APPLICATION NOTE 1 Effortless protein formulation with GRUNT Introduction Formulation development of biologics has not changed much over the last 20 years. You manu- ally prepare buffers, check the pH and then buffer exchange and concentrate your protein into your desired buffers. A traditional formulation screen can consume 2–4 days of a scientist’s time and can limit the number of formulations that can be evaluated. Designed through a partnership with Biopharma, Unchained Labs' GRUNT addresses this bottleneck (Figure 1). GRUNT combines buffer preparation, buffer exchange and a final concen- tration step into a single automated workflow. Au- tomation of manual processes like buffer prepara- tion and buffer exchange has typically come with complex hardware and software that requires a steep learning curve. GRUNT was designed with formulation scientists top of mind, making it an easy-to-use, powerful tool for the entire lab. Automated and streamlined workflow GRUNT automatically prepares up to 12 formu- lations at 1–8 mL each. First, GRUNT prepares 12 formulation buffers from stock solutions, then checks the pH and titrates if specified in the buffer recipe (Figure 2). Once GRUNT makes the buffer solutions, it then buffer exchanges a single protein into the prepared buffers to create up to 12 different protein formulations. GRUNT uses an innovative pressure-based micro UF/DF method that is ideal for small volume buffer exchange. Once 96% exchange is reached, the system can then add surfactant and additional buffer to reach the final volume (Figure 3). Innovative and versatile micro UF/DF process GRUNT’s unique micro UF/DF process combines pressure-based filtration and gentle orbital mixing to both buffer exchange and concentrate proteins. Multiple settings along with temperature control allow you to optimize the process to your molecule. The system has two main buffer exchange methods: 1. filter and refill and 2. dilute and filter which are described in Figure 4. GRUNT allows users to specify the amount of gentle orbital mixing as well as the temperature during the buffer exchange process. The buffer exchange module's temperature can be increased up to 25 °C to improve processing time or kept at 4 °C to better protect the protein. Once the buffer exchange is completed, prepared formulations can be kept at 4 °C overnight. Figure 1: GRUNT prepares up to 12 protein solutions automatically.
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ApplicAtion note
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Effortless protein formulation with GRUNT
IntroductionFormulation development of biologics has not changed much over the last 20 years. You manu-ally prepare buffers, check the pH and then buffer exchange and concentrate your protein into your desired buffers. A traditional formulation screen can consume 2–4 days of a scientist’s time and can limit the number of formulations that can be evaluated. Designed through a partnership with Biopharma, Unchained Labs' GRUNT addresses this bottleneck (Figure 1). GRUNT combines buffer preparation, buffer exchange and a final concen-tration step into a single automated workflow. Au-tomation of manual processes like buffer prepara-tion and buffer exchange has typically come with complex hardware and software that requires a steep learning curve. GRUNT was designed with formulation scientists top of mind, making it an easy-to-use, powerful tool for the entire lab.
Automated and streamlined workflowGRUNT automatically prepares up to 12 formu-lations at 1–8 mL each. First, GRUNT prepares 12 formulation buffers from stock solutions, then checks the pH and titrates if specified in the buffer recipe (Figure 2).
Once GRUNT makes the buffer solutions, it then buffer exchanges a single protein into the prepared buffers to create up to 12 different protein formulations. GRUNT uses an innovative pressure-based micro UF/DF method that is ideal for small volume buffer exchange. Once 96% exchange is reached, the system can then add surfactant and additional buffer to reach the final volume (Figure 3).
Innovative and versatile micro UF/DF processGRUNT’s unique micro UF/DF process combines pressure-based filtration and gentle orbital mixing to both buffer exchange and concentrate proteins. Multiple settings along with temperature control allow you to optimize the process to your molecule. The system has two main buffer exchange methods: 1. filter and refill and 2. dilute and filter which are described in Figure 4. GRUNT allows users to specify the amount of gentle orbital mixing as well as the temperature during the buffer exchange process. The buffer exchange module's temperature can be increased up to 25 °C to improve processing time or kept at 4 °C to better protect the protein. Once the buffer exchange is completed, prepared formulations can be kept at 4 °C overnight.
Figure 1: GRUNT prepares up to 12 protein solutions automatically.
