Advancing continuous biomanufacturing: Quality control and process intensification strategies for perfusion cell culture

Abstract

Emerging product classes, like fusion proteins, antibody-drug conjugates (ADCs),and bispecifics, are reshaping the landscape of biologics. Biosimilars have also entered the market in recent years. As competition grows and product pipelines expand,continuous manufacturing of biologics has gained momentum due to its potential for process intensification. A number of manufacturers are evolving or adapting their production platform to use perfusion cell culture and continuous downstream purification principles to benefit from more productive and faster production and purification capabilities.The present work aims at providing a product quality control framework for intensified steady-state perfusion processes, with the objective to build a time independent integrated continuous biomanufacturing platform for biologics in Chinese hamster ovary (CHO) cells. A prerequisite for such a continuous manufacturing platform is a steady-state perfusion cell culture yielding consistent product quality over time. This is especially challenging for highly intensified processes, almost indispensably requiring real-time process control to detect process excursions and initiate preventive actions to avoid product quality drifts. Process analytical technologies (PAT) such as multivariate spectroscopic sensors are promising tools to enable real-time process monitoring and control, yet direct measurement of critical quality attributes such as glycosylation remains extremely challenging due to relatively low concentrations and complex process matrices. Leveraging the understanding of the relationship between critical process parameters (CPPs) and critical quality attributes (CQAs), according to the Quality by Design (QbD) principle, appears to be a more tangible approach. This strategy involves estimating CQAs basedon real-time PAT measurements of CPPs. However, spectroscopic sensor calibrationremains a challenging and time-consuming task, especially for perfusion processes with inherently low process variability.The first step towards incorporating spectroscopic PAT tools into the manufacturing platform involved therefore the creation of a novel PAT sensor calibration method designed specifically for steady-state perfusion. This approach enables the development of exceptionally precise and robust prediction models within a matter of days, as opposed to the traditional duration of several months. With this method in place, a real-time Raman spectroscopy feedback control strategy was established,reducing ammonium accumulation 3-fold, which in turn stabilized CQAs of the bispecific antibody product. Various critical glycoforms, such as high mannoseglycans and fucosylation, were stabilized to yield standard deviations below 0.2% over the course of a 40-day perfusion process, qualifying the operation as time independentmanufacturing process. Additionally, promising glycosylation modulatorsto fine-tune antibody-dependent cell-mediated cytotoxicity (ADCC) were successfully screened and evaluated for applicability in continuous manufacturing,representing a particularly interesting tool to match reference product quality in the biosimilars field.This work further investigated potential process intensification strategies to boost productivity with a particular focus on their impact on product quality. Technologies for innovative bleed recycling applications were evaluated and scaled to manufacturing scale, with the intent to selectively concentrate the biomass and recycle the liquidfraction containing the product, which would otherwise be wasted. In conjunction with extended culture durations enabled through product quality stabilization by real-time control, a novel operating mode for frequently applied hollow fiber-based tangential flow filtration (TFF) was proposed. Additional harvest volumes gained through process intensification can be handled, thereby alleviating extensive productretention or premature filter clogging as experienced for standard TFF operation.In summary, the implementation of the developed quality control framework represents a significant contribution to the field of continuous biomanufacturing, as exemplified by an intensified CHO perfusion process producing a glycosylated bispecificantibody. QbD principles in combination with Raman spectroscopy as PATtechnology ensure time-independent product quality profiles under highly intensified manufacturing conditions, there by qualify the perfusion upstream process for seamlessincorporation into the integrated continuous biopharmaceutical manufacturingplatform.