Using Big Tuna to conduct automated platform buffer screening for multiple proteins

Screening formulation buffers to maximize stability is a crucial process that is laborious and tedious and is often considered a rate limiting factor in biologics development.

A protein’s chemical, conformational and colloidal stability is strongly impacted by the buffer solution. Modifying pH, buffer salts, excipients, ionic strength and surfactants might increase or decrease the molecule’s stability.

To ease some of the time needed to develop a new biologic molecule, a platform buffer screen is normally utilized to screen common formulation conditions to rapidly narrow down optimal buffer conditions. A platform buffer screen examines the stability of a new molecule with excipients, common buffers and surfactants in common pH ranges.

Traditional exchange methods are susceptible to discrepancies and are hard to handle in larger numbers. By contrast, automated buffer exchange systems have the ability to offer a highly uniform sample handling approach and degrees of process control that otherwise cannot be accessed through manual techniques.

The automation of a platform buffer screen can also reduce the time needed to enhance buffer conditions for new biologic molecules. Big Tuna automates the process while decreasing hands-on time and ensuring increased throughput. Furthermore, Big Tuna allows concentration to a new target following the completion of the exchange.

Big Tuna has been designed to increase throughput for low-volume, buffer exchange (Figure 1). Big Tuna removes buffer by employing pressure-based ultrafiltration or diafiltration (UF/DF) technique.

While the pressure-based filtration is in process, the plate is gently mixed, which guarantees a more highly uniform flow that is quicker than dead-end filtration techniques, and ensures that protein does not build up at the membrane surface.

By automating the buffer exchange process, Big Tuna decreases hands-on time and increases throughput.

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

Buffer exchange performed with Big Tuna is highly adaptable and flexible, enabling buffer exchange of up to 24 or 96 unique proteins and formulations in one experiment. For this process, Unchained Labs has created two filter plate formats.

The Unfilter 24 and 96 are filtration plates developed around the pressure-based UF/DF buffer exchange process (see Figure 2). Unfilter 96 can hold up to 96 samples in a volume range of 100 to 450 µL; Unfilter 24 can hold 24 samples ranging from 0.45 to 8 mL in one run. Both Unfilter types are available with Molecular Weight Cut Offs (MWCOs) of 10 kDa, 30 kDa, and 100 kDa. The Unfilter 96 is also available with a 3 kDa MWCO.

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

Prior to the run, the Unfilter 24 or Unfilter 96 is loaded with the samples to be exchanged and positioned in the exchange chamber. The new buffer is positioned on the deck.

During the process, Big Tuna shifts between filtration, volume measurement and new buffer addition to perform the buffer exchange process (see 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 discusses the use of Big Tuna to carry out platform buffer screening of four monoclonal antibodies (mAbs) in six special buffer formulations and to automatically concentrate the samples five-fold.

The platform buffer screen executed on Big Tuna included a single base buffer with one of five excipients for each of the four mAbs.

Solutions differed by flow rate, and Big Tuna tracked their volume to guarantee that no solutions dried out and everything reached the final concentration and exchange targets.

Methods

Protein and buffer preparation

The preparation of four stock mAbs (mAb A, mAb B, mAb C and mAb D) in their stock buffers was done at 10 mg/mL.

A base buffer of 10 mM histidine was made at pH 6.0 using one of five excipients: 150 mM sucrose, 150 mM arginine, 150 mM NaCl, 100 mM glycine, 75 mM mannitol, and no excipient control.

Each of the six special prepared buffers was manually pipetted into 2 mL, 96-well plates and positioned on the Big Tuna deck.

Each mAb was manually pipetted into six wells of a 10 kDa Unfilter 24 (8,000 µL/well) and was exchanged into the six buffers made for the platform buffer screen. Buffer exchange led to 24 biologic formulations, including four mAbs each in six unique buffers (Table 1).

Table 1. Formulation conditions studied on Big Tuna. A total of 24 conditions were run in a single experiment. Source: Unchained Labs

Condition Variable(s) tested
Buffer 10 mM histidine
pH 6.0
Excipients None, 150 mM NaCl, 150 mM sucrose, 75 mM mannitol, 150 mM arginine, 100 mM glycine
Antibodies mAb A, mAb B, mAb C, mAb D

 

Buffer exchange

Table 2 outlines the most important buffer exchange parameters. The buffer exchange protocol was fixed to 96% total exchange per well at a target volume removal of 66% per cycle. The final well volume was targeted to 1600 µL to achieve a final concentration of 50 mg/mL.

