Frankenbodies: How (mad) scientists are bringing new treatments to life

Sahana Mollah, senior manager, Global BioPharma Collaboration & CE Tech Marketing at SCIEX looks at how antibody variants are helping researchers develop new medicines. 

Over the past 35 years, biologic therapeutics have revolutionised the way we treat disease. Monoclonal antibodies (standard mAbs) have predominated in recent years as therapies for treating cancers and autoimmune diseases. While there are many more applications where mAbs can excel, researchers are finding better, smarter ways to expand beyond the limitations of these biologics. Intelligently designed antibody variants often referred to as “Frankenbodies” such as bi-specific and multi-specific mAbs, bi-specific T-cell engagers (BiTEs), peptibodies and nanobodies are showing promise.

Higher Performance Alternatives

Because of monospecific binding sites, standard mAbs can only interact with a single target on the cell surface. Their large size also hinders access to solid tissues and prevents them from crossing the blood-brain barrier. Frankenbodies are designed to overcome these limitations by including multiple binding sites and/or using specific fragments of conventional antibodies to provide greater in vivo stability, access to more targets and greater efficacy via multiple target binding. For example, one alternative to standard mAbs in cancer immunotherapy has been CAR T-cell therapies. However, multi-specific antibodies may provide a bigger advantage by being able to engage two or more antigens at once.

Merging Existing Technologies

Many of these new, more specific treatments are being generated by merging various components of existing biologic structures. These mAb variants can be divided into two major classes: immunoglobulin (IgG)-like and non-IgG-like.

IgG-like Frankenbodies include bi- and tri-specific antibodies, antibody-drug conjugates (ADCs), Fc-fusion proteins and more exotic types such as dual variable domain IgGs. They have at least one Fc region up to three Fab regions for binding multiple targets.

Non-IgG-like molecules do not have an Fc region. They include small fusion proteins, referred to as single-chain variable fragments (scFvs) or peptibodies, nanobodies, BiTEs, bi- and tri-specific killer-cell engagers (BiKes/TriKEs), and antibody fragments (Fab, F(ab)₂).

Greater Analytical Challenges

When Frankenbodies are manufactured, even the smallest changes in process conditions can impact the resulting product structure. Often many different variants – more than 15 in some cases – of the desired product are obtained in each batch, of which only one or two are therapeutically relevant. In addition, titers can be much lower (10–50%) for multi-specific Frankenbodies produced compared with those for mAbs.

mAbs and Frankenbodies face many similar challenges; however, these challenges are exacerbated for the Frankenbodies. The diversity and greater complexity of Frankenbody structures combined with the production of numerous variants and low titers create significant analytical challenges and thus a higher analytical burden, beginning at the clone selection stage through process development to commercial production. It is necessary to distinguish molecules with minor structural differences at low concentrations. Additional sensitivity and separation resolution are therefore essential when developing analytical methods for Frankenbodies.

Optimisation of Trusted Methods

To overcome these challenges, existing trusted methods are being adjusted for Frankenbody assays. Off-the-shelf mAb methods such as capillary electrophoresis (CE) and liquid chromatography-mass spectrometry (LC-MS) are being optimised for the development of effective solutions.

The major challenge is to adjust standard mAb methods for variants during assay development, which requires having the right sample preparation, the right separation method for analysis such as purity testing, and the correct method for glycan sequencing.

For instance, analytical systems that can run multiple characterisations for mAb assays such as CE-SDS (sodium dodecyl sulfate) for purity/heterogeneity, capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) for charge heterogeneity and fast glycan analysis for micro heterogeneity, can be very helpful. These methods can be optimised and extended for mAb-variants by increasing the percentage of reagents in the sample, using different reagents, lowering the pH, or changing the temperature and time of the analysis. Additionally, CE can provide information on the purity and structure (disulfide hinges and chain mismatching).

LC-MS is another technique that is popular for characterisation of standard mAbs and can also be optimised for mAb-variant assays. One such example is the characterisation of multi-specifics on the subunit level, using SCIEX’s TripleTOF 6600 LC-MS/MS System with SelexION differential mobility separation (DMS) technology. This technique enables separation of protein subunits and unambiguous characterization of each chain with a single injection and without the need for chromatographic separation, making data collection and analysis simple and reducing the overall time required to complete studies.

New Orthogonal Methods 

The industry has been working towards alternative orthogonal techniques (instead of just modified mAb methods) that can address the specific complexity of the variants and impurities in Frankenbodies. For example, in the development and manufacturing of nanobodies, which are approximately 10–15 kDa, intact protein analysis offers the overall picture of the protein product even offering the discovery of features as small as one–two Daltons. Unfortunately, even the ability to discriminate mass differences as small as one–two Daltons is insufficient for unambiguous identification of the actual molecular feature. Hyphenated techniques such as CE paired with mass spectrometry (CE-MS) can be ideal in these cases. Consider a two-Dalton mass shift, it could be due to a break in a disulfide bond or two deamidations. Charge variant separation via CE followed by online MS analysis can easily and unambiguously differentiate these isobaric modifications.

As advances in the space continue, companies are focused on collaborating across the biopharma, academia, and analytical sectors to find the best ways forward. These collaborations are crucial to continue innovating new applications and workflows to overcome analytical gaps and bring life-changing drugs to market faster.