Intact Mass and Cell Culture Media Workflows

Waters Bioprocess Walk-Up Solutions utilizes OneLab as a single-access platform for controlling sample preparation and LC-MS data acquisition and reporting

Cell culture or microbial-based inoculation for protein production is a time-consuming process, typically lasting about two weeks. It is increasingly desirable to routinely monitor critical process and product attributes such as changes in nutrient profiles and high-level glycoform information for the drug substance. Monitoring feed and metabolite components will aid in designing or optimizing feed strategies for nutrient replenishment; detect and quantify the formation of toxic metabolites; and can be used to elucidate what reactor conditions are conducive to optimal cell growth and bioproduction. Higher monitoring frequency of the protein formation in the process enables the determination of the most favorable cell culture duration; yield optimization; and most importantly ensures that the drug substance quality is within the specification criteria.

Moving these assays and technologies in the hands of bioprocessing groups facilitates faster access to more in-depth information for the process engineers.

This protocol, as part of a walk-up solution, enables robust, routine LC-MS analysis in bioprocess laboratories. A single interface based on OneLab software provides a seamless connection between sample input, automated sample preparation, and LC-MS data acquisition and analysis (Figure 1). 

Figure 1: Top-level flow chart for the walk-up solution using single access in the OneLab software platform

In addition, users of Sartorius Ambr® bioreactors can utilize the data interface to automatically upload all sample information and return their results back to the Ambr software for further interrogation. The interface, combined with predeveloped analytical workflows for key product and process quality attributes, are designed to allow bioengineers to perform assays generating high-quality data with minimal training in LC and MS technologies. 

Experimental

The protocol will process up to 48 clarified media samples in a 350 µL 96-well plate. The samples will be automatically diluted using a diluent that is the same as the starting mobile phase of the LC-MS assays to be run. At the end of the protocol, a single 350 µL 96-well plate is produced that contains ~180 µL samples that have been diluted to 1/20 suitable for intact mass analysis on the left side of the plate, and also 200 µL samples diluted to 1/400 on the right side of the plate that are appropriate for cell culture media analysis. Once the plate is prepared, the samples can be transferred to the BioAccord and acquisitions may be run.

Intact Mass Analysis

Once the plate has been transferred to the BioAccord, the 1/20 diluted samples will be automatically run and processed by waters_connect using the INTACT Mass application (Figure 2). For more details on application features and functionality, please refer to the application note (Reference 2).

Figure 2: Intact Mass Dashboard. Results are displayed in a simple layout containing: a simple data review workflow and a plate map (A); major glycoforms reported per sample (B); and optionally access to chromatographic integrations and spectral data (C, D, E) for each sample.

Cell Culture Media Analysis

Using the same plate as for the intact mass analysis, the 1/400 dilution samples are ready to be run and then processed using the UNIFI application. Samples will be screened against a comprehensive (built-in) library (Figure 3). For more details on application features and functionality, please refer to application note (Reference 3).

Figure 3: Example of overlaid trending plots (of choline and choline phosphate) across 5 bioreactors using the Cell Culture Media workflow.

This protocol provides automated sample preparation using the Andrew+™ Pipetting Robot and initiates LC-MS data acquisition for intact protein, and cell culture nutrient and metabolites monitoring in spent media. The combination of BioAccord LC-MS system and Andrew+ Pipetting robot provides the capability to process samples from bioreactor systems and process development teams quickly and easily to provide high-quality results with minimal user interaction.

Highlights and capabilities include:

• A single access and easy-to-use interface in OneLab, with predeveloped workflows, that will initiate sample preparation and subsequent data acquisition in a walk-up manner.
• Automated sample preparation and sample information transfer to maximize productivity and minimize human error.
• Acquire both intact protein and cell culture media data back-to-back based on column switching using a two-column compartment column manager. 
• Seamless collection of intact protein glycoprofiling and cell culture media composition will aid process monitoring and understanding. 
• The compact and user-friendly BioAccord LC-MS System produced excellent data quality to help support the development of more robust processes.

In conclusion, Waters Bioprocess Walk-Up Solutions will enable process engineers to easily and routinely obtain high-quality data to aid in process monitoring and optimization.