Table 2. Key buffer exchange parameters were user-defined in the Big Tuna software. Pressurization cycle duration was automatically adjusted to reach a maximum of 66% volume removed from any well. Source: Unchained Labs

Parameter Setting
Target exchange percentage 96%
Target volume removed per cycle 66%
Initial concentration 10 mg/mL
Initial well volume 8 mL
Target final concentration 50 mg/mL
Target final well volume 1.6 mL

 

During the entire automated buffer exchange, Big Tuna automatically adjusted the pressurization cycle duration to have the optimum volume eliminated per well roughly equal to the user-defined target volume.

Experimental design and execution were performed using Big Tuna Client. Data was exported to Excel directly from the Big Tuna Client for analysis of the results. Initial and final well volumes, average cycle duration and final percent exchange were estimated using Excel.

Protein concentration

The concentration of each of the 24 formulations and each of the four stock mAbs that resulted from buffer exchange using Big Tuna was analyzed using Lunatic (from Unchained Labs). Protein concentration was identified by using the A280 application on Lunatic with the help of the E1% specific to each of the four mAbs.

All proteins were exchanged in duplicate and average concentration was reported for all instances.

Protein stability

Uncle (from Unchained Labs) was employed to examine the stability of each of the 24 formulations for each of the four stock mAbs that resulted from buffer exchange using Big Tuna. Protein stability was determined on Uncle through dynamic light scattering (DLS) at 20 °C.

Results

Stock protein formulations

Preparation of each of the four stock mAbs in their stock buffer was set at a target concentration of roughly 10 mg/mL. The actual concentration and final well volume of each mAb before and after buffer exchange are illustrated in Table 3.

Table 3. Protein concentrations of each mAb formulation before and after buffer exchange as determined by the A280 application on Lunatic. Final volumes for each mAb formulation after buffer exchange as determined by the ultrasonic volume sensor on Big Tuna. Source: Unchained Labs

Protein   Before exchange After exchange Average
after
exchange
No excipient 150
mM NaCl
150
mM sucrose
75
mM mannitol
150
mM arginine
100
mM glycine
mAb
A
Conc.(mg/mL) 10.0 51.0 42.4 54.1 56.6 48.8 56.0 51.5
Vol. (μL) 8,000 1,593 1,928 1,621 1,586 1,765 1,573 1,678
mAb
B
Conc.(mg/mL) 10.0 51.6 55.0 42.4 51.9 57.3 58.7 52.8
Vol. (μL) 8,000 1,662 1,584 1,992 1,697 1,578 1,591 1,684
mAb
C
Conc.(mg/mL) 9.9 52.1 52.9 58.8 56.8 57.6 55.1 55.6
Vol. (μL) 8,000 1,548 1,586 1,546 1,550 1,576 1,543 1,558
mAb
D
Conc.(mg/mL) 9.7 50.5 54.3 52.9 55.6 57.9 56.8 54.7
Vol. (μL) 8,000 1,586 1,578 1,623 1,583 1,586 1,554 1,585

 

Buffer exchange

For each well, the target percent exchange of 96% was achieved after 10 buffer exchange cycles, averaging 27 minutes per cycle (range: 24–33 minutes/cycle).

Each cycle’s duration was automatically adjusted so the optimum volume eliminated from any well per cycle was roughly equal to the target of 66% exchange per cycle.

The full time taken to complete the buffer exchange of four proteins each in six buffer formulations on a single Unfilter 24 was roughly 8.5 hours.

Once buffer exchange was completed, three concentration cycles were required to concentrate the mAbs to 50 mg/mL. The average time taken for the concentration cycles was 20 minutes per cycle (range: 10–38 minutes/cycle).

The total time taken for five-fold concentration on Big Tuna was about 1.5 hours. The initial fill volume per well was 8000 µL. The ultrasonically quantified final fill volume per well was 1626 ± 115 µL, at the target of 1600 µL per well.

For every well, a target percent exchange of 96% was set and so it should reach an exchange of at least 96%. Due to the variations in the flow rate of solutions, certain wells were exchanged greater than 96% to guarantee that all wells were exchanged at a minimum of 96%.

