Stringent rules apply to process chromatography for the biopharmaceutical industry. Buffer in-line dilution is a popular way to obtain the required concentration of the fluid for the chromatography purification process.
One system, designed by Lewa, combines the chromatography process and buffer in-line dilution in a single platform. For this purpose, the company has developed a design combining multiple functions into a single system. This platform offers isocratic or linear gradient elution in addition to a built-in buffer in-line dilution capability. This functionality could benefit the user in process flexibility, higher quality and space savings. The Intellidrive mechatronic drive developed by the company, supplies a large, but precise pump adjustment range and allows the Ecoprime system to provide an industryleading single skid flow range and accuracy.
In many critical production systems, such as the biopharmaceutical sector, the process of metering and mixing liquids plays a central role. The same is particularly true of chromatography. "To ensure the ability to use concentrated buffers for chromatography processes with the precision required, the system needs to be designed accordingly," explains Joachim Regel, Sales Manager Bioprocess Systems Europe at Lewa. "The buffer dilution must take place before the chromatography so that the required concentration of the fluid can be achieved for this process step." The proprietary system design from Lewa combines chromatography with the buffer dilution in one system.
During the process, concentrated buffers are supplied and diluted to the exacting concentration required by the various chromatographic steps. The concentration value of these buffers may be five, ten or more times the required value. Concentrated buffers provide the user advantages in saving space, shipping costs and ease of handling in valuable cleanroom manufacturing space.
While two pumps are being used to supply buffers, the third pump delivers a diluent (typically purified water or WFI) that is supplied to the system until the desired fluid concentration is reached. Then, the diluted buffers can be used for the chromatography process. The entire process can be carried out within a single system, effectively eliminating large tank farms that had been a traditional holding area for low concentration buffers.
"When using buffers for chromatography, it is important to make sure that the concentrates do not precipitate before they are diluted," says Regel. "It is necessary to determine the solubility of each buffer precisely before it reaches the system to be used." In addition, the effects of the specific buffer concentrations on the materials must be taken carefully into account. The whole system is designed using wetted materials appropriate for both compatibility and strict regulatory demands.
The integrated buffer in-line dilution option offers the advantage of handling smaller liquid volumes. This allows buffer concentrates to be filled into single use bags which are another recent cost saving innovation in the biopharmaceutical industry. If single-use bags are used, however, the buffer bag tubing that feeds the Ecoprime system must be sized appropriately. At very extreme flow rates, bag tubing connections that are too small can result in pump performance issues such as pump cavitation. In such nuanced parameters, Lewa can provide the end user schemes that aim to provide optimal life-time performance.
Ecoprime provides for two unit operations on the same skid. Also, possible short comings of sensors (e.g. conductivity and/or pH) used to control the final mix ratio of buffers are taken into consideration. For instance, pH probes have the tendency to ‘drift’ over time resulting in degradation of read-out or continual intervention by operators skilled in re-calibration (which results in downtime). Additionally, sensors are only an indirect quality indication which does not directly measure the chemical composition of the blended components. The distance between the pumps and the sensors can cause time-delays to the pump feedback control. Time delays in monitoring generally depend on the placement of such in-line measurements a distance downstream of the pumps when used as feedback control devices. This delay can result in wasted (out of spec) buffer.
The Ecodos metering pumps combined with digital motors, provide the user with reliable volumetric blending. The pump is known to be more accurate and reproducible than in-line sensors. Adding digital motors (servo motors) further enhances the metering precision. A form of this combination of servo motors and metering pumps in chromatography systems has been patented by the company. Ultimately, the user of volumetrically controlled buffer in-line dilution systems achieves a more accurate, precise and reproducible measure of the buffer constituents without recalibration and/or sensor delay feedback.
The buffer in-line dilution process step is configured into the software. The company has designed different recipes that allow users to create alternate methods that suit their production requirements. An example is software allowing the determination of a fixed percentage with regard to the total flow rate and establishing the buffer concentration factor for regulating the supply and target concentration factors of the desired final fluid concentration. Such methods allow the same concentrate to be used in different process steps at different concentrations, based on the software configuration.
"In the case of isocratic elution, for example, two buffers are used for the (1:10) dilution, where the total flow rate is 5.0 l/min," explains Regel. "90% of this total flow rate is conveyed from the pump for diluting the two buffers, the configuration of the concentrate buffer percentage must correspond to the remainder of the total flow rate, giving us 10%, or 0.5 l/min.” A similar procedure is carried out for the gradient elution. During buffer in-line dilution, two buffers (e.g. a 1:10 dilution) are diluted for a three-step gradient elution. This gradient elution involves one linear segment followed by two isocratic steps. The total flow rate is 5.0 l/min here as well.
"The pumps installed in the systems operate along with the servo drive with maximum precision, allowing high reproducibility of the configured parameters," says Regel. "Compared with other systems, a considerably higher precision is achieved when the Ecoprime system with Lewa Intellidrive technology is used." The combination of ‘variation of the speed’ and ‘partial stroke mode’ offers an adjustment range of 1:150 or more. While conventional technology requires a frequency converter and an electrical stroke adjustment for this purpose, which is supplied via separate power and data connections, the Intellidrive only needs one connection. This allows a more advanced solution to be provided in a cost effective manner.
Techniques in piping design downstream of the pumps proprietary to the company enhance mixing and minimize hold-up, both very critical to proper chromatographic performance. In-line pH or conductivity probes, which tend to show discrepancies over time and require cumbersome calibration, are made obsolete by the conveying capacity, as are the associated unnecessary process variables brought into the mix by such sensors. The system is cleanroom-compatible and designed for use in GMP environments.