Integrated cell and bioprocess engineering for rapid and intensive continuous production of biopharmaceuticals

Continuous upstream processing is becoming increasingly popular for the industrial production of biopharmaceuticals. Despite this, cell line and process development remains reliant on batch-wise experimentation to acquire data; this approach is time consuming, costly and labour intensive as the demands put upon these processes increase. A shift to continuous experimentation, with perfusion utilised to keep cells sustained for longer periods with continual testing, may alleviate these demands. However, the lack of small-scale, high throughout perfusion systems makes this difficult to achieve. For the commercial systems that are available, the cell retention technology employed is frequently not compatible with familiar labware, instead requiring dedicated equipment for successful operation. This makes adoption with existing development protocols challenging. Microcarriers have been used previously to adhere CHO cells in suspension culture. Microcarriers are easily handled, scalable and compatible with many vessel formats. Their use as a potential cell retention device has been greatly overlooked. Commercial microcarriers are available, but they often require attachment proteins for cell adhesion, despite the discouragement of animal-derived products in biopharmaceutical processing. The work here has developed an in-house, microsphere-based cell retention device for a suspension-adapted, IgG-producing CHO cell line. An animal origin-free, inexpensive polymer, which has been neglected in the literature, has been shown to enable the attachment of these cells to vessel surfaces. Alongside this, acrylic-based polyHIPE microspheres with diameters from 270 to 1100 µm have been fabricated at negligible cost. A novel monomer has been included from 2 to 16 % to give the resulting materials increasing carboxyl functionality. These carboxyls have both increased the stability of microspheres in solution and enabled the rapid adsorption of this polymer. Cell loading, where high cell density suspensions are forced to interact with microspheres, has been introduced as a technique for guaranteed cell retainment. With this method, 30–100 million cells per mL of microspheres were retained within an hour, depending upon conditions used. To demonstrate the versatility of the cell retention device, a pseudo-continuous culture was performed for over 30 days using Erlenmeyer flasks, with continual IgG production demonstrated. A simple perfusion system, employing these microspheres within a modified spinner flask, was also tested. It has been shown here that sophisticated cell retention technology is not necessary for effective retainment of CHO cells. Because of this, continuous processing has been performed in the laboratory without costly equipment or prior experience. It is hoped the data obtained may inspire others to explore continuous operations for the pre-clinical development of biopharmaceuticals.