Two Decades of Publishing Excellence in Pharmaceutical Biotechnology

Abstract

Recombinant biological products have revolutionized modern medicine by providing both remarkably effective vaccines to prevent disease and therapeutic drugs to treat a wide variety of unmet medical needs. Since the early 1980s, dozens of new therapeutic protein drugs and macromolecular vaccines have been commercialized, which have benefitted millions of patients worldwide. The pharmaceutical development of these biological products presented many scientific and technical challenges, some of which continue today with newer candidates including recombinant protein-based vaccines with novel adjuvants, peptide and RNA-based drugs, and stem cellular therapies. Compared with small molecule drugs, the characterization, stabilization, formulation, and delivery of biomolecules share common hurdles as well as unique challenges. This area of drug development research has been referred to as “pharmaceutical biotechnology”, in recognition of the critical role that recombinant DNA technology plays in the design and production of most of these biological products. Current research focus areas in this field include (i) determination of structural integrity of the primary sequence, post-translational modifications, and higher-order three dimensional shapes, (ii) assessment of physicochemical degradation pathways and their effects on biological activity and potency, (iii) formulation design and development to optimize stability and delivery, (iv) evaluating and optimizing process development steps including lyophilization and fill-finish, (v) analytical method development and applications of new instruments and data visualization tools, (vi) design and development of drug delivery approaches, and (vii) studies of biological effects including pharmacokinetics, pharmacodynamics, and adverse immunogenicity. During the early days of pharmaceutical biotechnology research, there were numerous scientific challenges because the analytical characterization approaches needed for development of recombinant biological molecules in “real world” pharmaceutical dosage forms were essentially unknown. Furthermore, understanding critical drug product manufacturing issues (e.g., stability of biological compounds during processing, storage, and shipping as well as reproducibility of fill-finish production technologies) and behavior during and after patient administration was often achieved by “on-the-job” training. Fortunately, the pioneers in the field regularly presented research at key conferences and started publishing early in pharmaceutical sciences journals such as Journal of Pharmaceutical Sciences. Recognizing this critically important new field, the then Editor of the journal, Professor Bill Higuchi, instituted a new “pharmaceutical biotechnology” category for research papers. This insightful move was coupled with an equally wise decision to recruit Dr. C. Russell Middaugh as the new Associate Editor for the new research category. As will be detailed below, under Dr. Middaugh’s diligent and expert guidance, pharmaceutical biotechnology papers have grown in number, scope, and impact over the past 20 years, and these days, the Journal of Pharmaceutical Sciences is viewed by scientific leaders in the field as the “go to” place for publication of the most important results and descriptions of innovations in pharmaceutical biotechnology. The number of pharmaceutical biotechnology papers published in the Journal of Pharmaceutical Sciences from 1992 to 2013 is shown in Figure 1, both by year and cumulative number. These papers are categorized according to the pharmaceutical development of three different types of biotechnology- based product candidates: protein-based therapeutics, other biological molecules (including peptides, polysaccharides, DNA/RNA), and finally various macromolecular antigens (and adjuvants) being developed as vaccines. In 1994, Dr. C. Russell Middaugh joined the editorial board of Journal of Pharmaceutical Sciences as the first dedicated pharmaceutical biotechnology Editor. As can be seen in Figure 1, only a handful of biotechnology papers were published in 1993. From 1994 to the present, under Professor Middaugh’s ongoing editorial guidance, approximately 1000 pharmaceutical biotechnology papers have now appeared, with about half of the papers being published since 2007. For the first 6 months of 2014, 47 additional papers had been published (data not shown). This dramatic growth in pharmaceutical biotechnology papers in the Journal of Pharmaceutical Sciences parallels two major general trends in the biopharmaceutical industry over the past two decades: the emergence of therapeutic mAb drugs to address unmet medical needs for patients with a variety of disorders, especially cancer and autoimmune diseases, as well as the development of many new vaccines to protect both children and adults against a wide range of infectious diseases. To