Recent advances in (therapeutic protein) drug development

Protein engineering

The manufacturing and production of therapeutic proteins are highly complex processes^1–3. For example, a typical protein drug may include in excess of 5,000 critical process steps, many times greater than the number required for manufacturing a small-molecule drug⁴ (Figure 1a).

Figure 1. Complexity of therapeutic protein drugs.

(a) Graphical representation of the complexity of the manufacture of a therapeutic protein drug compared with a small-molecule drug. The number of batch records, product quality tests, critical process steps, and process data entries associated with small-molecule drugs (black) and therapeutic protein drugs (grey) as bars⁴. (b) Illustration depicting the differences in size and complexity of a protein therapeutic (recombinant (r) analogue of human coagulation factor VIII (FVIII); Novoeight, Novo Nordisk; molecular weight = 166,000 Da) and a small-molecule drug (ibuprofen; molecular weight = 206 Da) by molecular model.

Similarly, protein therapeutics, which include monoclonal antibodies as well as large or fusion proteins, can be orders-of-magnitude larger in size than small-molecule drugs, having molecular weights exceeding 100 kDa (Figure 1b). In addition, protein therapeutics exhibit complex secondary and tertiary structures that must be maintained. Protein therapeutics cannot be completely synthesized by chemical processes and have to be manufactured in living cells or organisms; consequently, the choices of the cell line, species origin, and culture conditions all affect the final product characteristics^5–7. Moreover, most biologically active proteins require post-translational modifications that can be compromised when heterologous expression systems are used. Additionally, as the products are synthesized by cells or organisms, complex purification processes are involved. Furthermore, viral clearance processes such as removal of virus particles by using filters or resins, as well as inactivation steps by using low pH or detergents, are implemented to prevent the serious safety issue of viral contamination of protein drug substances⁸. Given the complexity of therapeutic proteins with respect to their large molecular size, post-translational modifications, and the variety of biological materials involved in their manufacturing process, the ability to enhance particular functional attributes while maintaining product safety and efficacy achieved through protein-engineering strategies is highly desirable.

While the integration of novel strategies and approaches to modify protein drug products is not a trivial matter⁹, the potential therapeutic advantages have driven the increased use of such strategies during drug development. A number of protein-engineering platform technologies are currently in use to increase the circulating half-life, targeting, and functionality of novel therapeutic protein drugs as well as to increase production yield and product purity (Table 1)^{5–7,10–12}. For example, protein conjugation and derivatization approaches, including Fc-fusion^13,14, albumin-fusion¹⁵, and PEGylation¹⁶, are currently being used to extend a drug’s circulating half-life¹⁷. Longer in vivo half-lives are of particular importance to patients undergoing factor/enzyme/hormone replacement therapy, in which frequent dosing regimens can result in substantial negative impacts on patient well-being in terms of ease of administration and compliance, especially in young children¹⁸. Protein-engineering approaches have also been employed to target drugs through the addition of signaling peptides or the generation of antibody-drug conjugates¹⁹, thereby limiting toxicity and increasing drug efficacy. Additionally, exploiting particular functional characteristics of a protein drug can be accomplished through protein engineering. For example, influencing a protein’s glycosylation pattern through engineering strategies can impact the protein’s receptor-binding properties and overall effector function^20,21. In Table 1, we have highlighted a few examples of the many technological innovations and protein-engineering platform technologies incorporated by recently approved therapeutic proteins.

Overview of recently approved protein therapeutics (2011–2016*)

Since 2011, the U.S. Food and Drug Administration Center for Drug Evaluation and Review (CDER) and the Center for Biologics Evaluation and Review (CBER) combined have approved 62 recombinant therapeutic proteins (*January 1, 2011, through August 31, 2016; “Purple Book” list of licensed biological products, including biosimilar and interchangeable biological products²²) (Figure 2a). Of these 62 therapeutic proteins, almost half (48%) were monoclonal antibodies (for this analysis, we included antibody-drug conjugates and antibody fragment antigen binding in this group). Coagulation factors were the next largest class (19%) of approved protein drugs over this time period. Replacement enzymes comprised 11% of all approvals. Remaining approvals (22%) were divided among fusion proteins, hormones, growth factors, and plasma proteins (Figure 2b). These U.S. Food and Drug Administration (FDA)-approved therapeutic proteins are indicated for a wide variety of therapeutic areas. Over half of the approved therapeutic proteins were indicated for oncology (26%) and hematology (29%), whereas the remaining 45% had primary indications in cardiology/vascular disease (5%), dermatology (3%), endocrinology (6%), gastroenterology (2%), genetic disease (2%), immunology (6%), infectious diseases (3%), musculoskeletal (8%), nephrology (2%), ophthalmology (3%), pulmonary/respiratory disease (3%), and rheumatology (2%) (Figure 2c, left). Of the 16 oncology drugs, six were approved to treat hematologic malignancies, whereas the remaining therapeutics were indicated for dermatology (3), gastroenterology (2), obstetrics/gynecology (2), pediatrics (1), pulmonary/respiratory disease (1), and urology (1) (Figure 2c, right). For a complete listing of the approved products, see Table 2. It is evident that recently approved therapeutic proteins serve a wide spectrum of patient populations and are of great benefit to public health.