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12 Weak acid/base stock solutions
8 Stock excipient solutions
12 Buffers Measure pH Titrate (optional)
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Acid Base
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12 Buffers
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Figure 2: GRUNT prepares up to 12 formulation buffers from 12 weak acid/base stock solutions and eight excipient stock solutions. Once the buffers are mixed, the system checks the pH against the target, and will titrate if specified. Once the pH is reached, the system will top off with water to reach the target volume.
12 Buffers
1 Protein stock funls
12 Proteinformulations
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Addbuffer
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Figure 3: GRUNT can buffer exchange a single protein in up to 12 formulation buffers automatically. GRUNT delivers protein to each funl (filtration cup) and then uses a unique pressure-based micro UF/DF method to concentrate the protein. Once 96% exchange is reached, the system can then add surfactant and additional buffer to reach the final volume.
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STEP 2Measurevolume
STEP 3pressure-based
filtration
STEP 4Measurevolume
STEP 5Add new
buffer
STEP 1Add protein
STEP 6concentrate
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protein (mAb, etc) Buffer 1 Buffer 2
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STEP 1Add protein
STEP 2Measurevolume
STEP 3Add new
buffer
STEP 4pressure-based
filtration
STEP 5Measurevolume
STEP 6concentrate
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Protein (mAb, etc) Buffer 1 Buffer 2
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Figure 4: GRUNT’s innovative micro UF/DF process has two options. In both methods, the protein is added to the funl and the system measures the volume. The filter and refill method (A) then applies gentle pressure to concentrate the solution. After con-centration, GRUNT automatically measures solution volumes in each funl, calculates the volume that was removed, refills with new formulation buffer and calculates percent exchange. The system repeats filtration, volume measurement and refill cycles until each sample reaches greater than 96% buffer exchange. The second method, dilute and filter, (B) dilutes the protein with new buffer before the filtration step, but otherwise performs the same steps as the filter and refill method.
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Simple and easy setupGRUNT is an automated platform designed for scientists, not just automation experts. To en-sure everyone in the lab can readily use GRUNT, Unchained Labs designed an intuitive software interface that includes step-by-step instructions to simplify system setup. The software is designed to allow scientists to enter stock solutions, create buffer recipes and program formulation runs at their desk or at the instrument. The home screen of the software is divided into three common user action areas, Runs, Buffer Recipes and Solution Inventory (Figure 5).
Before programming the first formulation run, the user needs to input their buffer recipes. The Recipe Creation screen is shown in Figure 6. To fit in with multiple lab workflows, GRUNT offers two options for buffer creation. The first, Specify Volumes, allows scientists to translate their own buffer recipes into GRUNT by specifying exact components and volumes. The other option is System Calculate, where you simply enter your target concentration, select your stock solutions and GRUNT calculates the volumes for you. After specifying the mode, simply enter the target buffer
concentration, buffer type and target pH. The next step is to add your stock buffer solutions and then excipients. Click on the sign to add your new stock buffer. If you don’t see the stock solution, you simply need to create a new one in the same window. Enter your excipients, specify titration, calculate amount of water to add and save your recipe — you’re done.
Once your recipes are entered, you can set up a run within minutes. From the Select Run screen (Figure 7), select Create New Run. This will open a wizard that will take you step-by-step through creating a run, figuring out solution and labware requirements and loading the system. The first step is to name the run and specify the operation mode (Figure 7). GRUNT has three modes of operation: Full Run which is buffer preparation and buffer exchange, Buffer Only for buffer preparation or Buffer Exchange Only. To concentrate proteins after buffer exchange, simply indicate a target protein concentration and GRUNT will calculate what volume to target in the final buffer exchange step. The protein information is entered later. Once the operation mode is specified, click Next to move to the next step. In the Buffers screen, click Add Buffer Recipe
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Figure 5: GRUNT home page and global navigation. Both the home page and global navigation quickly guide you to your desired task. The home page (A) is divided into the three common user action areas. The global navigation win-dow (B) can be accessed in any screen by simply clicking the logo in the left hand corner.