illustrate the tremendous growth in development of therapeutic mAb treatments over the past two decades, we focus on United States Food and Drug Administration (US FDA) approvals, although similar are trends would be observed with worldwide regulatory approvals. The first therapeutic mAb product approved for human use by the US FDA was Orthoclone OKT®3 in 1986; a mouse IgG2a antibody against the CD3 receptor on T-cells for treatment of acute rejection of organ transplants. For the following 8–10 years, it was unclear whether therapeutic mAbs would live up to their potential as “magic bullet” pharmaceutical treatments, and no additional full-length mAbs were approved. During this time period, however, great advances were achieved in the area of antibody engineering allowing for the humanization of mouse antibodies resulting in the ability to produce chimeric, humanized, and fully human mAbs (approximately 75%, 95%, and 100% human amino acid sequences, respectively).1.Nelson A.L. Dhimolea E. Reichert J.M. Development trends for human monoclonal antibody therapeutics.Nat Rev Drug Discov. 2010; 9: 767-774Crossref PubMed Scopus (803) Google Scholar As shown in Figure 2, in 1994, the second mAb-based product was approved by the US FDA, a chimeric antibody fragment (anti-glycoproteinllb/IIIa Fab) used as a platelet aggregation inhibitor (ReoPro®). Starting in 1997 to the present, approximately 30–35 new therapeutic mAbs have been approved for commercial use with one to four new mAb approvals per year (except for no US FDA approvals in 1999 and 2005).2.Mullard A. 2011 FDA drug approvals.Nat Rev Drug Discov. 2012; 11: 91-94Crossref PubMed Scopus (138) Google Scholar, 3.Mullard A. 2012 FDA drug approvals.Nat Rev Drug Discov. 2013; 12: 87-90Crossref PubMed Scopus (109) Google Scholar, 4.Mullard A. 2013 FDA drug approvals.Nat Rev Drug Discov. 2014; 13: 85-89Crossref PubMed Scopus (93) Google Scholar, 5.Reichert J.M. Marketed therapeutic antibodies compendium.mAbs. 2012; 4: 413-415Crossref PubMed Scopus (328) Google Scholar When examining the overall trend over the past 20 years, mAbs have grown and evolved into the major category of protein-based therapeutics, and this trend is expected to continue as newer technologies such as antibody-drug conjugates, bispecific antibodies, and various types of antibody-based fragments and fusion proteins become available as therapeutic molecules. Approximately two-thirds of the therapeutic mAbs on the market are administered by intravenous (i.v.) injection, with most of the other mAb treatments injected subcutaneously, along with a few other administration routes including intramuscular and intravitreal (based on a review of online package inserts). In terms of pharmaceutical dosage forms, mAbs have been formulated as either liquid solutions (~2/3 of total) or freeze-dried powders (~1/3 of total) (based on a review of online package inserts). Liquid mAb formulations are filled and packaged into either glass vials or prefilled syringes (PFS), the latter can be used with auto-injectors as mAb drug-device combination products. Both pharmaceutical dosage forms and related administration procedures for therapeutic mAb treatments have become more sophisticated over the past 20 years. For example, in 1998 the first approved anti-tumor necrosis factor alpha (anti-TNF) mAb treatment, Remicade® produced by Centocor (now part of J&J), consists of a chimeric mAb which is lyophilized at 100 mg/vial, reconstituted with 10 mL sterile water for injection, and administered i.v. by medical professionals. In 2009, the same company introduced a newer anti-TNF mAb treatment (Simponi®) as a fully human mAb, formulated as a 100 mg/mL high concentration aqueous solution. A patient- convenient dosage form was utilized with the liquid formulation filled into a PFS, which in turn, is placed into an auto-injector allowing for self-administration at home/doctor’s office via subcutaneous (SC) injection.6.Shealy D.J. Cai A. Staquet K. Baker A. Lacy E.R. Johns L. Vafa O. Gunn 3rd, G. Tam S. Sague S. Wang D. Brigham-Burke M. Dalmonte P. Emmell E. Pikounis B. Bugelski P.J. Zhou H. Scallon B.J. Giles-Komar J. Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α.MAbs. 2010; 2: 428-439Crossref PubMed Scopus (199) Google Scholar The past 20 years have also witnessed a surge in development and commercialization of new vaccines, both in terms of new vaccine antigens and new formulations and delivery technologies. As shown in Table 1, there has been an impressive and diverse array of macromolecules and microorganisms developed as new vaccines including polysaccharide-protein conjugates (e.g., pneumococcal and meningococcal vaccines), killed and live, attenuated viruses (e.g., hepatitis A, rotavirus and shingles vaccines) as well as recombinant protein technologies including virus-like particles (human papillomavirus or HPV vaccine) and a recombinant hemagglutinin flu vaccine.7.US Food and Drug Administration