Overcoming the challenges in bioanalysis of oligonucleotide-based therapies and relevance to the clinical development plan

Interviews

hugh website-minHugh M. Davis
Chief Business Officer, Frontage Laboratories, Inc., Exton, Pennsylvania (PA, USA)

As CBO for Frontage Laboratories, Dr Davis oversees all aspects of business development, sales, marketing, strategic partnerships and alliances. Hugh has over 30 years of experience in the pharmaceutical industry in R&D. His most recent position was with Johnson & Johnson (NJ, USA) where he served as VP and Head of Biologics Development Sciences in the Janssen BioTherapeutics division of Janssen R&D, LLC since 2001. In this role, Hugh participated in the development and approval of many biologic therapies including: Remicade®, Stelara®, Simponi®, Sylvant®, Darzalex® and Tremfya®. Prior to J&J, Hugh led the Pharmacodynamics & Exploratory Research Laboratory in the Clinical Pharmacology Unit at GlaxoSmithKline (NJ, USA) from 1996 to 2001.

Hugh has published over 75 manuscripts in refereed journals, book chapters and invited review articles in areas of therapeutic drug discovery, clinical pharmacology and development in immunology, oncology, metabolic disease, bone metabolism and cardiovascular medicine. He obtained his PhD from Villanova University (PA, USA) and completed his Post-doctoral Fellowship at Centocor, Inc. (PA, USA) where he patented the characterization of the CA 125 cancer antigen.

Weip headshotWeiping Shao
Vice President of Biologics Services, Frontage Laboratories, Inc., Exton, Pennsylvania (PA, USA)

As VP of Biologics Services for Frontage Laboratories, Dr Shao oversees overall operations, scientific achievement and regulatory rigor of PK/TK, immunogenicity and biomarker analysis in support of innovative biologics, biosimilars, and new therapeutic modalities including gene therapy, nucleic acid therapeutics and CAR-T cell therapy. He also oversees CLIA compliant method validation and testing, genomic services including quantitative PCR and next generation sequencing (NGS). Weiping has over 20 years of experience in the pharmaceutical / biotech industry. His most recent position was with Regeneron Pharmaceuticals, Inc. (NY, USA) where he served as Director and Head of Bioanalytical Operations. In this role, he built and led the bioanalytical organization that supported numerous regulatory submissions including the approval of Zaltrap®, Eylea®, Praluent®, Kvzara®, Dupixent® and Libtayo®. Prior to Regeneron, Weiping led the development of safety assessment strategy for drug development and the development of safety biomarkers in Safety Assessment Department at Merck & Co. (PA, USA).

Weiping has published over 34 peer reviewed manuscripts, filed US patents and co-authored the worldwide accepted Best Practices for Biobank. He often chairs conferences and has given numerous presentations. He earned his PhD from Nanjing University (China) and completed his post-doctoral Fellowship in Biochemistry at University of California, San Diego (CA, USA).

Bioanalysis and gene therapy

We are in the midst of a very exciting time with respect to clinical success for many therapeutic asset platforms. Today we can expect cures in many cases rather than amelioration of signs and symptoms. Over the years we’ve seen the huge impact of small molecule, orally delivered medicines, followed later by the delivery of protein therapeutics, such as insulin and monoclonal antibodies. These protein therapeutics substantially reduce the severity of disease across many indications and in a number of cases, have given rise to complete responses. Research and clinical work on oligonucleotide-based therapeutics has recently shown great success, too [1,2]. Lately, gene therapy, either directly or through cell therapeutic approaches, such as CAR-T, not only has been successful clinically, but also has resulted in a number of approved products in recent years [3]. Gene therapy has been recognized as an important therapeutic modality that provides the promise of disease cure. With continuous progress in preclinical and clinical research and additional effort in delivery, formulation, and pharmaceutical development we can continue to deliver on the promise of curing diseases.

Oligonucleotide and gene therapies present unique bioanalytical challenges in development in that they require extraordinary specificity and sensitivity in bioanalytical methods in order to measure their exposure and biodistribution, characterize their pharmacokinetic/pharmacodynamic (PK/PD) responses, assess the risk of any immune reaction they create and, in some cases, determine viral shedding exposure. This is the case to a far greater degree than when working in small molecule research and even with other biologics.

Because these therapies need to be delivered intracellularly to bind their target, analyzing them requires a new set of skills, equipment, platform technologies and bioanalytical methods of characterization. It is necessary to determine not only the pharmacokinetics of these agents but also how these therapies are being metabolized, their functional residence time, and the reactions that they’re driving and mitigating within the body. More importantly, for preclinical and clinical development, the bioanalytical laboratory bench within a regulatory setting has had to keep pace with the discovery bench, and few facilities have the requisite capabilities to enable a full analysis of the preclinical and clinical effects of these agents, especially under GXP regulations.

