Industrializing Design, Development, and Manufacturing of Therapeutic Proteins

The authors discuss various approaches and related issues, including production of difficult-to express proteins using cell-free expression systems, scalability of protein expression, and site-specific chemical modifications.
May 01, 2011

From an industrial research and development (R&D) perspective, the design and development of protein therapeutics today appears somewhat akin to the rational design of small-molecule discovery back in the 1970s when lead compounds were generated from known physiological substrates or ligands. Facing a need to find novel and diverse small-molecule leads, attention in the 1980s centered on high-throughput screening (HTS) technologies and compound libraries. Those libraries, albeit large, were hardly diverse, with most therapeutic agents coming from a few target protein classes. Complementation of libraries with natural products, the development of combinatorial chemistry, and application of focused-library sets followed. This evolution, together with automated methods for content-rich assay systems and fast make-test cycles, enhanced discovery of novel, potent, and diverse lead series.

Contrast this with the present analagous processes for protein therapeutics: the discovery and development of novel biologics is hardly diverse, efficient or rapid. State-of-the art protein discovery and development use multiple expression hosts (e.g., mouse, E.coli, Chinese hamster ovary (CHO), and NS0) and several reformatting steps between hosts are often necessary during testing, scale-up, and production. The process of developing cell-based protein expression systems that are efficient, consistent, and scalable often is difficult and sometimes impossible using currently available technology.

To date, more than 150 protein drugs have been approved for clinical use, nearly all of which are produced in cell-based expression systems, such as E. coli, CHO cells, and Saccharomyces cerevisiae (S. cerevisea). These cell-based systems have several limitations, and many biologics can't be developed in these systems. For example, these systems only allow the overexpression of proteins that don't affect the physiology of the host cells. For many expression systems, identifying cell lines that stably synthesize high protein titers of the desired product is a time-consuming and labor-intensive process. Ideally, the same production host for rapid variant discovery, production for animal testing, and manufacturing of a clinical candidate would be used.

Ideally, one would want to emulate the huge leap made in iterative drug design seen in small-molecule discovery, namely, rapid make-test cycles and generation of multiple parallel libraries of drug candidates with diverse structural elements to optimize activity while maintaining feasibility for manufacture. An ideal system would do the following:
  • Make fast make-test cycles a prerequisite for re-iterative design on the order of three to five days, similar to those for focused small-molecule libraries
  • Create efficient and rapid expression and purification that allows for libraries of hundreds to thousands of protein-sequence variants to be simultaneously tested per make-test cycle using standard off-the-shelf robotics equipment
  • Incorporate preferred sequences defined from selection technologies, such as ribosome or phage display, into whole protein therapeutics for testing
  • Enhance the diversity of chemical structures by expanding the library of available amino acids at specifically targeted points in the protein sequence from 20 natural to many hundreds of non-natural amino-acids
  • Optimize several properties (e.g., agonist or antagonist, affinity, stability, and predictive manufacturability) simultaneously through rapid high-throughput make-test cycles
  • Create processes that are not only rapid but amenable to rapid scale-up and cGMP manufacturing once the desired therapeutic construct has been identified.

As ambitious as such a system would seem, several exciting technologies are emerging that improve expression systems and enhance diversity to enable modification of intrinsic properties of proteins, such as enzyme catalytic efficiency or binding. Others combine different properties in single therapeutics by conjugation chemistries. Further emerging technologies can lead to more rapid and parallel expression of many protein drug candidates. Getting all of these desirable technologies into a single amenable platform that has the flexibility to be scaled and support cGMP manufacturing is in sight.

Advances in development

Early improvements in endogenous protein-based therapeutics produced new, commercially successful therapeutics with desirable properties by simply extending sequence incorporating fusion to proteins such as the constant fragment of antibodies (Fc) or by PEGylating to increase half-life. Beyond these early approaches, considerable effort to produce ever-more elegant constructs that combine two separate functions have been made. One promising approach, antibody drug conjugates (ADCs), involves using a targeting antibody to known tissue selective cell-surface antigens or receptors to target conjugated toxins or cytotoxic drugs and so enhance selectivity over normal tissue.

Successful design of effective ADCS is complex and requires linking cytotoxic drug payloads to tumor-targeting antibody constructs. The selection of an ideal antigen target for optimal internalization and specificity for tumor tissues is critical. The design of linkers that are stable in circulation, but cleave when internalized in tumor cells to release the cytotoxic drug, adds to the complexity, but the other major technical hurdle has been to define how the cytotoxic payload with linker are conjugated to the targeting antibody. The biopharmaceutical companies Seattle Genetics and Immunogen have developed robust platforms that depend on conjugation of linkers and cytotoxic warheads to available cysteine or lysine residues, respectively, in the tumor targeting antibody sequence.

Despite successes with ADCs, there are many examples where seemingly optimized functional components (i.e., antigen-binding motif, linker, and drug-conjugate) do not translate into a developable therapeutic candidate. ADCs produced using conjugation chemistries to endogenous cysteines and lysines inevitably lead to the production of multiple species of the ADC with the drug conjugated in varying payloads of between one and nine molecules per immunoglobulin G (IgG). Furthermore, all sites of conjugation are not equal. Some conjugations interfere with antigen-binding epitopes, thereby reducing binding affinity and/or drug half-life (1). All too often, poor efficacy is revealed in the clinic only after significant investment in cell-based expression systems and scale-up.