Website, “Biological Approvals by Year”. Accessed July, 2014, at: http://www.fda.gov/BiologicsBloodVaccines/DevelopmentApprovalProcess/BiologicalApprovalsbyYear/.Google Scholar In addition, new formulations and delivery technologies have been introduced to improve and expand the utility of vaccines such as influenza (e.g., nasal and intradermal delivery, new adjuvants). Finally, new formulations containing mixtures of older, already approved vaccines have also been developed to decrease the complexity of the vaccination schedule and better ensure compliance (e.g., measles, mumps, rubella and varicella; and, although not listed in Table 1, diphtheria, tetanus toxoid, acellular pertussis, hepatitis B and inactivated poliovirus vaccines).Table 1Examples of New Vaccines and New Vaccine Formulations Approved for Human Use in the Past 20 Years (1993-2013)Vaccine AntigenYearProtection AgainstVaccine ProductVaccine FormulationNameManufacturerDosageAdjuvantAdminRoutePolysaccharideprotein conjugates1993Haemophilus influenzae type bActHIBSanofi PasteurLyoNoneIM2000/2010PneumococcalPrevnar/Prevnar 13PfizerLiquidAluminumIM2005/2010MeningococcalMenactra/MenveoSanofi Pasteur/NovartisLiquid/LyoNoneIMLive, attenuated virus1995Varicella (Chicken Pox)VarivaxMerckLyoNoneSC2005Measles, mumps, rubella and chickenpoxProQuadMerckLyoNoneSC2006Varicella (Shingles)ZostavaxMerckLyoNoneSC2006/2008RotavirusRotateq/RotarixMerck/GSKLiquid/LyoNoneOralInactivated virus1995/1996Hepatitis AHavrix/VaqtaGSK/MerckLiquidAluminumIM2009Japanese encephalitisIXIARONovartisLiquidAluminumIMRecombinant protein virus-like-particle2006/2009Human papillomavirus (HPV)Gardasil/CervarixMerck/GSKLiquidAluminum/AS04IMNew flu vaccines (live vaccine)2003InfluenzaFluMistMedimmuneLiquidNoneNasal(intradermal delivery)2011FluzoneSanofi PasteurLiquidNoneID(cell culture)2012FluarixNovartisLiquidNoneIM(recombinant protein)2013FlublokProtein ScienceLiquidNoneIM(biodefense stockpile only)2013Influenza A (H5N1)Influenza A (H5N1) virus monovalent vaccine, adjuvantedGSKLiquidAS03IMThis table is not a comprehensive list of approved vaccines by regulatory agencies, but rather provides illustrative examples from US FDA approvals of vaccine development trends in terms of types of new vaccine antigens, formulations, and delivery routes (7). Open table in a new tab This table is not a comprehensive list of approved vaccines by regulatory agencies, but rather provides illustrative examples from US FDA approvals of vaccine development trends in terms of types of new vaccine antigens, formulations, and delivery routes (7). From a pharmaceutical dosage form perspective, live attenuated viral vaccines tend to be lyophilized and do not require adjuvants. This can be attributed to the inherent complexity and instability of microorganisms along with their ability to replicate upon administration and thus better mimic a natural infection. One exception in the 1993–2013 time period is the successful development of a liquid formulation of the orally administered rotavirus vaccine, which required identification of stabilizers to ensure stability of a pentavalent virus mixture when stored at 2–8°C in a plastic squeeze tube, and at the same time, provide sufficient acid neutralizing capacity to protect the viruses from gastric acid degradation during oral administration.8.Clark H.F. Burke C.J. Volkin D.B. Offit P. Ward R.L. Bresee J.S. Dennehy P. Gooch W.M. Malacaman E. Matson D. Walter E. Watson B. Krah D.L. Dallas M.J. Schodel F. Kaplan K.M. Heaton P. Safety, immunogenicity and efficacy in healthy infants of G1 and G2 human reassortant rotavirus vaccine in a new stabilizer/buffer liquid formulation.Pediat Infect Dis J. 2003; 22: 914-920Crossref PubMed Scopus (54) Google Scholar Another trend to note in Table 1 is that inactivated viral and subunit vaccines (e.g., polysaccharide-protein conjugates, recombinant protein virus-like particle vaccines) tend to be formulated as liquid solutions containing adjuvants to enhance their immunogenicity. Aluminum salts have been used as adjuvants for decades, and most new vaccines still contain this conventional adjuvant. It is interesting to note that despite decades of research to identify and develop new adjuvants, US FDA approvals have occurred only relatively recently including GSK produced HPV vaccine with AS04 (aluminum adjuvant and monophosphoryl lipid A) in 2009, and a biodefense flu vaccine in the US stockpile with AS03 (emulsion containing squalene, polysorbate 80 and DL-alpha-tocopherol) in 2013. Additional vaccine formulations containing new adjuvants have been approved by European regulatory agencies including oil-in-water emulsions as adjuvants for flu vaccines (not shown). One interesting pharmaceutical biotechnology-related case study for new protein-based vaccines is the stabilization and formulation of the human papillomavirus virus-like particles (HPV VLPs). When the recombinant viral surface protein was recombinantly expressed in yeast