High Titers and Perfusion Processes Challenge Cell Harvesting Systems

Cell harvesting is a crucial step in biopharmaceutical manufacturing that can have significant impacts on product quality and the design of remaining downstream processes. Growing titers and viable cell densities, due to improved media and cell lines, and growing use of perfusion cell culture are making this downstream purification step more challenging. In addition, manufacturers desire to achieve more efficient, cost-effective processing with a smaller footprint.

“The biopharmaceutical market is now facing increasing demand to be quicker to market, with lower production costs and a smaller industrial footprint while improving productivity,” says Alain Lamproye, president of the biopharma business unit of Novasep. New filtration and separation technologies, including single-use systems for larger scale harvesting operations, are helping manufacturers meet these needs.

The move to single use
Current interest in monoclonal antibody (mAb) therapeutic candidates produced in CHO cell expression systems is dominating development pipelines at major drug producers, according to Timothy D. Hill, director of upstream process development for FujiFilm Diosynth Biotechnologies USA. “One major shift in mAb production is the replacement of stainless-steel reactors with single-use bioreactors in order to streamline operations, decrease changeover times, eliminate cleaning validation, and enable rapid capacity expansion as product demand rises,” he notes.

In addition to lowering upfront capital costs and offering a higher level of flexibility, single-use technologies for cell harvesting have also addressed one of the major issues concerning this process step--the risk of cross contamination, according to Frank Meyeroltmanns, head of product management for purification technologies, Sartorius Stedim Biotech. Single-use technologies also provide cost-effective drug development options for products under investigation. “Cell removal using conventional centrifugation drives up cost due to the high capital outlays required for the equipment and operating costs--which are primarily incurred due to required maintenance and cleaning-in-place--are avoided with single-use technologies,” Meyeroltmanns adds.

Impact of perfusion
Continuous-processing technologies for both cell culture and downstream purification steps are some of most advanced latest developments in terms of outcomes, according to Lamproye. “In many cases, continuous processes enable high productivity while keeping production costs low. These types of processes also have smaller industrial footprints and constant productivity and benefit from on-line control that automatically adjusts processing parameters to maintain optimal settings,” he observes.

Improvements in both fed-batch and perfusion processing have contributed to increasing viable cell densities and titers and are driving suppliers to provide improved clarification technologies so that recoveries are in line with the higher outputs of the upstream process, according to Joe Codamo, senior project manager for biologics at Patheon. He adds that continuous processes-both upstream and downstream-are also attractive as an approach for reducing unit operations in order to lower manufacturing costs, shorten timelines, and minimize product losses.

High CHO cell-density cultures achieved using perfusion bioreactor technology are, in fact, becoming more standard to minimize the upstream production scale and reduce the footprint of reactors in the manufacturing facility, thus lowering overall capital investment and operating costs. “Typical peak CHO cell densities have been reported publically by manufacturers experienced with perfusion culture to range from 30-50x106 viable cells/mL culture medium, with some reports of peak cell densities over 100x106 viable cells/mL in experimental bench scale systems. Furthermore, mAb titers are reported to exceed 10g/L in production cycles of 2-3 weeks,” Hill says.

Thoughts about harvest technology have also shifted in response to adoption of perfusion cell culture in the industry, according to Hill. “The goal of harvest has broadened to include cell and product retention during production, as well as separation of product from cells during the end of production,” he observes.

Challenge of higher titers
Current single-use depth filters need to handle ever-increasing cell concentrations yet deliver continuously higher yields, but they often suffer from blockage at lower loading capacities, particularly when such volumes have high biomass concentrations.

Major process-relevant parameters for cell cultivation with respect to clarification are particle-size distribution and particle quantity, according to Meyeroltmanns. “For typical cell-culture applications, particle sizes vary between 0.1 µm and 25 µm, whereas the quantity of total biomass-relevant particles ranges from 10 million/mL to more than 100 million/mL. These levels present a challenge for conventional clarification techniques, and often multi-step filtration regimes are required that use up large numbers of filter units and entail extensive case-based adaptations,” he explains.

Centrifuges are predominantly used to harvest cells from process volumes above 500 L to reduce cost and waste, according to Meyeroltmanns. “Large-scale unit operations up to 2000 L require both scale-up and scale-down concepts, as well as linear scalability,” he asserts.
Hill does note that 3M has made significant improvements to its line of commercially available traditional depth filters for cell removal, and Codamo adds that advances in depth-filter media have also contributed to significant improvements in filter capacity, flux, and product recoveries. Hill agrees, however, that depth filtration becomes less cost effective compared with centrifugation at CHO cell densities of approximately 30x106 cells/mL or greater.

New large-scale, single-use filtration technology
Sartorius Stedim Biotechnologies has adapted “body-feed filtration,” (BFF) technology from the blood and plasma fractionation industry for cell harvesting to enable large-scale, single-use filtration. The new Sartoclear Dynamics system, which was introduced earlier in 2015, is designed to replace both centrifuges and depth filters with a single-step process that achieves cell clarification of up to 2000-L batch volumes.

“The main principle of BFF technology involves the addition of a filter aid. This pharmaceutical-grade diatomite, a fine diatomaceous earth (DE) is highly porous, increasing the permeability of the filter cake that builds up as clarification progresses in the filtration system connected downstream. As a result, the DE prevents filters from becoming blocked, providing a double advantage of significant time savings and high flow rates,” says Meyeroltmanns. He also notes that special single-use bags containing ultrapure DE are also available for attachment using new, patented quick connectors and dust-free DE transfer directly into the cell-culture fluid in a bioreactor.

