A new class of therapeutics, monoclonal antibodies (t-mAbs), makes up a large and rapidly growing market (1). In this article, part two of this month’s focus on t-mAbs, we discuss different applications of mass spectrometry (MS) in analyzing t-mAbs, including strategies for sample enrichment, quantitation, and detection, as well as internal standard requirements (Part 1).

Strategies for Sample Enrichment

Because serum is a complex protein matrix, laboratories need to enrich serum samples for the analyte of interest and remove other protein fractions, mostly albumin. T-mAbs are modeled after human immunoglobulins and so must be enriched along with the human immunoglobulin (Ig) repertoire. Many extraction/enrichment options are available (Table 1) (Figure 1).

As the extraction/enrichment options become more specific for a particular t-mAb, they also become more complicated and expensive. Ammonium sulfate precipitation, a traditional method for partially purifying antibodies, is affordable and easy. At about 35% saturation, t-mAbs along with all Igs will precipitate out of solution leaving the majority of albumin and other proteins in the supernatant to be discarded.

Other techniques also enrich for all immunoglobulins, such as protein G or Melon Gel. Simplistic and economical, Melon Gel slightly favors the IgGs.

Options exist for subtype or class purification, but they add complexity and cost. For example, CaptureSelect Antibody Affinity Resins have binding specificity for an Ig class, an IgG sub-class, or lambda or kappa light chain. For an IgG1 kappa, the difference between these choosing a subtype technique over Melon Gel or ammonium sulfate precipitate is minimal, mainly cost and ease of use in a clinical laboratory or automated setting.

On the other hand, for the few t-mAbs that are of IgG4 subclass, such as eculizumab, CaptureSelect IgG4 affinity matrix en-riches for the IgG4s and allows for the 96% of other Igs to be washed to waste, enhancing sensitivity by at least 10-fold (3).

Alternatively, an anti-drug antibody may be generated to enrich specifically for a given t-mAb. This strategy needs careful crafting to capture free t-mAb and t-mAbs bound to targets, especially when the target is present in large quantities in serum. Laboratories may also use Stable Isotope Standards and Capture by Anti-Peptide Antibodies after trypsin digestion to remove unwanted peptides from the mix. This can clean up the sample prior to chromatography and detection. However, it can be difficult to find a unique peptide for the mAb of interest (4).

Choosing an Internal Standard

Before whole protein stable isotopes were commercially available, they were prohibitively expensive to have made and time consuming to wait on. As an alternative, some laboratories have used stable isotopically labeled tryptic peptides. We found these worked great as retention time standards and gave confidence to technologists in peak picking.

However, there was concern about the robustness of the assay, as the labeled peptides were not undergoing the trypsin digestion step and so would not take into account issues or differences in digestion.

Consequently, we chose a surrogate protein level internal standard, horse IgG1, to account for the digestion variation (5). Isotopically labeled full length t-mAbs are now available through Promise Proteomics and Sigma and may overcome this challenge, but they come with a high price tag.

Tryptic Peptide LC-MS/MS

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become a common platform in clinical laboratories and is an obvious choice for t-mAb assay development. Triple-quadrupole mass spectrometers are extremely sensitive, specific, and stable instruments with a proven track record of providing precise and accurate quantitative results. Triple quads are ideally suited for quantifying peptides produced after trypsin digestion of proteins, such as the ones generated from t-mAbs. When coupled to an ultra high performance LC system, the combination allows for high throughput and multiplexing of assays.

Our first lab-developed t-mAb assay was a quantitative assay for infliximab. Our development strategy was to detect and quantitate unique peptides by targeting the murine variable region of the light and heavy chains of this chimeric IgG1κ t-mAb. We utilized a saturated ammonium sulfate precipitation, followed by a trypsin digest, reverse phase chromatography, and MS/MS detection (6).

T-mAb Test Development Challenges

Tryptic peptide MS becomes challenging when looking at a t-mAb with fewer or no murine sequences. We had been working with a chimeric t-mAb, which had 35% murine sequence that could be targeted for signature unique peptides. The next challenge was to develop assays for humanized t-mAbs with <5% of animal sequences.

While finding unique peptides was possible in silico, development would eventually be stalled due to specificity issues: When running con-trol samples from hundreds of patients for robustness testing, many would give false-positive values. This was due to similar sequences in their polyclonal, normal human Ig background, which may be an issue if limits of quantitation similar to immunoassays are desired.

Intact Light Chain LC-MS

To overcome the challenges of the tryptic peptide MS method, laboratories can conduct studies detecting the intact light chain of the t-mAb by utilizing a time-of-flight (TOF) or QE-Orbitrap MS (QE) method (7–9) (Table 2). Pharmaceutical researchers routinely use this technique for t-mAb quality control, and we hoped it could be adapted to the more complex serum matrix (10). The intact method allows for simplification over the more tedious tryptic digest. Serum is enriched for Igs and then denatured to give separate light and heavy chains.

While we implemented our first t-mAb clinical assay as a tryptic peptide method, we have implemented subsequent t-mAbs as an intact MS method on the QE to allow for high throughput capabilities needed in our clinical lab environment. For eculizumab, a hybrid IgG2/4κ, we took advantage of its IgG4 characteristic and utilized CaptureSelect IgG4 affinity matrix to enrich for IgG4s specifically. This increased sensitivity more than 10-fold in comparison to the Melon Gel enrichment.

Moreover, eculizumab targets complement C5, which is present in high amounts in serum. The method required a more specific enrichment to capture free and bound drug, and a harsh elution to quantify total t-mAb concentration.

We also used the QE for vedolizumab, an IgG1κ. It is enriched utilizing Melon Gel denatured. With vedolizumab being a humanized IgG1κ, the mass accuracy of the instrument and robustness of the quantitation were pushed to their limits to differentiate the t-mAb from endogenous immunoglobulins, particularly for a test with expected high volumes, low limit of quantitation, and short turnaround times.

Overall, every t-mAb will have unique characteristics that make test development challenging. By traveling on this journey, we added to our tool box insights on many different enrichment and detection techniques to facilitate future t-mAb test development.

While assessing for t-mAbs is not a one-size-fits-all approach, MS has proven to be a versatile technique capable of multitasking. With a triple quad or a quadrupole time-of-flight, a clinical laboratory could perform testing for differentiation between a t-mAb and a disease-causing clone, as well as quantitation of t-mAbs that require therapeutic drug monitoring for improved patient outcomes.

Paula M. Ladwig, MS, MT (ASCP), is a development technologist coordinator in the department of laboratory medicine and pathology at Mayo Clinic in Rochestor, Minnesota. +Email: ladwig.paula@mayo.edu

Maria Alice V. Willrich, PhD, DABCC, FAACC, is an assistant professor of laboratory medicine and pathology and medicine in the College of Medicine of Mayo Clinic in Rochester, Minnesota. +Email: willrich.mariaalice@mayo.edu


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CLN's Focus on Mass Spectrometry is supported by Waters Corporation.