and then assembled into virus-like particles in vivo, the purified VLPs were observed to form a mixture of fully and partially assembled particles. An in vitro large scale disassembly and reassembly procedure was developed and implemented to ensure the formation of correctly assembled virus-like particles, resulting in enhanced potency and improved stability (accelerated and long-term storage) for a quadrivalent, aluminum adsorbed HPV VLP vaccine formulation.9.Mach H. Volkin D.B. Troutman R.T. Wang B. Luo Z. Jansen K.U. Shi L. Disassembly and reassembly of yeast-derived recombinant human papillomavirus virus-like particles (HPV VLPs).J Pharm Sci. 2006; 95: 2195-2206Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar After three decades of diligent research, pharmaceutical scientists now know that the safety and efficacy of therapeutic protein drug products can be compromised not only via post- translational modifications in the cell, but also by well-defined physical and chemical degradation pathways. It has also become apparent that sometimes even trace amounts of modified or degraded protein can result in suboptimal to adverse effects in patients (see immunogenicity section below). We have learned that proteins can be extremely susceptible to such degradation and that such damage can occur at all stages of a protein product’s life history, from fermentation, purification, formulation/storage to patient administration (and perhaps in vivo within the patient). Furthermore, the starting bulk drug substance can contain a wide range of molecular variants, e.g., subpopulations with different glycosylation patterns or charged isoforms, which can have different pharmaceutical properties such as receptor binding, pharmacokinetic profiles and propensity to aggregate. Currently, the study of protein physical stability is understood to encompass characterization of degradation products such as “soluble” aggregates (e.g., oligomers), “particles” (submicron, subvisible and visible) and larger precipitates. In addition, a particular concern for low dose drug products is the loss of potency due to adsorption of protein molecules to the container/closure (e.g., wall of glass vials or rubber stopper) and/or the delivery system (e.g., bags for i.v. administration). In addition, physical stability can refer to the key properties of the native protein under various solution conditions; e.g., the effects of pH or ionic strength on a protein’s conformational and colloidal stability. In turn, it is now better understood that these physical properties can govern the rates of degradation of a given protein (e.g., aggregation rate). Furthermore, pharmaceutically unacceptable physical properties can include opalescent appearing solutions, liquid-liquid phase separation and high solution viscosity. These properties are particularly problematic with the development of high concentration (e.g., 100–200 mg/ml) formulations of mAbs. Moreover, it has been well established that there are exposures to numerous stresses during a protein product’s life history that readily induce aggregation, particle formation and/or loss of protein molecules from solution due to adsorption at interfaces. These include freeze-thawing, exposure to extremes of pH, filtration steps, pumping during fill-finish and fluid transfers, exposure to various surfaces and interfaces in primary containers and delivery systems, as well as agitation and other stresses during shipping. Many of the stresses result in exposure of protein molecules to interfaces to which they can adsorb, resulting in assembled networks of native and/or structurally altered protein molecules on the interface. Disruption of the assembled films or gels formed at the interfaces (e.g., during agitation) can result in protein aggregates or particles in the bulk solution. In addition, extrinsic, foreign particles can be shed from essentially any material to which protein solutions are exposed, (e.g., silicone oil-in-water, stainless steel particles-in-water, glass delamination) and protein molecules can readily adsorb to the foreign particle-liquid interfaces resulting in heterogeneous particle formation. These may, in turn, stimulate further protein aggregation and particle formation, especially upon exposure to pharmaceutically relevant stresses (e.g., freeze-thawing or agitation). In concert with the efforts to delineate the causes of protein physical degradation, tremendous progress has been made in understanding mechanisms by which proteins can be stabilized against such damage and in developing effective means to minimize degradation. This work includes both theoretical and experimental advances, which have led to much more rationale design of protein stabilizers, high-throughput formulation development approaches to optimize protein formulation composition, and to more powerful data processing/data visualization methods. Also, approaches to reduce the detrimental impact of processing