A current focus of biopharmaceutical research and production is seed train process intensification. This allows for intermediate cultivation steps to be avoided or even for the direct inoculation of a production bioreactor with cells from cryovials or cryobags. Based on preliminary investigations regarding the suitability of high cell densities for cryopreservation and the suitability of cells from perfusion cultivations as inoculum for further cultivations, an ultra-high cell density working cell bank (UHCD-WCB) was established for an immunoglobulin G (IgG)-producing Chinese hamster ovary (CHO) cell line. The cells were previously expanded in a wave-mixed bioreactor with internal filter-based perfusion and a 1 L working volume. This procedure allows for cryovial freezing at 260 × 10⁶ cells mL⁻¹ for the first time. The cryovials are suitable for the direct inoculation of N−1 bioreactors in the perfusion mode. These in turn can be used to inoculate subsequent IgG productions in the fed-batch mode (low-seed fed-batch or high-seed fed-batch) or the continuous mode. A comparison with the standard approach shows that cell growth and antibody production are comparable, but time savings of greater than 35% are possible for inoculum production.

Modern production processes for biopharmaceuticals often work with very high cell densities. Moreover, there is a trend towards moving from fed-batch to continuous perfusion processes; a development that is influencing the requirements for bioreactor design and process control. In this study, the transfer of fed-batch and perfusion experiments between different cylindrical stirred lab-scale bioreactors and Thermo Scientific’s^TM (Waltham, MA, USA) cubical HyPerforma^TM DynaDrive^TM Single-Use Bioreactor was investigated. Different scaling parameters were used, which were selected based on the requirements of the respective processes. Peak cell densities of up to 49 × 10⁶ cells mL⁻¹ and antibody titers of up to 5.2 g L⁻¹ were achieved in 15- to 16-day fed-batch experiments. In 50-day perfusion cultivations, a viable cell volume of >100 mm³ mL⁻¹ was maintained and more than 1 g L⁻¹ d⁻¹ of antibodies were harvested. The perfusion processes were automated with both cell bleed control and glucose concentration control. Cell retention was performed using Repligen’s (Waltham, MA, USA) XCell^® ATF perfusion systems and single-use devices. In summary, approaches for successfully scaling highly productive fed-batch and perfusion processes between geometrically dissimilar lab and pilot scale bioreactors were demonstrated. The advantages of perfusion in comparison to fed-batch processes were also observed.

Single‐use technology is now an indispensable part of biopharmaceutical production processes. This not only applies to research and development, but also to commercial processes. The manufacturers of single‐use systems are working on improving them for use in intensified processes as well as on new developments. One of the bottlenecks was the availability of single‐use disk stack separators whose stainless steel variants are widely used in industry. In this study, the suitability of the new single‐use disk stack separator kytero 500 was investigated for the first time for cell separation in pilot‐scale Chinese hamster ovary cell‐based antibody production. The cells were completely separated over a wide range of separator settings with nearly no release of intracellular substances and no loss of the antibody.

Various approaches to process intensification are currently being investigated to ensure time and cost savings when producing biopharmaceuticals. In the present study, a one‐step inoculum production was established based on an ultra‐high cell density working cell bank with immunoglobulin G producing Chinese hamster ovary cells. Cryovials were used for direct inoculation of 1‐L wave‐mixed perfusion bioreactors. When reaching around 180 · 10⁶ cells mL⁻¹ in N−1 perfusion mode, low‐seed and high‐seed fed‐batch experiments in shake flasks were inoculated, as was the case for 250‐mL stirred single‐use bioreactors. Additionally, proof‐of‐concept runs at 50‐L and 200‐L scale were successfully performed. The intensification approach presented allows manufacturing capacity to be increased by up to 50 %.

The perfusion mode has become increasingly important in biopharmaceutical production in recent years. A bioreactor system used in many laboratories for the development of monoclonal antibodies (mAbs) production processes is the Sartorius’ Ambr. 250 system. Vessels designed for perfusion mode are only available for its high throughput version, while the modular version of the Ambr 250 is not designed for perfusion mode. In this study, perfusion processes for the production of a mAb with Chinese Hamster Ovary (CHO) cells were realized in the Ambr 250 Modular in combination with Repligen’s ATF 1 single-use device for the first time, to the authors’ knowledge. After testing a semi-perfusion setup in well plates and the Ambr 250, an N−1 perfusion process was developed to produce ultra-high cell densities of more than 150 Å~ 106 cells mL−1 for the inoculation of subsequent mAb production processes. In a second step, continuous mAb production was successfully realized over 23 days in a proof-of-concept experiment, achieving a volumetric productivity of 0.65 g L−1 d−1. The results of the N−1 and continuous perfusion processes were comparable to a 3 L HyPerformaTM Glass bioreactor (Thermo Scientific) with an ATF 2 (Repligen).

There are several ways to make antibody production processes more efficient and less expensive through process intensification. In this study, an ultra‐high cell density working cell bank was used to inoculate production bioreactors directly with cells from cryovials. After lab‐scale experiments, a fed‐batch pilot‐scale experiment was successfully conducted as a proof‐of‐concept. In addition, cells were cultivated in perfusion mode at 2 L scale. The intensification approach presented here allows the complete elimination of the inoculum production outside the production bioreactor and the creation of additional production capacity by saving time and space. In perfusion mode, an increase in bioreactor productivity of over 180% can be achieved compared to fed‐batch mode.