26 February / March 2016
Improving Quantification of Protein Therapeutics by Standardising the Sample Preparation Approach to LC-MS/MS Analysis: High-sensitivity Bioanalysis of Infliximab, and Total Antibody Quantification of the Antibody-drug Conjugate Trastuzumab Emtansine
by Erin E. Chambers, PhD, Director of Technology Advancement, Consumables, and Principal Scientist, Waters; Mary E. Lame, Senior Scientist, Waters; and Karen Haas, Marketing, Waters.
Biotherapeutics are dominating the new pharmaceutical landscape. While liquid chromatography and mass spectrometry (LC-MS) technologies that enable robust characterisation of a protein therapeutic are available, their bioanalysis remains a challenge for laboratories moving a drug product through development. Many techniques can be used for protein quantification; researchers recognise the importance of LC-MS/MS, particularly as it’s become a standard for small-molecule pharmacokinetics.
One of the hurdles of protein quantification is sample preparation. Here we demonstrate a new approach combining pre-measured reagents and proven protocols that can simplify and standardise protein quantification methods. We provide two working examples. First, infliximab is used to demonstrate the achievement of a 10 ng/mL detection limit from 35 µL of plasma, with a step-by-step method that prepares the samples for analysis in less than six hours. Second, we use an antibody-drug conjugate, ado-trastuzumab emtansine, to demonstrate total antibody quantification. These methods ensure both the sensitivity and the transferability required for protein quantification.
In any bioanalytical assay, one of the greatest sources of variability arises from the sample preparation. This can be of particular concern when assays are transferred from sponsor to CRO or from lab to lab, whether within a single company or across multiple sites.
As more drug development efforts focus on large molecule biotherapeutics, such as monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs), traditional ‘small molecule’ bioanalytical scientists find themselves challenged not only by the complexity and time-consuming nature of heterogeneous large molecules, but also by the multitude of potential workflows that exist for protein quantification by LC-MS/MS. Method development time and expertise required are significant. Additionally, in many organisations, the expertise in peptide and protein bioanalysis, if it exists at all, tends to be a rare and
valuable skill, held by only a few individuals.
Protein therapeutics such as mAbs and ADCs are on the rise due to their target specificity, lower toxicity, and higher potency. Historically, they have been quantified using ligand binding assays (LBAs), such as the gold-standard ELISA. While these immuno-based methods are sensitive and simple to execute, poor reagent reproducibility, lack of standardisation, cross-reactivity, limited linear dynamic range, and other shortcomings have led the drive to convert to LC-MS/MS. In contrast, mass spectrometry-based methodologies offer many advantages over traditional LBAs, such as multiplexing, broad dynamic range, superior selectivity, and shorter method development times.
Complexities in the protein quantification workflow
LC-MS/MS bioanalytical workflows encompass a multitude of processing segments, each having many steps, and just as complex can be the sample preparation steps that will optimise a large molecule’s quantitative separation and detection. Decisions about specific reagents, as well as the time, temperature, and concentration of the reagents or steps can all impact analytical sensitivity, making it difficult to quickly arrive at a method that produces the desired detection limits.
The difficulty in executing or replicating bioanalytical assays is further aggravated
by the complexity of the troubleshooting required when analytical goals such as acceptable accuracy and precision guidelines or reliability and reproducibility standards are not met. Problems that arise in any of those attributes can negatively impact an organisation’s ability to make critical research and discovery-stage decisions.
Opportunities with surrogate peptides
One of the current trends in quantification of mAbs is to use surrogate peptides. Due to its high sensitivity and specificity, and the increased use of triple quadrupole mass spectrometers in bioanalytical laboratories, the surrogate peptide approach has become the most commonly used strategy for MS-based protein quantification. With this approach, the target protein is digested into smaller peptides. Then one or more unique peptides, surrogate analytes of the target protein, are then analysed by LC-MS/MS.
Standardising sample preparation can ensure consistent analyses
Proper sample preparation of the target protein and its surrogate peptides is critical to developing a reliable and robust LC-MS/ MS assay. By applying a standardised workflow to digest the protein into smaller peptides, laboratories can ensure reproducibility and successful bioanalytical
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