Key challenges

As the field of bioanalysis of oligonucleotide-based and gene therapies is still relatively nascent, scientists are faced with a number of challenges in preclinical studies such as determining the safety index, the therapeutic window, the maximum recommended starting dose, potential toxicities, and untoward effects such as exaggerated immune reactions. Drug developers need to be aware of, and knowledgeable about, these challenges in order to better design the clinical development program. In the bioanalytical area, scientists have a number of tools and technology platforms available for quantifying and characterizing the lead asset in preclinical studies and to characterize their effects such as PK/PD responses and safety. It is helpful to be able to quantify and understand the duration of active, delivered dose through surrogate measures in different matrices including blood, urine and tissues. A bioanalytical strategy should be developed to consider assay sensitivity, specificity, matrix interferences, and pre-existing immunogenicity. Hybridization ELISA and MSD-based immunoassays allow for extremely high sensitivity (pg/mL range) with very little sample clean-up [4]. Unfortunately, there is a need for specific reagents, and the techniques don’t provide metabolite information. For determination of specific metabolism, LC/MS/MS provides the highest specificity, with good precision and reproducibility, albeit, with the need for extensive sample clean-up. The sensitivity is good for smaller oligos (<25 mers). For a complete understanding of active concentration at the tissue level, HPLC-MS has been employed with good results. This entails tissue homogenization, liquid-liquid extraction, SPE extraction and desalting, and then HPLC-MS analysis. Understanding the concentration of the lead asset at the tissue level provides important information in determining First-in-Human (FIH) starting dose, the Maximum Recommended Starting Dose (MRSD) and the Safety Index. Active concentrations of intact lead in the plasma and tissue as well as the impact of immunogenicity on active concentration are all important considerations when putting together a minimal Physiologically-based PK (mPBPK) model and mPBPK/PD model that will enable an efficient, safe and robust FIH trial design. The application of qPCR assays have been a standard assay approach to study biodistribution and viral shedding. Additionally, cellular immunity can be characterized by detecting T-cell produced cytokines through ELISPOT although it is challenging to perform. The use of PD markers and downstream biomarkers of activity are key to developing a Recommended Phase 2 Dose (RP2D), as PK/PD and safety will drive that analysis and trial design.

Advice to Biotech and Pharma companies seeking to outsource their R&D

  • Engage your R&D partner early so that the proper bioanalytical strategy can be put in place for assay development, validation, and analysis. Ideally, some steps can be performed in parallel (for example, you might start developing the tools you’ll need in human studies while you are in the process of conducting animal testing.) Also, the data produced need to make sense in a continuum from in vitro to in vivo to the clinic, so working with a partner early can help ensure that the data are developed in a consistent and continuous way.
  • Appreciate what you don’t know, which includes determining what questions to ask of your R&D partner or what questions to be asked by regulatory agencies. Sometimes, companies, out of scientific interest, explore aspects that are not necessary at that stage of research or development, wasting time and money. In other cases, developers define their research focus too narrowly, thus missing out on important information that will be required as the asset progresses through development. The right R&D partner can help you determine the key questions that must be answered in the analysis.
  • If your company is a start-up or small- to medium-sized Biotech, be ready to discuss your exit strategy, as it will be a factor in your analysis plan. Is the plan to take the asset to FIH and then sell it? Or, is the plan to take it further and derive more value out of it? If the latter, you will need a more solid foundation around the tools/measures you develop.
  • Ensure that your assays will be robust enough to stand the test of time for the drug development, meaning that they will work to provide reliable and consistent results over time. For instance, there might be matrix effects when you move between disease states in human. There might be a different immune reaction, or a homologous sequence might cause a nonspecific reaction.

In conclusion

Oligonucleotide-based therapies provide another therapeutic modality that gives patients options and hope in the amelioration of their disease. Gene therapy provides the hope of actual cures rather than only attenuation of signs and symptoms. These therapies still have many challenges to overcome before they will become widely developed therapeutic options. Discovery and clinical researchers in partner with an R&D partner can help to bring these to fruition through the development of good science and quality data. For both oligonucleotide-based and gene therapies, bioanalytical challenges and strategies should be developed and assessed prior to and at each stage of pre-clinical and clinical development. An R&D partner that has extensive knowledge and experience across all aspects of the bioanalytical tools and methods including PK, DMPK, PK/PD and immunogenicity will enable your programs to progress efficiently and effectively.

References

1. Lundin KE, Gissberg O and Smith CI. Oligonucleotide therapies: the past and the present. Gene Ther. 26(8), 475–485 (2015).
2. Krishna M. From petunias to the present- a review of oligonucleotide therapy. Clin. Epigenet. 3(4:38), 1–4 (2017).
3. Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K and Sadelain M. Gene therapy comes of age. Science. 359(6372), eaan4672 (2018).
4. Pan C, Chen X, Wang S, Erol H, Zhang Z, Citerone D and Lin Z. Quantitation of anti-sense oligonucleotides in biological samples using a one-step SPE method and LC-MS/MS. Abstract W1030-03-019, AAPS Annual Meeting, Nov 2018; https://www.eventscribe.net/2018/PharmSci360/PosterTitles.asp?pfp=PosterTitles