Use automated buffer exchange on Big Tuna to fast track formulation pH screening

Screening formulation buffers for optimizing stability is a crucial process that requires extensive human resources and consumes time. This is a common limitation in biologics development.

The conformational, chemical and colloidal stability of a protein is deeply influenced by the buffer solution. Changing the pH, buffer salts, excipients, ionic strength and surfactants may raise or lower the stability of a molecule.

A common procedure in the process of optimizing buffer conditions for a biologic molecule is screening across a pH range.

Traditional exchange techniques are slow and laborious, prone to inconsistency, and challenging to manage in larger numbers. Automated buffer exchange systems offer more uniform sample handling techniques, increase throughput and reduce operational and hands on time.

Big Tuna was created for automating the buffer exchange process. This facilitates high throughput and offers degrees of process control that otherwise cannot be accessed by manual techniques (Figure 1). Big Tuna employs a pressure-based ultrafiltration/diafiltration (UF/DF) technique to eliminate the buffer.

Big Tuna automates buffer exchange for up to 96 unique samples with Unfilter 96 or up to 24 unique samples with Unfilter 24.

Figure 1. Big Tuna automates buffer exchange for up to 96 unique samples with Unfilter 96 or up to 24 unique samples with Unfilter 24. Image Credit: Unchained Labs

While performing pressure-based filtration, the plate is mixed gently to ensure that the protein does not accumulate at the membrane surface, while maintaining a more uniform flow and a pace quicker than dead-end filtration methods.

Buffer exchange performed with Big Tuna is highly customizable and adaptable, enabling buffer exchange of up to the range of 96 unique proteins and formulations in one experiment. Unchained labs have developed two filter plate formats for this process.

The Unfilter 96 and Unfilter 24 are regenerated cellulose (RC) filtration plates built to work with the pressure-based UF/DF method during the buffer exchange process (Figure 2). Both Unfilters are available with a 10 kDa, 30 kDa, and 100 kDa Molecular Weight Cut Off (MWCO). Unfilter 96s is also available with a 3 kDa MWCO.

Unfilter 96 is capable of processing up to 96 samples ranging from 100 to 450 μL. Unfilter 24 can process 24 samples varying from 0.45 to 8 mL in one run. Before executing the process, the Unfilter 96 or Unfilter 24 is filled with the protein to be exchanged and positioned in the exchange chamber.

Big Tuna can accommodate both Unfilter 96 and Unfilter 24. (A) Unfilter 96 allows for up to 96 samples to be buffer exchanged simultaneously at volumes of 100 μL - 450 μL per well. (B) Unfilter 24 allows for up to 24 samples to be buffer exchanged simultaneously at volumes of 0.45-8 mL per well.

Figure 2. Big Tuna can accommodate both Unfilter 96 and Unfilter 24. (A) Unfilter 96 allows for up to 96 samples to be buffer exchanged simultaneously at volumes of 100 μL - 450 μL per well. (B) Unfilter 24 allows for up to 24 samples to be buffer exchanged simultaneously at volumes of 0.45-8 mL per well. Image Credit: Unchained Labs

The new buffers are mounted on the deck. During the process, Big Tuna changes between volume measurement, filtration and new buffer addition to buffer exchange proteins, as shown in Figure 3.

Big Tuna uses a pressure-based UF/DF method with gentle orbital mixing to buffer exchange proteins with the Unfilter 96 and Unfilter 24.

Figure 3. Big Tuna uses a pressure-based UF/DF method with gentle orbital mixing to buffer exchange proteins with the Unfilter 96 and Unfilter 24. Image Credit: Unchained Labs

This article shows that Big Tuna can be used for pH screening on a monoclonal antibody (mAb) in 20 mM histidine over a pH range of 6.3–7.4. Each of the 12 formulations was exchanged in duplicate at 4 mL per well using an Unfilter 24.

Methods

Protein and buffer preparation

A stock mAb was prepared in its proprietary stock buffer at roughly 45 mg/mL. The 20mM histidine was prepared over a pH range of 6.3–7.4, at 0.1 pH increments. This adds to 12 buffer formulations. The buffers were positioned on the deck of Big Tuna before the buffer exchange.

The mAb was pipetted manually into 24 wells of a 10 kDa Unfilter 24 (4 mL/well). The mAb was exchanged into the 12 buffers prepared for pH screening. This led to 12 formulations, in 20 mM histidine over a pH range of 6.3–7.4, each sample in duplicate.

Buffer exchange

Table 1 represents important buffer exchange parameters. The buffer exchange protocol was set to 96% total exchange per pool with a target volume removal of 33% per cycle. The final well volume was set to 4 mL to maintain the same concentration before and after buffer exchange.

