The application of conjugation chemistry to biopharmaceutical drug development is becoming ever more popular and sophisticated. Generally, this development strategy blends the functions of different molecular entities to enable specific targeting of therapeutic payloads. Such combinations have included protein-pharmaceutical, protein-peptide, protein-oligonucleotide and peptide-oligonucleotide.
Along with this merging of the fields of biochemistry and chemistry comes the need for special considerations in the analytical approaches taken as a demonstration of veracity of the final product which can be particularly challenging when such diverse molecular types are linked. Any therapeutic conjugate, of which antibody drug conjugates (ADCs) are just one example, require not only the same in-depth structural analysis that any non-conjugated therapeutic would require but also come with a whole set of other structural considerations because of the conjugation process itself.
So how do we tackle this? Firstly, it must be realized that, since a protein scaffold is involved, the product requires the same areas of structural investigation laid out in ICH Q6B as would a non-conjugated protein (1). Thus, the primary structure must be analyzed, along with any cell-derived post translational modifications, glycosylation, physicochemical properties, higher order structure, and aggregation.
This analytical focus must also be applied to other conjugate structures such as oligonucleotides which also require confirmation of structural integrity such as sequencing. This is overlaid with considerations that must be borne in mind because of the chemical process used to generate the conjugate. This can be summed up in the two key questions:
Whilst, for proteins, much of this can be covered as part of the ICH Q6B investigational work, pharmaceutical considerations, such as documented in ICH Q6A (2) and other more specific guidance, will also be required for investigation of linker and drug.
To address the first of these questions, one aspect we must consider is the ratio of therapeutic to the carrier. For ADC's, this is more familiarly seen as the drug:antibody ratio (DAR). This is a measure of how much drug is conjugated to each protein molecule and is a measure of efficiency and success of the conjugation process.
Mass spectrometry in the form of LC-MS along with protein chemistry protocols is a powerful tool in the analysis of this drug:protein ratio. Since conjugation can often be a multistep process involving initial linker addition prior to conjugation of the drug to the linker, the use of mass spectrometry also allows identification of the other partially conjugated forms and can therefore provide an overall profile of the state of conjugation and the degree of completion of the conjugation process. This concept can be extrapolated to other conjugate types although often requiring bespoke procedures.
Confirmation that the correct product has been manufactured also includes an assessment of the linkage position in terms of abundance and specificity. The somewhat agnostic application of techniques such as mass spectrometry and NMR is ideally suited to these investigations but often requires sample manipulation prior to analysis.
Answering the second question regarding potential modifications as a result of conjugation can often be satisfied using methods such as peptide or oligonucleotide mapping which provide a detailed "image" of the primary structure.
A detailed investigation of the backbone is essential to assess any adverse modifications caused by the conjugation process(es). These may include:
For many protein-based products, changes to glycosyl structures should be evaluated, and this can also extend to glycan-modified oligonucleotides.
This may sound like a very large list of assessments that need to be borne in mind, and indeed careful consideration needs to be given to the effects that the chemistry may have on the conjugate. However, with appropriate high mass accuracy investigations of the overall product as well as detailed mapping studies, a clear picture can be built up of the reaction products.
The application of mass spectrometers such as Q-TOF instrumentation or other mass spectrometers that can generate fragment ions in parallel with mass data provides a means to clearly identify unknown peaks in a chromatogram or track changes through the chemical stages of product formation (see Figure 1). For ADCs, which incorporate smaller, pharmaceutical moieties, techniques such as GC-MS may also be employed as investigative tools for impurities and residual conjugates.
Beyond primary structural investigations, we must also consider physicochemical properties such as charge profiles through the use of icIEF, electrophoretic profiles (e.g. via CE-SDS), and higher order structure investigations as well as aggregation. The higher order structure and aggregation have been discussed in one of our previous blogs for ADCs, and this theory extends to the wider field of conjugates in general.
When carrying out the comprehensive characterization of any conjugated final product, it is very important to analyze the native protein or other carrier alongside the final conjugate. Furthermore, during the development of the process, the intermediate products from each conjugation step should also be analyzed using selected investigations focusing on key risk areas to assess how each chemical step is affecting the various components of the conjugate. This may cover, for example, intact mass and mapping as fundamental assessments. Data can be used to feed back to any necessary process modifications.
Figure 2 - Peptide mass and confirmatory fragmentation data for an ADC. (A) – Peptide observed in the parent mAb. (B) – Peptide observed in the intermediate product. (C) – Peptide observed in the fully conjugated ADC. The observed peptide masses are correct for the predicted conjugation events, and the fragment ions in each case confirm the peptide. The fragment ion masses are the same between the samples since the site of conjugation is towards the N-terminus of the peptide and thus not represented in the fragment ion masses observed which are derived from the C-terminal region. Nonetheless, the identity of the conjugated peptide is confirmed. Samples supplied and work carried out in collaboration with Iksuda Therapeutics.
Conjugation chemistry is becoming ever more sophisticated with the boundaries constantly being pushed as newer and more complex conjugation strategies are developed, such as multidrug, nucleotide and timed-release conjugates. Nevertheless, the basis for detailed and informative structural investigations remains the same: Use state-of-the-art instrumentation for assessment of your product through each stage of the production process and finally for a full understanding of the structure of the final product. In this way, chemistry can be monitored and processes controlled to generate the desired product.
Whether you are progressing through early development or preparing for regulatory submission, BioPharmaSpec provides expert-led analytical characterization of complex therapeutic conjugates, including ADCs, peptide conjugates, and oligonucleotide-linked modalities.
Contact our scientists today to discuss your conjugate program and receive a customized analytical approach designed around your molecule.