Figure 2. U.S. Food and Drug Administration (FDA)-approved therapeutic proteins (2011–2016*).

(a) Bar graph showing the number of therapeutic protein FDA approvals by year (2011–2016*). (b) Pie chart showing the distribution of FDA-approved therapeutic proteins (2011–2016*) by drug class. (c) (Left) Pie chart showing the distribution of FDA-approved therapeutic proteins (2011–2016*) by therapeutic area. (Right) Pie chart showing the distribution of secondary therapeutic area for oncology drugs. *January 1, 2011, through August 31, 2016.

Pathways for the development of novel therapeutics

The rapid advances in biomedical science and technology to address unmet medical needs also require that regulatory agencies ensure that such products are safe and effective. Several new pathways have emerged or have been finalized since 2011 and are summarized below (Table 3).

Emerging trends, challenges, and opportunities

As protein-engineering technologies and regulatory frameworks evolve over time, so do protein therapeutics. Optimized versions of existing therapies can be achieved through better drug targeting as well as enhancing potency and functionality. By understanding the mechanism of action as well as the structure-function relationship of a protein, rational design and engineering strategies allow the modification of its activity or the introduction of new activities, leading to customization of existing proteins or the generation of novel therapeutics for specific clinical applications. Here, we will highlight just two examples of rational modifications to existing protein drugs achieved through protein engineering that have led to the approval of novel second-generation therapeutics (see Box 1 and Box 2).

Over this examination of the recently approved therapeutic protein drug landscape, several emerging trends have become apparent. We anticipate that the inclusion of pharmacogenetics information in drug labeling and the importance of “companion diagnostics” will become the focus of increased attention. The FDA has been encouraging drug developers to collect and submit pharmacogenomics data through a guidance, “Pharmacogenomic Data Submissions”, issued in March 2005³⁶. Pharmacogenetics can play an important role in identifying responders and non-responders to medications, avoiding adverse events, and optimizing drug dose. Drug labeling may contain information on genomic biomarkers and can describe drug exposure and clinical response variability, risk for adverse events, genotype-specific dosing, mechanisms of drug action, and polymorphic drug target and disposition genes. Therefore, pharmacogenetic profiling is of particular importance when potential drug candidates exhibit highly variable safety, efficacy, or pharmacokinetics profiles. In January 2013, the FDA issued a guidance, titled “Clinical Pharmacogenomics: Premarket Evaluation in Early-Phase Clinical Studies and Recommendations for Labeling”, to assist drug developers with conducting exploratory pharmacogenomic investigations, enrichment strategies for clinical trials, adaptive trial designs, or companion diagnostics³⁷. Pharmacogenetic information and changes in drug labeling can lead to drugs targeted for different populations, personalized dosing regimens, and companion diagnostics. There have been 11 therapeutic proteins approved since 2011 that have included pharmacogenetic biomarkers in their drug labels (Table 6). For a complete listing of drugs with available pharmacogenetics information, see the FDA’s Table of Pharmacogenomic Biomarkers in Drug Labeling³⁸. The FDA’s review process will continue to adapt as the incorporation of pharmacogenetic information becomes more commonplace. This will also require a coordinated cross-center review to incorporate the companion diagnostic/sequencing in the drug development/licensure process.

It is reasonable to anticipate that proteins will be more extensively engineered in the future. This means that the new generation of therapeutic proteins will carry neo-sequences not found in nature. Thus, the potential risks of immunogenicity (undesirable immune responses to therapeutic proteins)³⁹ will also increase and, in turn, demand new technologies for immunogenicity risk assessment and mitigation^39,40. Protein engineering is no longer restricted to altering the primary sequence of proteins. On the other hand, the rapidly growing trend of codon optimization involves the substitution of synonymous codons to improve protein synthesis and increase protein production^41,42. A growing scientific literature suggests that although synonymous codons do not alter protein sequence they can have profound effects on protein folding and function^43–45. Consequently, these therapeutic proteins designed by using such strategies will have to be carefully evaluated.

Finally, a confluence of computational and high-throughput experimental methods for protein-engineering and “off the shelf” platform technologies has ushered in unprecedented opportunities to develop safe, effective, and more convenient protein therapeutics. These opportunities do come with risks but rapid advances in new technologies as well as the underlying science suggest that these risks can be managed.