Fully integrated hybrid system for mAb harvest clarification — Harvest systems for 2K single use bioreactors are a border area between single use technology and stainless steel technology, and home-made equipment set up can be found in most cases. A consistent hybrid system design with the best fit of both technologies is the better solution which transfers the responsibility for a workable system from the user to the equipment vendor.
An increasing number of commercial mAb plants is utilizing single use equipment, mainly because of the high flexibility. In most cases, the reactor size is 2000 l (the so-called 2K bioreactor), which is a standard in the industry. Up to now, 2K is the largest volume with proven track records for single use bioreactors. Up to four reactors can be operated in overlapping mode to feed one purification suite. At a typical cultivation time of two weeks, there will be two harvest operations per week. However, those facilities claiming single use are in reality hybrid systems that also utilize stainless steel equipment. This is the case for nearly all facilities with 2K bioreactors, as harvest and primary clarification will be performed by centrifugation with a separator. At a 2K-scale, harvest by centrifugation is common due to its comparable lower cost per batch compared to clarification by depth filter. Even though 2-stage depth filtration offers cost advantages at volumes below 1000 l, clarification at volumes above 2000 l will be more cost-effective by centrifugation followed by secondary depth filtration, once the utilization rate exceeds 40 %, see reference.
However, we often find a “home-made” set up in the harvest room, in which individual process equipment from different vendors is simply interconnected in a suboptimal way. Further, the user has the sole responsibility for the system.
A commonly used equipment set up for mAB harvest clarification is shown in a simplified Process Flow Diagram (page 37), with the equipment located in different rooms: The single use bioreactors in the bioreactor room, the separator and depth filter in the harvest room and the harvest bag in the purification room. A peristaltic pump is used to feed the cell culture broth from the bioreactor to the separator. The separator outlet feeds a depth filter with a break bag in between, which is used for balancing the flow of the separator and the depth filtration skid. The outlet of the filtration skid is directly connected to the harvest bag.
As both separator and main flow path of the filtration skid are made from stainless steel, cleaning in place (CIP) is required. A complete separate CIP station adds too much cost to the system, therefore very often the separator will come with an add-on CIP tank. CIP of the depth filtration skid can be done using a mobile CIP unit or simply by using cleaning buffer from a bag. All connections between the individual equipment will be done via single use hoses, in case of connections to the stainless steel equipment special designs are needed to make sure that there will be no stainless steel sections which are not included in the CIP flow path.
Using flexible tubing for the connection of the main equipment can be a problem, as there may be long distances between bioreactor, harvest and purification room with wall penetrations in between. Furthermore, the high flow rates that are required for WFI flushing of the depth filter may cause problems with collapsing of the tubing at the suction side of the feed pump. The described simple system needs many manual operations and cannot be automated to a higher degree which should be the case for commercial production systems. Last but not least it should be mentioned that the equipment set up as shown must be elaborated by the user himself, which includes also the coordination with the individual equipment vendors, which could be a cumbersome task.
Smart Process Design
The concept for a fully integrated system is shown in the detailed Process Flow Diagram on page 38, which uses consistently hybrid technology. This overcomes the problems with home-made systems and offers additional advantages. The basic concept is based on three principles:
- Isolation of the CIP tank at the separator into a stand-alone process tank as well as utilization of the tank for other purposes.
- Elongation of the inlet and outlet at the separator into stainless steel transfer lines to the bioreactor and to the single use harvest bag.
- Connection of the single use components (bags, filters) along the stainless steel transfer lines by using steam-to valves as a sanitary interface.
A proper design of an integrated harvest clarification system must take into account the necessary parameters for the main equipment, per table on page 36, which should be aligned properly in order to avoid bad design with compromises or over-engineered designs. The selected parameters are applicable to a CSC 20 separator from Gea and to a Millistak+ Pod disposable depth filter system from Merck, which are widely used for mAb harvest clarification in the industry. However, it should be noted that comparable equipment from other vendors will have process parameters in the same order of magnitude.