Table 1. Key buffer exchange parameters used for this experiment. Pressurization cycle duration was automatically adjusted to reach an average of 33% volume removed from any well. In total, 12 conditions in duplicate were run in a single experiment. Source: Unchained Labs

Parameter Setting
Target exchange percentage 96%
Target volume removed per cycle 33%
Initial concentration 45 mg/mL
Initial well volume 4 mL
Target final concentration 45 mg/mL
Target final well volume 4 mL

 

Big Tuna automatically adjusted the pressurization cycle duration throughout the whole automated buffer exchange process to maintain an average volume removed per well roughly equal to the user-defined target volume.

Big Tuna Client was employed for experimental design and execution. The outcomes were studied by exporting data to Excel directly from the Big Tuna Client. Excel was used to calculate the initial and final well volumes, average cycle duration and final percentage exchange.

Protein concentration

The concentration of the stock mAb and the 12 exchanged formulations was analyzed using Stunner (from Unchained Labs). Protein concentration was quantified using the E1% that is specific to the stock mAb. Every measurement was executed in triplicate and average concentration was reported in all instances.

Results

Stock protein formulations

The stock mAb was prepared in its stock buffer at a target concentration of about 45 mg/mL. The actual mAb concentration before and after buffer exchange and the average final volume of each formulation are listed in Table 2.

Table 2. Protein concentrations of each formulation before and after buffer exchange as determined by Stunner. Final volumes for each formulation after buffer exchange as determined by the ultrasonic volume sensor on Big Tuna. Source: Unchained Labs

Buffer
target pH
Initial
volume (μL)
Average final
volume (μL)
Initial
conc (mg/mL)
Average final
conc (mg/mL)
Average %
exchange
6.3 4,000 4,022 45.8 44.7 99.1
6.4 4,000 4,026 45.8 45.0 98.9
6.5 4,000 3,991 45.8 45.2 98.8
6.6 4,000 4,010 45.8 45.6 98.5
6.7 4,000 3,987 45.8 45.4 98.3
6.8 4,000 4,020 45.8 45.4 98.2
6.9 4,000 3,999 45.8 45.0 96.9
7.0 4,000 3,990 45.8 45.3 97.8
7.1 4,000 4,018 45.8 45.2 97.3
7.2 4,000 3,988 45.8 45.6 97.3
7.3 4,000 4,005 45.8 45.5 96.5
7.4 4,000 3,989 45.8 45.7 96.9
Average 4,000 4,004 ± 15 45.8 45.3 ± 0.3 97.9

 

Buffer exchange

A total of 10 buffer exchange cycles averaging 21 minutes per cycle (range: 19-22 minutes/cycle) were needed to achieve the target percent exchange of 96% for each well.

The time taken for each cycle was adjusted automatically to ensure that the average volume removal from any well per cycle was roughly maintained at the target level of 33% exchange per cycle.

It took 7.1 hours to complete the buffer exchange for this pH screen on an Unfilter 24. The initial fill volume per well was 4,000 μL. The final fill volume per well was ultrasonically quantified as 4,004 ± 15 μL, at the target of 4,000 μL per well.

Every pool was designated a target percent exchange of 96%, so every pool must reach a minimum of 96% exchange. Due to the changes in the flow rate of solutions, some pools exchanged to more than 96% to ensure that every pool exchanged to at least 96%.

The average percent exchange for each pool over the Unfilter 24 was 97.9%, with a minimum percent exchange of 96.5% and a maximum of 99.1%, which is illustrated in Table 2.

Although each of the 12 formulations exchanged at slightly different rates, even with those differences, the buffer exchange was achieved across the Unfilter 24, thanks to Big Tuna’s real-time optimization of the pressurization cycle duration.

Final protein concentration

Post buffer exchange on Big Tuna, the target final concentration was found to be the same as the initial concentration of about 45 mg/mL. The actual protein concentration before and after buffer exchange as quantified by Stunner is shown in Table 2.

The final concentrations were roughly the same as the target concentrations of 45 mg/mL. Final concentrations combined with final well volumes indicated that protein recovery was high for every well, even when variations were observed in the concentration.

Conclusion

Big Tuna has the ability to conduct high-throughput pH screens with very little hands-on time. Big Tuna exchanged a mAb into 12 buffer formulations over a pH range of 6.3–7.4.

An automated pH screening performed using Big Tuna enables researchers to make informed decisions to enhance biologic formulations with considerably less hands-on time.

The ability of Big Tuna to tweak the duration of the pressurization cycle after every cycle provides efficiency and accurate volume control during the exchange process.

Initial and final protein conditions, like well volume, concentration and target percent exchange, were found to be consistent over the Unfilter 24, despite the differences in formulations.

About Unchained Labs

Unchained Labs is all about helping biologics researchers break free from tools that just don’t cut it. Unleashing problem-tackling products that make a huge difference in the real science they do every day.

That’s their mantra, their promise and they own it. They live by an unconventional strategy for a start-up: they're buying commercial businesses and developed technologies, adding their magic touch to turn them into breakthrough products, investing massively in customer-facing teams and then selling those products like gangbusters.


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Last updated: Sep 17, 2021 at 10:31 AM

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