Balancing Confidence and Accuracy
Debadeep Bhattacharyya

Balancing Confidence and Accuracy

LC-MS-based methods for biologics are still evolving, but better workflows are bringing us closer to the payload. The first of a three-part blog series.

Antibody Drug Conjugates (ADCs), also known as immunoconjugates, aim to combine the potency of cytotoxic drugs with the high specificity of a monoclonal antibody (mAb). This class of therapeutics are becoming increasingly important, especially as a treatment against cancer. [1] Two ADCs, brentuximab vedotin (Adcetris®) for the treatment of Hodgkin's lymphoma and ado-trastuzumab emtansine (Kadcyla®) for breast cancer, have been recently approved by the US Food and Drug Administration (FDA), and 30 others are currently in clinical trials. [2, 3, 4, 5] ADCs are created by chemically attaching highly potent cytotoxic agents to monoclonal antibodies (mAbs) that are specific to tumor-related antigens with cleavable and non-cleavable linkers. Depending on the conjugation chemistries, different ADC structures have been developed. The drug payloads can be randomly attached to surface-exposed lysine residues distributed on both light and heavy chains (8-95 for every IgG). Such conjugation can be observed in the case of Kadcyla®. [6] ADCs can also be created by site-specific coupling to two or more of the eight cysteine residues involved in inter-chain disulfide bridges of chimeric, humanized, or human IgG1 after mild reduction, as noted in the case of Adcetris®. [7] The glycan moiety can also be subjected to mild oxidation and used for site-selective conjugation via a hydrazine linkage. [8] In third-generation ADCs, antibodies are designed to contain free surface-exposed cysteines for site-specific drug linkage and optimal drug loading. Site-specific conjugation to antibodies results in more homogenous drug loading and avoids ADC subpopulations with altered antigen-binding caused by cross-linking in the complementary determining regions, or altered pharmacokinetics caused by cross-linking at Fc domain binding sites of FcRn. Several third-generation ADCs have recently been described, including the addition of two cysteines in the antibody variable domains [9] or in the constant domains. [10, 11,12]

Conjugation of drugs to mAbs increases the structural complexity of the resulting molecule, which triggers the need for improved characterization methods. [13] In addition to the inherent complexity of mAbs, the chemistry of linkers, drugs and their biotransformation pathways complicate the characterization and bioanalytical workflows for ADCs. Moreover, the liquid chromatography-mass spectrometry (LC-MS) based platforms typically used in analysis of small molecules, are still evolving when it comes to the field of biologics. While triple quadrupole mass spectrometers are extremely efficient in performing sensitive bioanalytical assays of small molecules, they fail to reflect ADC drug load distribution changes. In certain cases drug payload can prematurely deconjugate from ADCs by multiple mechanisms, higher drug-loaded species can undergo faster clearance, the dynamic nature of the drug-load distribution can affect the PK/PD modeling, and the catabolism/metabolism related changes can have an impact on safety and/or efficacy.

While most ADC analysis can be addressed by immunoassays (e.g. ELISA), the use of tandem mass spectrometry (MS/MS) or High Resolution Mass Spectrometry (HRMS) can offer significantly improved information such as:

  • Identification and quantification of unconjugated (i.e. free) drug
  • Identification of catabolites/metabolites
  • Determination of the drug-antibody ratio (DAR)

From development and production workflows for characterization and quantification of ADCs, SCIEX has developed a host of robust, reproducible, accurate, and sensitive solutions in close collaboration with several leading pharmaceutical organizations. In this first of three blogs, we highlight two of these solutions and their components whilst demonstrating how their optimal combination enables ADC characterization.

Running Protein Interference

Heterogeneity and high molecular weight species pose challenges for analytical scientists and both of these problems converge with ADCs. Early in development, rapid feedback on how well a conjugation strategy may have worked is critical. Later, as process development accelerates, confirmation is required that the product has maintained its integrity (e.g. in formulation). Essential experiments in ADC characterization therefore involve the determination of:

  • Intact molecular weight of the construct
  • Information about DAR
  • Extent of drug linkage

Figure 1
Figure 1. ESI-MS spectrum of an intact ADC using a TripleTOF®5600+ Spectra

Figure 2
Figure 2. ESI-MS spectrum of an intact ADC using a TripleTOF®5600+ with a differential ion mobility applied demonstrating removal of interferences

While these determinations may appear straightforward, in reality, the task is significantly more complicated owing to the heterogeneity of the constructs, the number of impurities or fragments should synthesis be at an early stage of optimization, and the presence of interfering compounds such as those found in formulation. Overcoming these difficulties can require time-consuming sample preparation, involve reducing the construct to its component parts to simplify analysis, or may even necessitate fractionation. Such procedures risk sample modification thus making it difficult to draw firm conclusions regarding stability.

To illustrate some of the challenges in ADC characterization, Figure 1 shows the electrospray ionized high resolution time-of-flight (TOF) spectrum of an intact ADC based on an IgG1 molecule.

In the spectrum there are a number of interfering species, some of which are visible at the lower end of the mass to charge ration (m/z)scale (< m/z 2500). The deconvolution of this spectrum provides only a gross, uninformative reconstruction. Fortunately, the interfering species can be removed using SelexION™ technology. SelexION™ is a device that fits at the source of the mass spectrometer and provides an orthogonal separation based on differential ion mobility [a brief description of ion mobility might be prudent]. A number of attributes make this an ideal addition to the analysis:

  • Simple voltage changes are instantly applied
  • No complex tuning required
  • Separation occurs PRIOR to detection, eliminating interference with data processing
  • An identical LC method can be used for all cases

The Lysine-linked ADC in Figure 1 elutes from the SelexION™ device at a CoV (compensation voltage) around -5V, while the smaller contaminants and related fragments elute near positive CoV values. This difference in CoV allows for the acquisition of the ADC raw spectrum without interfering species (Figure 2), providing a much more informative deconvoluted MS Spectrum when processed using BioPharmaView™ software (Figure 3).

Figure 3
Figure 3. Data processed in BioPharmaViewTMSoftware for an ADC in development. The panels at the bottom show raw (left) and automatically deconvoluted (right) spectra.

The results presented in this first of three blogs demonstrate that for complex biologic species, the SelexIONTM differential ion mobility device coupled to a TripleTOFTM HRMS system provides a simple and elegant solution for the analysis of intact ADCs free of interference that:

  • Doesn't affect other experimental conditions
  • Uses identical processing informatics parameters
  • Is transferable across other SCIEX MS platforms supporting SelexIONTM(e.g. SCIEX QTRAP®Systems)

In the second installment of the application of LC-MS methods for ADC determination, we'll examine techniques used in peptide mapping to identify the exact location of the drug and linker, as well as elucidate the variety of constructs.


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