“We believe that as a linearly scalable filtration technology, BFF closes the biggest gaps by enabling single-use cell harvesting of fluids from 2000 L standard single-use bioreactors and eliminating the need for centrifugation technology for removal of cells from such volumes. This technology also provides a high level of flexibility while helping to significantly reduce upfront capital investments,” Meyeroltmanns states.

Technologies for cell harvesting from perfusion processes
Perfusion technology presents the challenge of retaining cells and product within the bioreactor until the end of the production run, when the product is separated from the cells. Hollow-fiber filters initially developed for the growth of adherent cells have more recently been used as physical barriers for cell and product retention, while allowing the passage of fresh and spent media for exchange in the bioreactor, according to Hill. He also notes that single-path tangential flow through hollow-fiber filters has been replaced by alternating path flow (ATF, alternating tangential flow) to prevent cell attachment and filter fouling. By selecting the appropriate filter pore size, the process can be highly selective for operation in the retention or permeate modes.

The combination of clarification and product capture into one unit operation also reduces timelines and improves product recoveries, according to Codamo. He points to Patheon’s expanded-bed adsorption chromatography technology as an example.

Meeting future needs
Going forward, market needs will be the main drivers of further cell-harvesting technology development, and particularly the need to improve the production economics for biological products, according to Lamproye. As the biologics industry continues to grow and competition within the industry also increases with the rise of biosimilars, Codamo agrees that advances in cell-harvesting technology will lead to improved processing efficiencies, reduced costs, and greater flexibility for use with all of the mammalian cell types used in the industry and the various recombinant proteins currently in the pipeline.

With respect to the technology itself, Meyeroltmanns expects growing mid-term and long-term adoption of both large-scale unit operations and single-use equipment that provides continuous processing capabilities for CMO environments and multi-product facilities. Automation will also be of increasing importance for biologics production, according to Hill.

“Particular challenges include the need to continually improve depth filter efficiency in single-use applications to deal with ever-evolving cell densities and productivities, and the need to reduce process timelines further, especially for products susceptible to degradation during cell harvest and in the presence of host-cell impurities,” Codamo observes. He does note, however, that these issues have been addressed to some extent by newer technologies, but further improvements will be needed. One example is the development of single-use technologies that are better suited for low-temperature harvesting and thus would support improved product quality.

For cell harvesting with perfusion processes, Hill believes that while ATFs currently provide a good level of automation, the equipment is still cumbersome to setup, sterilize, and operate. Cell fouling of hollow-fiber filters has been the most frequent problem at large scale (2000 L). “The concept of separation of product from cells in a continuous or semi-continuous manner is necessary on the front end of manufacturing to provide a constant process flow through purification,” he also notes. Presterilized single-use filter units would also greatly improve robustness and build efficiency for setup and operation of ATF systems, according to Hill.
He adds that technologies are also needed to increase the speed of product separation from cells through hollow fiber filters, because currently this process is much slower than continuous centrifugation equipment (up to 24 hrs for ATF compared with less than 2 hours for continuous centrifugation at the 2000-L scale). Repligen Corp., which manufactures ATF systems, expects to launch a single-use hollow fiber filter within the next 12 months, according to Hill, but he is not currently aware of any technology under development that would improve the harvest time of ATF systems.

Harvesting adherent cells for therapeutic applications
Cell-based therapies are still in the early stages of development, and one key unanswered question relates to harvesting of the cells at commercial scales. “Harvesting of adherent therapeutic cells is very different from the harvesting of suspension cells that need to be separated from the desired product. In the former case, the cell viability and activity of the surface proteins must be retained during the harvesting process in order to preserve the therapeutic properties of the cells,” according to Alun J. Fowler, commercial marketing manager for EMEA vaccines and biologics in the Laboratory Products Group of Thermo Fisher Scientific.

“Currently there is no platform technology available for the harvesting of therapeutic cells,” he notes. “One factor is the variety of cell-culture techniques being used; most have very different culture conditions and harvesting requirements, which makes the development of a platform technology quite challenging,” says Fowler. Some cells (e.g., stem cells) are grown as sheets while others are grown on microcarriers, and yet others are intended as scaffold systems for regenerative medicine. Harvesting of therapeutic cells on a large scale is also difficult because many of these techniques are not easy to implement for large quantities of cells, according to Fowler. One possible solution is massive parallel cell culture as a scale-up approach.

Alternatives to actual harvesting are also being explored, such as detachment technologies. In one example (the UpCell Surface), adherence to a surface is switched on and off with changes in temperature, thus allowing the grown cells to detach as sheets without the need to use enzymes that could affect their properties. It is possible that harvesting cells as sheets rather than individually may be cheaper and easier at larger scale.

A second alternative involves the use of biodegradable scaffolds for growth of therapeutic cells. The plant-based material can be digested by cellulase without impacting the viability and make-up of the product cells, according to Fowler. He also notes that enzymatic detachment from microcarriers is being investigated, but this method requires careful selection of enzymes that will not affect the crucial aspects of the cell structure.

Article DetailsBioPharm International
Vol. 28, No. 9
Pages: 28–31, 39

Citation: 
When referring to this article, please cite it as C. Challener, "High Titers and Perfusion Processes Challenge Cell Harvesting System," BioPharm International 28 (9) 2015.