conditions have been studied at lab scale, and resulting mitigation strategies have been scaled-up and implemented in commercial manufacturing settings. Thirty years ago the field was not aware of many of the numerous problems that can arise during the scale-up, manufacturing and shipping/handling of therapeutic proteins, because the development efforts for these products were in their infancy. Through rigorous and insightful research over the intervening years, and the publication of important results and theoretical insights, pharmaceutical biotechnology has made remarkable progress. Many of the key papers have been (and continue to be) published in Journal of Pharmaceutical Sciences. These publications include impactful Commentaries and Reviews, as well as numerous seminal research papers. For example, over the years, higher resolution, more sensitive and increasing reliable characterization and quantitation assays for monitoring protein aggregation and particle formation have been developed. This progress in developing improved analytical tools has greatly increased our insights into how protein physical degradation can readily occur and our understanding that if even minute fractions of a protein product become degraded, there may be detrimental impacts on subsequent protein stability and product quality, and on product safety and efficacy in patients (as discussed in more detail below). Since the inception of development of therapeutic proteins, in parallel with research efforts to understand and control protein physical stability, intensive research also has been focused on the chemical stability of proteins, which is also critically important for safety and efficacy of therapeutic protein products. Decades of advancements in the field were required to develop the theoretical and practical understanding - and the requisite advances in analytical capabilities - that we now have about chemical degradation oftherapeutic proteins. There are many different pathways for chemical degradation that have been elucidated in extensive mechanistic detail including oxidation, deamidation, and hydrolysis of certain amino acid residues. This in turn has led to a better understanding of chemical hotspots in protein molecules (e.g., Asn deamidation, Asp isomerization, Met oxidation, and Trp photo-degradation). In addition to chemical degradation, chemical heterogeneity in therapeutic protein products is also well-established, for example, disulfide isoforms, charged variants, C-terminal lysine and N-terminal pyroglutamate variants, and even proteolytic clippings. Early efforts often focused on investigations of causes and control of known chemical degradation pathways and discovery of new pathways. It was quickly realized that conditions encountered during processing, storage, shipping, and delivery could lead to rapid chemical degradation of specific amino acid residues and/or the polypeptide backbone. These conditions included exposures to: metals from processing equipment, primary containers and/or excipients; peroxides from surfactants; extremes of pH; and light. Current control strategies now include screening of excipient lots for metals and peroxides, and the replacement of stainless steel processing equipment with single-use plastic systems. But the plastic systems have not been without problems, for example, substances leaching from the plastics have been found in some cases to cause chemical degradation of therapeutic proteins. For some products, despite these efforts, chemical degradation is so extensive in solution that freeze-drying is required to ensure chemical stability and a multi-year shelf life. Light exposure during manufacturing is minimized with approaches such as running a side stream from a chromatography system through the UV detector while the main flow is not exposed to light. In addition, using secondary packaging helps to reduce light exposure in the final product container. But there are still unintended and poorly controlled situations in which light exposure and potential damage can occur to the protein product. For example, when patients are warming a PFS prior to administration at home, they may place it on the kitchen counter in direct sunlight. Similarly, during preparation and administration of i.v. products, there is exposure to room lights and potentially to sunlight. Such exposures can cause substantial photochemical (and physical) degradation of proteins. There also has been much effort on developing formulation approaches to minimize chemical damage to proteins in the drug substance and in the final formulated drug product. Some successful approaches that have been implemented include the selection of the optimal solution pH and the inclusion of free radical scavengers (e.g., methionine) and/or metal ion chelators in the formulation. It has also been discovered that other additives, such as the pharmaceutical anti-oxidant ascorbate, may actually accelerate protein