By following the first principle above, the process tank is used as CIP tank, delivering the CIP fluids via CIP supply pump to the objects to be cleaned. The CIP objects are the separator, the depth filtration skid and all stainless steel transfer lines. Secondly, the tank will also be used as a break tank for balancing the flow rates of separator and depth filter skid. With the third application, the process tank will be used as WFI holding tank for initial WFI flush of the depth filter. Depth filter WFI flush needs a high WFI flow rate, which may not be taken directly from the WFI point of use in the harvest room.
The Process tank will be equipped with a pump that serves three purposes: It is used as CIP pump at a high flow rate, as a feed pump for WFI flush at a moderate flow rate and as a feed pump for filtration at a low flow rate. A four-piston diaphragm pump, e.g. Quattroflow QF4400S, which offers a large flow range is well suited for this application.
By following the second principle above, stainless steel transfer lines will be installed from the harvest room to the single use bioreactor in the bioreactor room, as well as from the harvest room to the harvest bag in the purification room. The transfer line from the bioreactor room will also serve as the CIP supply line for the separator and will be cleaned together with the separator. The transfer line to the purification room will be cleaned in once-through mode to bio-waste. This integrated system is also suitable for steaming in place (SIP), which will cover the separator, the process tank, and all stainless steel transfer piping.
Compared with the commonly used home-made equipment set-up, the described integrated hybrid system needs less equipment but provides a higher automation level. Furthermore, the number of interfaces can be reduced if one vendor is responsible for the process tank, depth filtration skid and all stainless steel transfer lines.
Up-to-Date Equipment Design
The concept of using the same process tank and pump for three different purposes has been put into practice with an intelligent piping design based on valve ring technology, which is common practice for high-quality sanitary piping. Valve rings built from T-valves and Y-valves allow for full cleanability and drainability of all product wetted stainless steel piping sections without forming pockets or dead ends.
Following the third principle, all connections between single use and stainless steel equipment are done by so-called steam-to valves, which are available as reusable valves from Neumo or in a disposable version from Merck. Those valves have short distance connections to the piping and fulfil all criteria for SIP and CIP.
With the hybrid approach of the described concept, the filtration set up will consist of a stainless steel skid with full SIP/CIP capability, and disposable single use filter elements. The stainless steel skid contains the main product flow path with all instruments and valves needed for automated operation. The filter elements for depth filtration are disposable cassettes which are mounted at a movable pod filter holder. The sterilizing grade filter is a disposable capsule filter, which may be also positioned close to the harvest bag. Both filters are connected via flexible hoses with steam-to valves to the stainless steel skid piping. During facility design the quite large size for a depth filter skid must be taken into account, the picture on page 36 shows a neat skid design at which the filter holder is located underneath the skid assemblies.
Robust and Flexible
2000 l single use bioreactors are used in many projects for commercial mAb facilities nowadays. Even the bioreactor is single use technology, the primary clarification step in harvest is done in most cases by centrifugation with a separator which is stainless steel equipment. Very often the users handle by their own installation and interfacing of single use systems/components with stainless steel systems/components in a suboptimal way.
Compared to such home-made equipment set up the described approach for a fully integrated hybrid system for mAb harvest clarification provides the following features and benefits
- With the concept for a multi-purpose process vessel less equipment is needed in the harvest room.
- Transfer lines between the rooms are fix installed stainless steel pipes, no need to run long flexible hoses between the rooms.
- The requirements for large WFI volume/high WFI flow for depth filter flush can easily be covered with stainless systems.
- All product wetted stainless steel equipment/piping is designed for SIP and CIP.
- By using sanitary connections via steam-to valves a functional closed system can be realized.
- The system can be automated to a high level, only connections/disconnections of the single use filter elements are manual operations.
- The number of interfaces and vendors will be minimized by having two vendors only, one vendor for the separator and another vendor for process tank, depth filtration skid and all stainless steel transfer piping.
The basic concepts as described still allow for adaption to changing process requirements. Furthermore, the hybrid system as detailed in this article is a good example which proves that the right combination of stainless steel technology and single use technology will lead to robust, flexible and cost-effective solutions.
* * Wilfried Kappel is an Independent Consultant, Renfeng Sun Technical Department Manager, Muren Wuritu Bulk Production Process Lead, both at Chongqing Genrix Biopharmaceutical. Henry Yuan is Product Manager Single Use Technology at Austar.