An Integrated Approach to Buffer Dilution and Storage

Most testing was performed at blend ratios of 10x since this was the highest concentrate factor proposed for our new facility. 

Blend Accuracy and Precision:

The accuracy of blended solutions produced by the inline dilution system was quantified by comparing to precise hand-made dilutions. We produced 10x blends on the skid using a concentrated acetone solution and purified water, meaning that the flow rate of acetone solution was 10% of the combined flow rate and water was 90%. Samples were taken from the outlet of the inline dilution system and the optical density (A280-A320) was measured on a benchtop UV meter. Using a sensitive benchtop scale and a volumetric flask, 100mL of concentrated acetone solution was combined with 900mL of purified water to create the hand dilutions. The optical densities of these samples were measured and average values are shown in Table 2. Blends from the inline dilution system differed by only 0.6%-1.2% from the hand dilutions. These results gave us confidence that 10x concentrates could be used in our new facility.

Impact of Pressure Disturbances:

During the transition of flow from divert to process or vice-versa, a pressure disturbance is generated due to inherent pressure differences between the two lines and from the timing of discrete valves on those lines. The sudden change in pressure will impact the flow rates of solutions controlled by the pumps and valves in each line. For example, if the pressure suddenly drops, the resistance to flow will also drop and the flow rate will increase.

Adjusting the proportional-integral-derivative (PID) tuning parameters for each pump and valve can significantly affect the time required to respond to a pressure disturbance. If flow rates are out of specification for an extended period of time, the blend ratio will be impacted and any buffers produced may also be out of specification. This can lead to significant buffer wastage and prevent successful transitions between the divert and process lines. Our goal was to optimize the tuning parameters for each pump and valve to respond quickly to pressure disturbances without compromising flow rate stability. We also tested the various flow control strategies to identify the most robust option.

The diagram in Figure 4 shows the setup used to generate artificial pressure disturbances on the inline dilution system. By toggling automated discrete valves on the divert and process lines, we could redirect flow between the two lines. Back-pressure was controlled through manipulation of a pneumatic valve on the divert line and a hand valve on the process line. By creating a pressure differential between the two lines at any given flow rate, a pressure disturbance was created once the flow transition took place. We quantified the effects of pressure disturbances on 10x blends (acetone solution and water) by measuring OD downstream of the filter housing. We leveraged this to tune our flow control pumps and valves by observing the response to pressure disturbances. We adjusted the proportional (P) and integral (I) tuning parameters to minimize the pressure disturbance effect while still maintaining flow stability. The derivative (D) constant was not changed.

(Click to enlarge image) Figure 4.

Figure 5 shows data from a 10 psid pressure disturbance test on a 10x blend with 60 LPM total flow. We examined three control strategies: 1) valve controls flow; pump at fixed speed; 2) pump controls flow; valve at fixed position; and 3) pump controls flow; valve controls backpressure in the line. The pumps and valves were well-tuned in each case. The data shows that pump control alone is not very responsive to pressure disturbances compared to other control options. We saw a 70% change in the blend ratio using pump control compared to only 10% for valve control and pump/valve control.

(Click to enlarge image) Figure 5.

We highly recommend incorporating control valves on all buffer lines in the design of an inline dilution system. This will enable more robust and sophisticated flow control strategies and provide flexibility to maximize buffer concentration factors to 10x and possibly greater.

Chromatography Gradients and Multi-step Sequences:

One requirement for the inline dilution systems in our new facility was the ability to produce gradients with concentrated buffers. A chromatography gradient involves a linear change in either conductivity or pH at constant total flow rate. To produce this type of a gradient, the inline dilution skid must be capable of blending three streams simultaneously (tertiary blend). One stream is water and the other two are concentrated buffers. The pumps, valves, and flow meters on the concentrated buffer lines must have an adequate turndown ratio to accommodate the relatively low concentrate flow seen at the beginning or end of the gradient. Flow stability at these low flow rates is critical.