Engineered Proteins as Tools in ADC Development and Manufacture

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There has been a growing interest in antibody-drug conjugates (ADCs) as cancer treatments because of their ability to enhance the selectivity of chemotherapeutic agents while limiting systemic exposure and adverse effects. The development and manufacture of these complex molecules, however, can be a challenge.

“The typical manufacturing process for a monoclonal antibody (mAb) begins with a production cell line and progresses through filtration and chromatography steps to capture and polish the final drug substance,” says Deepan Shah, team leader at Orla Protein Technologies. He explains that an ADC manufacturing process, however, involves additional steps of linker coupling and toxin coupling, followed by ultrafiltration/diafiltration to remove excess toxin and linker. “The antibody, linker, and toxin each must be characterized both structurally and in terms of impurities before they can be added to the process,” he adds, stressing that it is crucial to ensure the absence of antibody-degradation products, critical contaminants, and drug/linker-related impurities such as degradation products, residual solvents, and heavy metal ions. ADC development begins with the selection of target antigen and antibody, mAb production, payload-linker synthesis, followed by ADC optimization of conjugation and purification conditions, analytical method development, and formulation and stability studies. Production is then scaled up for GMP manufacturing.

“Therapeutic antibodies are generally targeted to antigen with high abundance in tumor cells and low expression on normal tissue to limit toxicity and maximize efficacy,” Shah explains. He adds that linkers with attachment sites for both the antibody and drug are used to join the two components. “Numerous techniques and linker chemistries targeting different sites on antibodies have been developed,” he notes. “Most methods rely on conjugation to exposed cysteine or lysine residues resulting in populations with heterogeneous drug-antibody ratios (DARs).” Shah points out that because low drug loading reduces potency and high drug loading can negatively impact pharmacokinetics (PK), DARs have a significant impact on clinical efficacy. “The linker must remain stable in systemic circulation to minimize adverse effects, yet rapidly cleave upon endocytosis,” he says. “Once inside the cell, the drug is released through hydrolysis or enzymatic cleavage of the linker or via degradation of the antibody. Typically, the unconjugated drug should demonstrate high potency, ideally in the picomolar range, to enable efficient cell killing upon release from the ADC.” Shah highlights that method development to optimize all these characteristics is, therefore, vital prior to manufacture and clinical use of ADCs. Advances in bio-orthogonal chemistry and protein engineering have facilitated the design of optimal ADCs. Shah spoke to BioPharm International about the tools used in ADC development and manufacture, including the application of the OrlaSURF protein engineering platform.

Assays for ADC production

BioPharm: What are the different assays used in ADC production?

Shah: There are several important quality attributes that require careful monitoring during the design of an ADC production process.

• The DAR and the drug distribution (i.e., the number of antibody molecules containing 0, 1, 2, 3, etc… drug molecules) are key parameters since individuals in a heterogeneous population may have differing PK properties. These parameters are most commonly monitored by ultraviolet-visible (UV/VIS) spectroscopy and mass spectrometry; but many other methods (1) are available depending of the specific properties of the antibody, linker, and drug. DAR and drug distribution can be driven to narrower distribution ranges by careful optimization and control of the reaction conditions, such as temperature, solvent, reagent ratio, pH, and reaction time.

• The ability of the ADC to bind to its target antigen, become internalized, and release the toxic cargo are all crucial steps that determine the efficacy of the ADC. The first step in this process (antigen recognition and binding) is the most likely to be adversely affected by the treatments necessary for ADC production. Organic solvents, pH, and temperature shifts can all cause damage to an antibody. Therefore, the ability of the antibody to recognize and bind to its target needs to be monitored throughout the development. Commonly used assays are dot blots and enzyme-linked immunosorbent assay (ELISA) to assess binding, and cell kill assays that test the ability of the ADC to recognize and kill the target cell.

In addition to these key attributes, further downstream in the production process, the purity and stability of the ADC are also important. The presence of contaminants such as buffer excipients, unreacted toxin, and endotoxin must be tested. Stability in circulation must be investigated. Tolerance to freeze thaw and other ambient temperature shifts during storage must be determined.

The industry, therefore, requires cost-effective analytical tools that allow rapid decision-making during the development process to facilitate progress of the ADC to the clinic and beyond. In particular, the ability to monitor the binding of antibody to its target during ADC method development and manufacture is key to its success.

Protein engineering platform

BioPharm: How does the OrlaSURF protein engineering platform work?

Shah: The immobilization of polypeptide target molecules on microtiter plates, biosensor matrices, and microcarriers and resins is key to the sort of binding assays described previously. Current methods rely on adsorption or chemical coupling of peptides or proteins directly to a surface; it is difficult to control the structure and orientation of proteins when immobilized. In many cases, this leads to inaccuracies in the analytical determination of functional product. OrlaSURF technology enables the production of binding proteins that can be used to develop ELISAs, analytical biochips, and other tools for rapid in-process assays. The basic technology involves the fusion of the ADC target antigen to a proprietary surface-binding unit (SBU). The SBU is a small protein entity that has inherent ability to self-assemble as a monolayer on gold surfaces but it also binds tightly to many other substrata such as glass and plastic and biological polymers. The SBU is highly amenable to modification by fusion of other proteins and peptides. Orla has constructed a toolkit of vectors to enable a variety of fusions to be generated (N-or C-terminal, within loops), allowing the display of the antigenic target moiety in a structural milieu that most closely resembles that of its natural state. Subsequent immobilization via the SBU results in the presentation of the antigen in the correct orientation with near 100% preservation of its structure and function. Once a fusion of the ADC target antigen with an OrlaSURF SBU has been created, it can be attached to ELISA plates, surface plasmon resonance (SPR) biochips, quartz-crystal microbalance (QCM) biochips, bio-layer interferometry tips, and any other substrata by a simple apply-and-wash process requiring no chemical reactions.

BioPharm: What are the applications of OrlaSURF in ADC process development? Can you provide some examples or case studies?

Shah: OrlaSURF technology can be used for the development of target-binding assays to monitor the binding of ADC to its antigen during method development and optimization, and also for process monitoring.

One example is the development of a binding assay for Roche’s Herceptin (trastuzumab) in collaboration with Glythera, an ADC company, to facilitate their design of experiment process for evaluation of reaction conditions in ADC production. We created a fusion of the binding site for Herceptin on Her2 protein into a loop structure of an OrlaSURF SBU. The new protein, named ORLA254, was produced as inclusion bodies in E. coli, purified, and refolded in vitro using proprietary redox refolding techniques. The binding of trastuzumab to ORLA254 immobilized on ELISA plates was superior to that on native Her2-Fc fusion. The ORLA254 assay was identical on cheap, untreated polystyrene plates and also on more expensive treated plates (Nunc Maxisorp and Polysorp), whereas the “native” Her-2 Fc fusion did not show binding at all on untreated polystyrene. ORLA254 protein and ELISA protocols were transferred to Glythera where they have been in routine use for the method development for ADC production. Glythera have demonstrated a clear correlation between binding to ORLA254 and cell kill ability of treated antibodies and this correlation adds a valuable analytical tool to inform and speed up their ADC development process.

Reference

1. A. Wakankar et al. mAbs 3 (2), 161-172 (2011).

Article Details

BioPharm International
Vol. 30, No. 4
April 2017
Pages: 20-21

Citation

When referring to this article, please cite it as A. Siew, "Engineered Proteins as Tools in ADC Development and Manufacture," BioPharm International 30 (4) 2017.