Meet Big Tuna: versatile, automated buffer exchange

Screening of formulation buffers to improve the stability of biologics is a crucial process that is time-consuming and labor-intensive. This has been often considered a rate-limiting factor in biologics development.

The chemical, conformational and colloidal stability of a protein is highly impacted by the buffer solution. Varying buffer salts, pH, excipients, ionic strength and surfactants might increase or decrease the molecule’s stability.

Traditional exchange methods are laborious, susceptible to discrepancies and hard to control in bigger numbers. The current methods available for buffer exchange exhibit numerous limitations, all of which are aggravated at higher throughput.

Automated buffer exchange systems have the ability to allow highly uniform sample handling and degrees of process control that are hard to achieve through manual methods. Big Tuna has been designed to bridge the gaps in low-volume and high-throughput buffer exchange (Figure 1).

Big Tuna automates buffer exchange.

Figure 1. Big Tuna automates buffer exchange. Image Credit: Unchained Labs

Big Tuna makes use of a pressure-based ultrafiltration/diafiltration (UF/DF) technique to remove buffer.

At the time of pressure-based filtration, the plate is gently mixed, which ensures that sample flow remains uniform,  and that protein is not accumulated at the membrane surface. Both advantages contrast with centrifugation filtration methods, where flow may decrease as protein concentrates at the membrane surface.

The buffer exchange process has been automated by Big Tuna while decreasing hands-on time and increasing throughput. Big Tuna also allows sample concentration to a new, specific target following the completion of the exchange.

Buffer exchange with Big Tuna is highly tailorable and adaptable, thereby enabling buffer exchange of up to 96 proteins and formulations in a single experiment.

Unchained Labs has designed two filter plate formats for this process. The Unfilter 96 and Unfilter 24 are filtration plates engineered for the pressure-based UF/DF buffer exchange process (Figure 2).

Unfilter 96 can process up to 96 samples varying from 100 to 450 µL; Unfilter 24 can process 24 samples ranging from 0.45 to 8 mL in one run. Both Unfilter types are used with 10 kDa, 30 kDa, and 100 kDa Molecular Weight Cut Off (MWCO) membranes. The Unfilter 96 also has a 3 kDA MWCO version available.

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 100–450 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 100–450 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 process of Buffer exchange can be unpredictable. The exchange rate is highly governed by solution viscosity, which in turn is governed by the sample concentration, formulation, protein and volume. At higher throughput, it is more likely that samples have different viscosity and have different exchange rates.

Uniformity is maintained by stopping the pressurization and measuring the volume for every sample using an ultrasonic sensor. Using this measurement, the amount of eliminated volume and the amount of the new buffer to be added back prior to the next cycle are estimated. The flow rate is calculated for each sample, and the pressurization time for the next cycle is adjusted to maintain sample integrity. This improves the buffer exchange process in real-time.

Users can set the degree of exchange for every cycle, or percent buffer removal, to make sure that a protein is not over-concentrated at the time of the exchange. This measure also guards against making drastic changes to the formulation that would lead to other aggregation events.

High percent removal per cycle can be used wtih low protein concentrations, or exchange into similar buffers to complete the exchange faster. A lower percent removal can be used with high concentrations of protein, proteins sensitive to aggregation or large formulation changes. This exchange occurs slower than the fastest possible method but it is more gentle for proteins that may require more delicate handling.

Through automation, Big Tuna reduces the hands-on time. Prior to the run, the Unfilter 24 or Unfilter 96 is loaded with protein to be exchanged and positioned in the exchange chamber, and new buffers are positioned on the deck.

When the process is in progress, Big Tuna shifts between volume measurement, filtration and new buffer addition (Figure 3).

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

This article describes proof-of-concept experiments run on Big Tuna, showing its ability to execute buffer exchange in several formats and meet final concentration targets that cannot be met through manual methods.

Methods

Protein and buffer preparation

Human IgG (hIgG) was made nominally at 10 mg/mL or 100 mg/mL in PBS, pH 6.8. Proteins were manually pipetted into a 10 kDa Unfilter 96 or Unfilter 24 before each buffer exchange. Proteins were buffer exchanged into PBS with a pH of 6.8.

Protein concentration

The Lunatic (Unchained Labs) was employed for studying the concentration of all samples before and after buffer exchange. The concentration of protein was identified with the A280 application on Lunatic with the help of the E1% unique to hIgG.

For each well, final concentrations were quantified and reported as the average concentration over the Unfilter 96 or Unfilter 24 ± standard deviation.

Buffer exchange capabilities

With Big Tuna, the operator can fix a total percent exchange of up to 99.99%. The exchange percent was set to 96% total exchange for all experiments reported in this article. The total exchange for the pool was set to 96% when sample wells were determined as duplicates.

Big Tuna enables the operator to select the percent volume removal per cycle. Apart from experiments to compare Dilute and Filter (50% removal/cycle) and Filter and Refill (25% removal/cycle), 66% removal per cycle was used in all other experiments.

The other abilities for buffer exchange methods have been explained within their respective experiments. Experimental design and execution were performed using Big Tuna software. After logging the results, further concentration analysis was performed offline.