Assay notes

As part of the Bioprocess Walk-Up Solution, this protocol:

1. Manages sample information coming from bioreactors and passes the metadata through to the analytical system, removing transcription errors
2. Executes automated sample preparation using the Andrew+ Pipetting Robot
3. Dynamically scales sample preparation to the number of samples provided
4. Automatically creates a LC-MS sample run list
5. Initiates the BioAccord LC-MS data acquisition for glycoform determination using the INTACT Mass application
6. Optionally sends back intact protein (relative glycoform levels) back to be visualized as variables in Ambr® systems via a data interface
7. Creates samples to be run for cell culture media analysis

Clarified bioreactor samples (centrifuged and 0.2 uM filtered samples) are prepared for analysis via multiple sample dilution steps, creating a plate that contains samples ready for intact mass analysis (the determination of major relative glycoforms) and samples ready for spent cell culture media analysis (the determination of levels of feed components and metabolites).

For this protocol and assays, the samples are required to be centrifuged and filtered prior to plating for sample preparation and executing the program.

Figure 4: Andrew+ OneLab deck setup for the Intact Mass and Cell Culture Media workflows. Samples and reagents locations on the Andrew+ deck: [1-2] Tips, [5] Samples in 96 well microplate, [6] 96 well microplate will transferred in the BioAccord, [9] 8-channel reservoir contains 0.1% Formic acid reagent

Ordering information

Andrew+ System Components: Dominos, Devices, Electronic Pipettes & Tips

• Andrew+ Pipetting Robot
• OneLab software Enterprise deployment
• OneLab Intact Mass Integration module | p/n 716012207
• 2× Tip Insertion System Domino | p/n 186009612
• 2× Microplate Domino | p/n 186009600
• 8-Channel Pipette Reservoir Domino | p/n 186009613
• Andrew Alliance Bluetooth Electronic Pipette, 1-ch 10 μL | p/n 186009769  
• Andrew Alliance Bluetooth Electronic Pipette, 8-ch 10 μL | p/n 186009768
• Andrew Alliance Bluetooth Electronic Pipette, 1-ch 300 μL | p/n 186009606
• Andrew Alliance Bluetooth Electronic Pipette, 8-ch 300 μL | p/n 186009607
• 2× Pipette Adaptor Single Channel | p/n 186009590
• 2× Pipette Adaptor Multi-Channel | p/n 186009591

Additional suggested items

• 2 additional Tip Insertion System Domino | p/n 186009612; ℹ️ It will remove the manual refill of tip boxes during the execution with over 24 samples.

Consumables

• Waters 350 µL 96-round well collection plate (Pk100) | p/n 186002643
• Polypropylene Cap Mat Round Well for 96-well Plate (Pk50) | p/n 186002483
• INTEGRA 10 mL multichannel reservoir | p/n 4332
• BH Tip 0.1-10 μl, Refill (10x96) Pk10 | p/n 700013293
• BH Tip 5-350 μl, Refill (10x96) Pk10 | p/n 700013297

Columns

• ACQUITY Premier HSS T3 Column 1.8 µm, 2.1 x 150mm, 1/pk | p/n 186009469
• ACQUITY Premier Protein BEH C4 300 Å 1.7 µm, 2.1 x 50 mm, 1/pk | p/n 186010326

Certificates

• BioAccord System Install Certificate | p/n 741000510
• Andrew+ Robot System Installation Cert. | p/n 741000516
• Remote Andrew+Robot Sys IMPL Certificate | p/n 741000521

References

1. Waters Application Note 720008062EN - Simplifying Bioreactor In-Process Monitoring With Waters Bioprocess Walk-Up Solutions.
2. Waters Application Note 720007547 - INTACT Mass™ - a Versatile waters_connect™ Application for Rapid Mass Confirmation and Purity Assessment of Biotherapeutics (https://www.waters.com/nextgen/ee/en/library/application-notes/2022/intact-mass-a-versatile-waters-connect-application-for-rapid-mass-confirmation-and-purity-assessment-of-biotherapeutics.html).
3. Waters Application Note 720007359 - Monitoring Nutrients and Metabolites in Spent Cell Culture Media for Bioprocess Development Using the BioAccord LC-MS System With ACQUITY Premier (https://www.waters.com/nextgen/us/en/library/application-notes/2021/monitoring-nutrients-and-metabolites-in-spent-cell-culture-media-for-bioprocess-development-using-the-bioaccord-lc-ms-system-with-acquity-premier.html).