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to add your recipe (Figure 8). You can use all 12 channels or just a few. Once you’ve added your buffers, click next to go to the Protein Formulation screen (Figure 9). In this screen you enter the protein name, concentration and volume, along with any surfactant you would like to add after buffer exchange. If you want to concentrate during the final step, you would specify a target concentration higher than the initial. Once your formulations are set, you can move on to the Instrument Parameters screen and select your pH calibration method, temperature control and buffer exchange settings (Figure 10). Once you’ve entered your instrument parameters, the Run Summary screen lets you review your run in one simple table (Figure 11).
Once you’ve reviewed and confirmed your parame-ters in the Run Summary screen, GRUNT will create a shopping list for you (Figure 12). The Shopping List screen provides a list of all the solutions, vol-
umes, containers, pipette tips , wicking discs and funls (filtration cups) needed to run the system. The shopping list also provides handy maps that tell you exactly where each item needs to be placed.
Print the shopping list, gather your solutions and labware and then it’s time to load up the system. When you are ready to start the run, simply follow the instructions in the Pre-Run Guide screen (Figure 13). This guide provides step-by-step instructions to make sure you load the system correctly. Once you are finished, hit Start Run and GRUNT does the rest. GRUNT calibrates the pH meter and verifies pH accuracy before moving on to buffer preparation and buffer exchange. During the run, the Status Screen provides easy visibility to the status of the system (Figure 14). At the end of the run, the system will generate a report to give you complete visibility and traceability to system operation and ultimately your protein formulations (Figure 15).
Figure 6: GRUNT offers two modes of creating a formulation buffer recipe. You can specify the exact volumes of your stock solutions (Specify Volumes) or you can simply select stock solutions, enter your target concentration and GRUNT can calculate the volumes for you (System Calculate). To select or enter a stock solution, simply click the button and select from current inventory or create a new stock solution instantly.
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Figure 7: Select Run or click Create New Run to open the wizard. The first step is to enter the run name and select the operation mode.
Figure 8: Specify the formulation buffers by clicking Add Buffer Recipe for each channel.
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Figure 9: Protein information is entered into the Protein Formulations screen. Enter protein name, initial concen-tration, target concentration and volumes. You can also specify the addition of a surfactant post buffer ex-change. To concentrate as a final step, simply enter a target concentration higher than the initial concentration.
Figure 10: The fourth step in the wizard is entering your process parameters (Instrument Parameters). The user can specify 2- or 3-point calibration, along with standard solutions. Buffer exchange settings provides tempera-ture control options for both stock protein solution and the buffer exchange module. You can set the temperature during buffer exchange to 25 °C to speed up exchange of viscous proteins and then after processing, decrease the temperature to 4 °C to protect the protein.
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Figure 11: The Run Summary screen allows you to view final formulations and instrument parameters in a convenient table so you can easily review and confirm your run parameters.
Figure 12: GRUNT Shopping List provides a detailed list of all solutions and labware required to run the system. The list comes with maps and labels so you know exactly where you need to load solutions. You can also enter a lot number and expiration date for any solution if desired.
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Figure 13: GRUNT Pre-Run Guide provides step-by-step instructions on loading the system from start to finish so you don’t miss a beat.
Figure 14: The easy-to-read and color-coded Status Screen provides the critical information you need to ensure the system is running as it should.
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Unchained Labs 6870 Koll Center Parkway Pleasanton, CA 94566 Phone: 1.925.587.9800 Toll-free: 1.800.815.6384 Email: [email protected]
Effortless formulationUnchained Labs’ GRUNT brings formulation de-velopment into the modern age. The streamlined, automated workflow, innovative micro UF/DF process and fast and easy setup make protein formulation feel effortless. Any scientist or analyst can easily program a run and set up the system in less than one hour of hands-on time. Accurate formulation preparation combined with
Figure 15: GRUNT report provides detailed information on exact volumes delivered, stock solutions used, pH calibration info, time of buffer exchange and total volume of buffer used during buffer exchange. If you need more information, just click View Run Log from the Report screen.
precise pH measurement guarantees consistency. Automated buffer exchange and sample concen-tration allow you to evaluate more formulations. With GRUNT, you can finally spend your time focused on solving critical problems and formu-lation challenges while GRUNT takes care of your formulation prep work.