Results

Buffer exchange with Unfilter 96

Buffer exchange of a stock of hIgG (10.5 mg/mL) was performed into PBS, pH 6.8. Each well of an Unfilter 96 was filled with 450 µL manually. For each pool, the target percent exchange of 96% was achieved in three cycles of about 20 minutes each.

As shown in Table 1, the average percent exchange over the Unfilter 96 was 96.3%.

Table 1. Big Tuna buffer exchanges human IgG into PBS in 96 wells of an Unfilter 96 at 450 µL per well. Source: Unchained Labs

Variable Initial Target final Actual final
Conc. (mg/mL) 10.5 10.5 10.4 ± 0.2
Well fill vol. (μL) 450 450 457 ± 9
% exchange >96 96.3

 

It took 2.6 hours of total run time to finish the buffer exchange of one protein exchanged into one buffer on an Unfilter 96. For each well, the final fill volume was similar to the initial fill volume, at 457 ± 9 µL.

This shows uniformity over the Unfilter 96 and no considerable variation between the initial and final fill volume for each well. After the buffer exchange, the average concentration across the Unfilter 96 was 10.4 ± 0.2 mg/mL, which met the target (Table 1).

Buffer exchange and 3x concentration with Unfilter 96

In this experiment, the exchange of protein was exchanged into buffer and then concentrated three-fold following the completion of the exchange. Buffer exchange of a stock of hIgG (9.7 mg/mL) was performed into PBS pH, 6.8. Protein at 450 µL was added to each well of an Unfilter 96.

For each pool, the target percent exchange of 96% was achieved in four cycles lasting roughly 18 minutes each. Each cycle’s duration was automatically adjusted to ensure that the average volume eliminated per cycle was roughly the target of 66% exchange per cycle.

The average percent exchange over the Unfilter 96 was 98.6% (Table 2).

Table 2. Big Tuna buffer exchanges human IgG into PBS in 96 wells of an Unfilter 96 at 450 µL per well, followed by three-fold concentration to 150 µL. Source: Unchained Labs

Variable Initial Target final Actual final
Conc. (mg/mL) 9.7 29.1 31.9 ± 2.5
Well fill vol. (μL) 450 150 146 ± 5
% exchange >96 98.6

 

Following buffer exchange, human IgG was concentrated three-fold with a target final concentration of 29.1 mg/mL. A single concentration cycle was needed after buffer exchange to concentrate the protein to meet the target of 29.1 mg/mL. The concentration cycle lasted for about a minute.

The final average concentration over the Unfilter 96 was slightly above the target at 31.9 ± 2.5 mg/mL. Due to the three-fold concentration step, final fill volume per well was targeted to 150 µL. The final fill volume per well, which was at 146 ± 5 µL, is similar to the target (Table 2).

It took a total run time of 3.5 hours to finish the buffer exchange of one protein exchanged into one buffer followed by a three-fold concentration step on an Unfilter 96.

Buffer exchange with Unfilter 24

Buffer exchanged of a stock of hIgG (10.8 mg/mL) was performed into PBS, pH 6.8. To each well of an Unfilter 24, 8 mL was manually transferred. For each pool, the target percent exchange of 96% was achieved in three cycles that lasted approximately 58 minutes (56–60 minute range).

The average percent exchange over the Unfilter 24 was 97.7% (Table 3). A total run time of 4.3 hours was needed to complete the buffer exchange of one protein exchanged into one buffer on an Unfilter 24.

For every well, the final fill volume was quite similar to the initial fill volume per well at 8,001 ± 73 µL. This value was also consistent throughout the plate with no considerable difference between the initial and final fill volume.

The average concentration across the Unfilter 24 after buffer exchange was 10.9 ± 0.1 mg/mL, which met the target (Table 3).

Table 3. Big Tuna buffer exchanges human IgG into PBS in 24 wells of an Unfilter 24 at 8,000 µL per well. Source: Unchained Labs

Variable Initial Target final Actual final
Conc. (mg/mL) 10.8 10.8 10.9 ± 0.1
Well fill vol. (μL) 8,000 8,000 8,001 ± 73
% exchange >96 97.7

 

Buffer exchange and 3x concentration with Unfilter 24

This experiment was performed by exchanging protein into buffer and then concentrating it three-fold following the completion of the exchange. Buffer exchange of a stock of hIgG (9.8 mg/mL) was performed into PBS, pH 6.8. To each well of an Unfilter 24, 8 mL was manually added.

For each pool, the target percent exchange of 96% was achieved in three cycles of about 60 minutes (58- to 62-minute range). Each cycle’s duration was automatically adjusted to make the average volume removed per cycle roughly the target of 66% exchange per cycle.

The average percent exchange over the Unfilter 24 was 97.0% (Table 4).

Following buffer exchange, Human IgG was concentrated three-fold, with a target final concentration of 29.4 mg/mL. The average concentration over the Unfilter 24 after buffer exchange was 32.2 ± 0.5 mg/mL, which is slightly above the target.

As a result of the three-fold concentration step, the final fill volume per well as quantified by the on-deck volume sensor was targeted to 2,667 µL. The final fill volume per well was quite similar to the target at 2,680 ± 15 µL (Table 4).

