High Cell Density Cryopreservation: A Valuable Approach To Seed Train Intensification

By Jochen Sieck, Head of Perfusion Systems R&D, Mona Bausch, Lead Scientist for HCDC, and Habib Horry, BioContinuum™ Upstream Platform Marketing, Merck KGaA, Darmstadt, Germany*

As the rise of advanced therapeutics continues to drive an evolution in the biopharmaceutical market, companies must seek innovative new ways to produce drugs. Established approaches to process development and manufacturing are currently being challenged with novel technologies and methods designed to improve drug quality and increase process flexibility and efficiency. Implementing new process strategies has become fundamental to ensuring the industry can meet the rising demand of small batch and/or multi-product manufacturing while minimizing costs of manufacturing.

Single-use technology, advanced automation and process controls, and intensified processing are key enablers for the facility of the future. They allow for closed processing, which reduces the environmental requirements for a manufacturing site, but a smaller footprint, making biopharmaceutical manufacturing facilities quicker and cheaper to build as well as easier to maintain and operate. As the focus on intensified processing grows, special attention has recently been given to upstream with the renewal of perfusion-based processes, especially for seed train expansion.

For a fed-batch process, the seed train starts with a one-milliliter (ml) vial of cells thawed from the working cell bank that are then expanded to 15,000-liters stainless steel bioreactors or 2,000-liter single-use bioreactors, taking up to five weeks to complete. This traditional method based on open and manual handling steps under laminar flow is time consuming and poses substantial contamination risks and reproducibility issues.

An alternative is High Cell Density Cryopreservation (HCDC), which offers a valuable approach to traditional seed train expansion. HCDC allows for expanding cells of interest and preparing frozen seed train intermediates in bags up to 150 ml working volume. By freezing cells at larger volumes and higher densities obtained from a bioreactor running in perfusion mode, the expansion process can begin later, jump-starting the manufacturing campaign by several weeks and removing a large part of the seed train from the critical path to production.