 2: Solvent Refractive Index by Concentration


Figure 3: Max Refractive Index Range During Processing for Each Parameter