Table 4. Big Tuna buffer exchanges human IgG into PBS in 24 wells of an Unfilter 24 at 8,000 µL per well, followed by three-fold concentration to 2,667 µL. Source: Unchained Labs

Variable Initial Target final Actual final
Conc. (mg/mL) 9.8 29.4 32.2 ± 0.5
Well fill vol. (μL) 8,000 2,667 2,680 ± 15
% exchange >96 97.0

 

A total run time of 4.4 hours was taken to complete the buffer exchange of one protein exchanged into one buffer followed by a three-fold concentration step on an Unfilter 24.

Filter and Refill buffer exchange method

Filter and Refill is a default method that initially concentrates the protein to the percent volume removal target and then adds new buffer back to the original volume.

Buffer exchange of a stock of hIgG (99.6 mg/mL) was performed into PBS, pH 6.8. To each well of an Unfilter 24, 4 mL was manually transferred. For each pool, the 96% target percent exchange was achieved in 12 cycles lasting about 110 minutes per cycle (range: 99.4–126.6 minutes/cycle).

For each cycle, the duration was automatically adjusted so the average volume eliminated per cycle was roughly the target of 25% exchange per cycle. The average percent exchange over the Unfilter 24 was 96.9% (Table 5).

The final fill volume per well was similar to the target at 3,993 ± 25 µL. The average measured concentration over the Unfilter 24 after buffer exchange was 100.9 ± 1.6 mg/mL, which met the target (Table 5).

Table 5. Big Tuna buffer exchanges human IgG into PBS in 24 wells of an Unfilter 24 at 4,000 µL per well, using the Filter and Refill method. Filter and Refill first concentrates protein down to target, then refills wells with the new buffer in each cycle. Source: Unchained Labs

Variable Initial Target final Actual final
Conc. (mg/mL) 99.6 99.6 100.9 ± 1.6
Well fill vol. (μL) 4,000 4,000 3,993 ± 25
% exchange >96 96.9

 

The complete run time to finish the buffer exchange of a high concentration protein in an Unfilter 24 through the Filter and Refill method was 24.6 hours.

Dilute and Filter buffer exchange method

The Dilute and Filter method involves diluting the starting protein using the new buffer and then commencing the exchange. Dilute and Filter might allow higher concentration exchanges to process quickly.

Buffer exchange of a stock of hIgG (99.7 mg/mL) was performed into PBS, pH 6.8. To each well of an Unfilter 24, 4 mL was transferred manually. Each well was diluted to a total of 8 mL with 4 mL of PBS by Big Tuna.

For every pool, the target percent exchange of 96% was achieved in five cycles lasting about 115 minutes per cycle (range: 1.3–145.2 minutes/cycle).

For each cycle, the duration was automatically adjusted so the average volume removed per cycle was roughly the target of 50% exchange per cycle. The average percent exchange over the Unfilter 24 was 97.0% (Table 6).

Table 6. Big Tuna buffer exchanges human IgG into PBS in 24 wells of an Unfilter 24 at 4,000 µL per well, using the Dilute and Filter method. Dilute and Filter first dilutes each well with new buffer then concentrates protein down to target in each cycle. Source: Unchained Labs

Variable Initial Target final Actual final
Conc. (mg/mL) 99.7 99.7 98.7 ± 1.3
Well fill vol. (μL) 4,000 4,000 4,009 ± 18
% exchange >96 97.0

 

A total run time of 11.4 hours was taken to finish the buffer exchange of a high concentration protein by making use of the Dilute and Filter method in an Unfilter 24. This time is less than half the time of the Filter and Refill method.

Viscosity is reduced by diluting the sample before filtering it, thus enhancing the flow rate. A diluted protein sample flows faster and ensures a higher percent volume removal per cycle. This helps further reduce the total buffer exchange time.

For exchanging a larger volume of protein, the default Filter and Refill method will efficiently exchange protein over a longer period of time (Table 7).

Table 7. High protein concentration buffer exchange using Filter and Refill versus using Dilute and Filter on Big Tuna. At high protein concentrations, viscosity slows buffer exchange. The Dilute and Filter method dilutes proteins before buffer exchange, decreasing viscosity and total buffer exchange time. Source: Unchained Labs

Variable Filter & refill method Dilute & filter method
Total run time (hours) 24.6 11.4
Time under pressurization (hours) 22.1 9.5
Number of cycles 12 5

 

Conclusion

Big Tuna enables automated buffer exchange of up to 96 samples of 100–450 µL, or up to 24 samples of 0.45–8 mL in one run. It is the only automated buffer exchange platform with the flexibility to handle both low- and high-volume samples.

Big Tuna can execute high-throughput buffer exchange in various formats with the least hands-on time. It enables a controlled buffer exchange procedure by allowing users to choose their preferred percent exchange, buffer exchange method, volume removed per cycle and final concentration.

Initial and final protein conditions — like concentration, well volume and percent exchange — were found to be consistent over 10 kDa Unfilter 96 and Unfilter 24 in all six experiments performed in this article.

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 10:29 AM

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