An Integrated Approach to Buffer Dilution and Storage

Figure 6 shows a successful gradient that we produced on the inline dilution system. The two flow rates in the plot represent 10x concentrated buffers, while the water flow rate is not shown. While there was slight flow instability at the lowest flow rates, as seen by the oscillations on the trend, this was acceptable because it did not impact the resulting final blend as seen by the yellow OD trend, which was smooth and linear, mainly because the 25 liter filter housing chamber effectively mixed the oscillations completely. We were very pleased with these results and felt comfortable implementing gradients with concentrated buffers at our new facility.

(Click to enlarge image) Figure 6.

Multi-step Sequences:

Using TotalPlant Batch software from Honeywell, we were able to string together multiple chromatography phases to test multi-step sequences on our inline dilution system-steps that were fully representative of an actual chromatography operation. The plot in Figure 7 depicts a three-step sequence. Again, the “concentrated buffers” were acetone solutions and the water flow rate, which is constant for each step, is not shown. 

Step 1- binary blend with 10x concentrate and water

Step 2- tertiary (3 stream) blend gradient with 10x concentrates and water

Step 3- binary blend with 4x concentrate and water

The gaps between the dashed lines show the time required to transition from one blend to another. During this time, the buffer is “out of specification” and is diverted to drain. Once the buffer has reached its target, the flow is transitioned through the process line and directed to the chromatography column. The “divert time” is a function of the total flow rate and size of the filter housing, since the housing must be completely flushed to establish a new blend ratio. One general rule of thumb is that it takes three filter housing volumes worth of liquid to achieve 95% removal of the previous solution.

(Click to enlarge image) Figure 7.

Inline Dilution and Disposable Bags in Our New Facility

Satisfied with the performance of the inline dilution systems tested, we gave the final thumbs up to approve installation of three inline dilution systems for chromatography operations in our new facility (see one of the installed systems in Figure 8). Each system is capable of blending 10x concentrates with potentially higher concentrations in the future. The inline dilution systems were coupled to a large-scale disposable bag system that housed 12 2500-L bag holders (fabricated by ConeCraft; Figure 9) used primarily for buffer hold operations.

Capital equipment costs represent a large percentage of the cost of a new biomanufacturing facility⁶ so the capital savings from this setup were tremendous. Instead of purchasing 12 Hastelloy tanks up to 25,000L in size, we were able to construct a very compact bag holder system. A reduction in buffer prep tank size led to further capital savings. Use of disposable bags, especially for buffer storage, is widespread in the biopharmaceutical industry⁷ and we felt comfortable using bags at such a large scale.

The use of disposable bag technology will provide process efficiencies during production by eliminating clean-in-place (CIP) and steam-in-place (SIP) operations plus reduce the water for injection (WFI) used in the facility¹. Reducing WFI demand reduced the size and utility requirements for the WFI production system in the facility. Coupled with a reduction in the number and size of chemical storage tanks, we saw further capital savings as a direct result of implementing inline dilution.

Figure 8.	Figure 9.

Summary and Recommendations

Through this extensive engineering project, we confirmed our belief that inline dilution could be a valuable tool for chromatography operations in a large-scale biomanufacturing facility. We showed that an inline dilution system equipped with precise pumps, valves, and flow meters could produce extremely accurate 10x blends at large scale, including gradients where the concentration is changing linearly over time. We determined that inline control of pH and conductivity is not necessary to guarantee accurate blends since tracking flow rate through a Coriolis mass flow meter is inherently more accurate. Careful tuning of the flow control parameters is vitally important to minimizing the effects of pressure disturbances on the system.