Want a less stressed QC lab? Discover how automation can brighten your days.
Maybe you’ve experienced this scenario a few too many times.
Your boss comes into the office in the morning and causes immediate stress. He needs the results of your antibody analysis on his desk THAT EVENING.
Ugh! You can’t help but wonder, maybe he forgot how long such an analysis takes and how many manual steps are necessary. After all, assessing the quality attributes of a monoclonal antibody (mAb) sample is not a simple task. As you can see in Figure 1, the process involves enzymatic digestion to fragment the large protein into smaller peptides. The samples are subsequently analyzed via LC-UV-MS, and the results must be evaluated, reported, and signed.
Figure 1. Schematic of a workflow of mAb impurity analysis, including sample preparation, data acquisition, data processing, and reporting.
With this highly manual process, there’s plenty of room for human errors to creep in — first preparing your standard, then during digestion, and data interpretation.
Even one small pipetting error can set your analysis back an entire day, which won’t make you or your boss happy.
Although you can’t fully control what happens inside your vials or instruments, you can help reduce the likelihood of manual errors by automating your workflow. Here’s a look at ways to automate your mAb workflow to save time and minimize errors.
Automate your sample digestion to improve reproducibility.
Anyone who analyzes proteins in the lab knows sample preparation is complex and time-consuming. And the more complex your preparative pathway, the more error-prone your method becomes.
Not to mention, manual digestions are more susceptible to human error than automated techniques. Subtle differences in pipetting techniques and enzyme reaction conditions can hinder repeatability. The combined effects of many small errors may lead to inaccurate peptide quantitation and poor method precision.
Eliminating imprecisions from manual digestion through automated sample preparation is one practical way to improve method reproducibility. You can also look at implementing a double digest protocol instead of a single digest to help reduce the likelihood of missed cleavages that can lead to inaccurate quantitation.
A typical double digest protocol includes:
- Mix buffer, sample, and reducing agent in a reservoir
- Digest at 70°C for 15 min and shake at 1400 rpm
- Cool down the heater & shaker to 40°C
- Transfer the entire solution to a fresh reservoir
- Digest at 40°C for 30 min and shake at 1400 rpm
- Centrifuge the sample and then cool to 10°C
- Add formic acid to stop the reaction
Automation of the sample preparation for the digestion of mAbs is easily done with the KingFisher Duo Prime Purification System in combination with a commercially available SMART Digest Kit with magnetic beads. The benefits are simply a fast and simple standardized protein digestion.
In no time, all the components of your kit are put together and the digestion process runs completely automatically, allowing you to enjoy a coffee break.
And if you’re anything like me, coffee makes you happy!
Automate your data acquisition, processing, and reporting to save time and reduce processing errors.
To ensure your instrument and method work as expected, you should always start with a control sample for a system suitability test (SST) before running your target samples.
A typical sample analysis involves:
- Inject and process blank samples to check for interferences in the retention time range where the target analytes elute
- Inject SST sample(s) then process and evaluate if SST criteria passed (peak asymmetry, resolution, relative standard deviation of retention time, and peak area)
- Inject and process blank samples to check for carry-over from previous injections
- Inject target sample(s)
- Inject and process blank samples to check for carry-over from previous injections
- Inject check sample(s)
- Process target and check samples based on requirements
- Create a report of the results
- Sign report and jump for joy
This multi-step manual data evaluation process is another step you can automate in your Chromatography Data System (CDS).
To begin, you enter all the necessary SST criteria, such as peak asymmetry, resolution of critical peak pairs, peak retention time, area precision limits, and an interference/ carry-over test case into your processing method and start your analysis.
When using Thermo Scientific Chromeleon CDS, your SST samples are automatically injected and compared against these criteria because an intelligent software feature allows “on the fly” data evaluation before the next injection.
All you have to do is check whether your instrument is still running because that tells you all test criteria are successfully met. Then, your target samples can run while you have another cup of coffee, and you’ll still have time to enter the test criteria for the target samples into the processing method.
After each injection, the software automatically checks if all the target compounds are detected and if the relative abundance of your peptides is in the expected range. The last thing you’ll need to do is check the report and see if the columns of “Test Results” (Figure 2A) and “Relative Abundance CHECK” pass or fail (Figure 2B).
Figure 2. Reporting: Confirmation of the presence of target peaks in a certain RT window (A) and report of relative quantitation results for glycopeptides in the sample (B).
If your analysis is successful, you can electronically sign and send the report to your boss.
Now you and the boss are happy and enjoying a coffee break together.
Be happier by boosting your productivity with automation (and coffee!)
Automating your antibody analysis is one example, and you can create automated analytical workflows in research and development, manufacturing quality control, and academia.
Fully using the features in your chromatographic software allows manual data evaluation and report creation steps to be automated quickly and easily in a fully compliant way. Download our technical note to see the workflow and data presented here.
Figure 3. Chromatograms of MS Quantitation channel (A) and UV channel at 214 nm (B) using automated workflows. Peak numbers correspond to respective peptide sequences and retention times.
Additional references:
- Performing automated system suitability testing and intelligent run control
- Automated system suitability test with intelligent run control for peptide mapping QC assays
- An integrated LC-MS system performance evaluation test for peptide mapping and monitoring
- Automating the Thermo Scientific SMART Digest Trypsin Kit with the Thermo Scientific KingFisher System
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