The average percent exchange per well over the Unfilter 24 was 99.0%, where the minimum percent exchange was 96.9% and the maximum was more than 99.9% (Table 4).

Although there was a slight difference in the rates at which each of the four mAbs exchanged, notwithstanding these discrepancies, buffer exchange was successful across the Unfilter 24 as Big Tuna enhanced the pressurization cycle duration in real-time.

Table 4. Actual percent exchange for each sample after automated buffer exchange on Big Tuna range from 96.9% to >99.9%. Target percent exchange per pool was user-defined as 96%. Source: Unchained Labs

Protein No
excipient
150 mM
NaCl
150 mM
sucrose
75 mM
mannitol
150 mM
arginine
100 mM
glycine
mAb A 99.5 97.5 98.9 99.5 98.0 99.6
mAb B 97.8 99.0 96.9 97.5 99.0 97.8
mAb C >99.9 99.5 >99.9 >99.9 99.7 >99.9
mAb D 99.9 98.6 99.4 99.8 99.2 99.9

 

Final protein concentration

While performing buffer exchange on Big Tuna, for each protein, the target final concentration was five times the initial concentration, of roughly 50 mg/mL.

Table 3 displays the actual concentration of each mAb before and after buffer exchange as quantified by Lunatic.

Actual final concentrations were roughly equal to the target of five-fold concentration. Final concentrations coupled with final well volumes indicate that protein recovery was high for all wells, although there were changes in concentration.

Protein stability

DLS measurements were performed to identify antibody sizing and quality before and after exchange. There were no considerable variations in size or aggregation.

For instance, a comparison of the DLS results of mAb C stock with that mAb C exchanged into 10 mM histidine pH 6.0 with 100 mM glycine indicates no major differences in size or aggregation before and after the exchange (Figure 4).

Also, mAb D exchanged into 10 mM histidine pH 6.0 without any excipient indicated no variations after buffer exchange (Figure 5).

DLS of mAb C before and after buffer exchange on Big Tuna.

Figure 4. DLS of mAb C before and after buffer exchange on Big Tuna. Image Credit: Unchained Labs

DLS of mAb D before and after buffer exchange on Big Tuna.

Figure 5. DLS of mAb D before and after buffer exchange on Big Tuna. Image Credit: Unchained Labs

Conclusion

Big Tuna has the ability to perform high-throughput platform buffer screens of various proteins and different buffer formulations with very little hands-on time. Big Tuna was able to exchange four mAbs into six buffer formulations and concentrated the proteins five-fold following the exchange.

The potential of Big Tuna to adjust the pressurization cycle duration following every cycle ensures efficiency and avoids over-concentration during the exchange process.

Initial and final protein conditions — like well concentration, volume and target percent exchange — were found to be uniform over the Unfilter 24, despite the variations in proteins and 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.


Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: Sep 17, 2021 at 10:26 AM

Citations

Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Unchained Labs. (2021, September 17). Using Big Tuna to conduct automated platform buffer screening for multiple proteins. News-Medical. Retrieved on October 22, 2021 from https://www.news-medical.net/whitepaper/20210828/Using-Big-Tuna-to-conduct-automated-platform-buffer-screening-for-multiple-proteins.aspx.

  • MLA

    Unchained Labs. "Using Big Tuna to conduct automated platform buffer screening for multiple proteins". News-Medical. 22 October 2021. <https://www.news-medical.net/whitepaper/20210828/Using-Big-Tuna-to-conduct-automated-platform-buffer-screening-for-multiple-proteins.aspx>.

  • Chicago

    Unchained Labs. "Using Big Tuna to conduct automated platform buffer screening for multiple proteins". News-Medical. https://www.news-medical.net/whitepaper/20210828/Using-Big-Tuna-to-conduct-automated-platform-buffer-screening-for-multiple-proteins.aspx. (accessed October 22, 2021).

  • Harvard

    Unchained Labs. 2021. Using Big Tuna to conduct automated platform buffer screening for multiple proteins. News-Medical, viewed 22 October 2021, https://www.news-medical.net/whitepaper/20210828/Using-Big-Tuna-to-conduct-automated-platform-buffer-screening-for-multiple-proteins.aspx.

Other White Papers by this Supplier