chemical degradation by catalyzing the generation of free radicals in solution under certain conditions. In other cases, inclusion of appropriate excipients has been documented to inhibit light-induced degradation in some proteins, whereas in other studies they have been found to be ineffective for another protein. The difference might be due to the locations of the damage-sensitive amino acid residues within the protein molecule. For example, residues located on the protein surface may be protected to some degree by stabilizing excipients in the solution, whereas residues that are degraded because of photon absorption in the interior of the protein may not be protected by components in the formulation. Chemical damage and physical stability are often linked. For example, oxidation of amino acid residues within a protein may also lead to protein aggregation, perhaps including covalent crosslinks. Another example is creation of a protein species with reduced solubility via fragmentation or proteolytic clipping of the polypeptide backbone, leading to protein precipitation. Conversely, perturbation of the tertiary structure of a protein molecule may result in more rapid degradation of amino acid residues that were previously buried in the most compact species in the native state ensemble. The exact nature of such linkages between chemical and physical stability cannot be predicted - nor can the consequences of the damage. Consequently, Arrhenius kinetics cannot be relied on to give accurate predictions of degradation rates or shelf life, adding complexity to formulation development for biologics compared to small molecules. Careful studies are needed to characterize the degradation profile for each given protein and to develop formulations to minimize both physical and chemical degradation pathways. Often times, a compromise is required to identify conditions that lead to optimal overall protein stability, conditions which in turn, may not be optimal for every individual physical or chemical degradation pathway. As has been the case for publications on physical stability of therapeutic proteins, many of the key research papers, Reviews and Commentaries have been, and continue to be, published on the topic of chemical stability of protein drug candidates in Journal of Pharmaceutical Sciences. These papers are further evidence of the important roles that the journal has played in the advancement of the field for the past two decades. For millions of patients, therapeutic protein products are miracle drugs that save and improve lives. However, for many patient populations these miracle drugs, which are initially highly effective, eventually fail in a fraction of the initial responders. Depending on the product and patient group, the fraction of these so-called “secondary non-responders” may reach 50% or higher, with many patients developing treatment failure in less than a year or two. In some cases, (e.g., rheumatoid arthritis patients treated with anti-TNF therapies) patients can be switched to another product in the same class with a restoration of therapeutic effectiveness. But even these patients may experience subsequent treatment failures. For other patients there is no alternative therapy or they have already used all of the approved biologics. In these cases, they suffer from loss of treatment with the protein miracle drugs, resulting in morbidity or even death. It is now widely documented that treatment failure is usually due to adverse immunogenicity caused by the protein drug product. Since the 1960s, many clinical investigations and animal studies have shown that a major contributing factor to immunogenicity is the presence of protein aggregates and particles. Even trace amounts of these degradation products can stimulate an immune response leading to generation of antibodies that neutralize the drug’s activity and/or promote its rapid clearance from the body. Furthermore, immune response to aggregates and particles can be greatly enhanced if the protein molecules are absorbed onto pharmaceutically relevant foreign particles (e.g., glass from vials or stainless steel from filling pumps) and/or are chemically degraded (e.g., oxidized). Therefore, major research efforts are now devoted to understanding and controlling the levels of such protein aggregates, particles and chemically degraded species. Concurrently regulatory expectations in this area are becoming increasingly more stringent. As with the other categories considered in this Commentary, many of the most innovative and influential research papers, Reviews and Commentaries in the area of immuno- genicity of protein drugs, its causes, mechanisms and related regulatory expectations have been published in the Journal of Pharmaceutical Sciences. In the 1980s and early 1990s, some of the new therapeutic protein products were being developed in traditional “small molecule” pharmaceutical companies. Often there was a philosophical, as well as a phy