Author: Vanaja Raguvaran
Read Time: 7 min
Biopharmaceuticals are very diverse compounds. Their development is challenging and demands very flexible quantitative methods.
There are different LC-MS/MS approaches to the bioanalysis of large protein-based molecules.
A typical bottom-up approach involves enzymatic digestion before the quantification of signature fragments. This method shows significant strengths in sensitivity, quantification performance and multiplexing capability. Yet, it comes with some challenges. For instance, unknown biotransformation in matrices may cause misinterpretation of actual concentrations. And the extensive sample preparation may introduce artifact or additional variation.
To lower these risks, scientists have considered an alternative, top-down approach. This one focuses on intact protein quantification with accurate mass spectrometry.
Key advantages are:
- High throughput, accurate quantification and differentiation of major modifications at the intact level
- More information with the analysis of the protein as a whole molecule, overcoming the limitation of the bottom-up approach to the peptide level
- More straightforward analysis approach, avoiding potential artifacts introduced by the proteolytic digestion step
- Unique advantage to investigate biotransformation, PTM and major changes in protein forms (for example, for monitoring mAb glycoform distribution)
- For ADC analysis, evaluation of drug attachment and release at the intact level
However, this approach comes with some challenges for the data analysis:
- The signal distribution spreads over many charge states, resulting in low peak intensity
- There may be interferences from the matrix proteins
- The signal-to-noise ratio can be poor, especially at low concentrations (as a result, selecting the target m/z ion at MS spectrum is difficult, and the peak integration becomes inaccurate due to high background noise)
- The intensity of charge state ions may shift due to concentration, or pH, which might cause inaccurate quantification
Therefore, quantification using the m/z for an ion of specific charge state should not be the method of choice.
To overcome these problems, SCIEX introduces a new workflow which is an add-on module that requires a separate license to enable this feature within SCIEX OS Software 1.7.
Intact Quantification is a new protein quantification workflow based on reconstructed masses.
It can be set up to meet the required criteria for Good Laboratory Practice (GLP) and 21 CFR Part 11 compliance.
New workflow in SCIEX OS Software 1.7 - Intact Quantification:
The Analytics workspace now provides an Intact Quantification workflow that uses deconvoluted peak area of the molecule for quantification. This workflow is supported for MQ4 and Summation integration algorithms.
For large molecules, a charge state spread is typically observed in an MS full scan spectrum.
The Intact Quantification workflow provides a faster and easier-to-use quantification approach for these molecules than the alternatives:
- quantification using the m/z for an ion of specific charge state,
- or enzymatic digestion and quantification of signature fragments
It also offers a unique ability to quantify therapeutic or clinically relevant metabolites or PTM in the intact level.
To use the Intact Quantification workflow, users create a processing method that uses the Quantification workflow and then enable the Mass Reconstruction feature. The Intact Quantification feature allows users to perform these tasks:
- Change the default settings for Mass Reconstruction Processing in the Project default settings
- Optimize peak finding, reconstruction and integration parameters in the processing method
- View the XIC, Average and Reconstruction graphs in both the processing method and the Peak Review pane for the results
- Generate a calibration curve and view statistics using the deconvoluted area
- Calculate and compare the ratios of different components using the calculated column feature in the Analytics workspace
- Generate an intact quantification report or transfer Intact Quantification results to a LIMS
- Audit Intact Quantification processing activities
To read more about the Intact Quantification workflow, please download the technical note:
What else is new in SCIEX OS Software 1.7?
Mixed mode security:
A new security feature allows the user who is logged on to SCIEX OS to be either a different user than or the same user as the user who is logged on to Windows. The user logged on to SCIEX OS can be assigned to a specified role in the same way as in Integrated mode. This feature supports a group logon for Windows with a common password, while requiring users to log on to SCIEX OS with a unique user name, password, and if required, domain. If Mixed mode is selected, then the Screen Lock and Auto Logout features are available for use.
Watson LIMS support:
Users can now transfer processing results directly to a Watson LIMS from the Analytics workspace. The Watson LIMS software must be open on the processing computer. Batches can also be imported from a Watson LIMS. Export the batch from the LIMS as a text file, and then the text file can be imported into SCIEX OS.
SCIEX OS Software is our next-generation mass spectrometry software solution that offers fast data acquisition and the combined quantitative and qualitative data analysis tools that laboratory scientists need. Whether the workflows are basic or complex, from fully compliant routine analyses to totally flexible research workflows, the SCIEX OS Software platform can be configured to support them all.
SCIEX OS is available with three different feature sets:
- SCIEX OS-MQ – For quantitative processing. Replaces MultiQuant™ Software.
- SCIEX OS-Q – For qualitative analysis. Replaces the combination workflow using PeakView™ Software, MasterView™ Software, and LibraryView™ Software. It also includes the quantitative functionality of SCIEX OS-MQ.
- SCIEX OS for Acquisition – For acquisition on the X-series QTOF MS Systems. It includes all quantitative and qualitative processing functionality.
Interested in upgrading to SCIEX OS Software? And moving from from MultiQuant™ or MasterView™ Software to next-generation mass spectrometry software solutions SCIEX OS-MQ or SCIEX OS-Q to help you advance your data processing capabilities?
Contact us to learn more about the benefits of a software platform change.
Or request a free trial now.