Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.

Cynthia A. Challener

The FDA continues to approve drugs at a rapid pace, although fewer novel drugs (48) were approved by the Center for Drug Evaluation and Research in 2019 (1) compared with 59 in 2018 (2). Compared with other agencies around the world, however, FDA again lead other leading regulatory authorities in the number of approvals.

	According to the Kyoto Encyclopedia of Genes and Genomes Drug Database, 35 drugs with new active ingredients were approved in Japan by the Pharmaceutical and Medical Devices Agency (PMDA) as of Nov. 4, 2019 (3), while the total number of European public assessment reports (EPAR) authorized medicines as of Nov. 1, 2019 that were novel included 15 based on new molecular entities, approved under a biologic license application, and one cell therapy (4). In India, just 16 new drugs were approved by the Central Drugs Standard Control Organization as of mid-November (5).

	Looking at specific drugs, only one-Skyrizi (risankizumab-rzaa) from AbbVie for treatment of psoriasis-was approved in the United States, Europe, and Japan; the therapy was approved in Japan approximately one month in advance of approval in the US and Europe. Rozlytrek (entrectinib) and Evenity (romosozumab-aqqg) were approved in both the US and Japan, with the latter receiving approvals just days a part, and the former getting the okay in Japan two months before it received approval in the US (6).

	Interestingly, many more drugs in 2018 were approved in two or all three jurisdictions, and several of the drugs approved in the US and Europe in 2018 didn’t receive approval in Japan until 2019, including Erleada (apalutamide), more than one year later, Braftovi (encorafenib), three to six months later), Mektovi (binimetinib), three to six months later, and Onpattro (patisiran sodium), 10 months later.

	Many other drugs approved by FDA in 2018 did not receive approval by the European Medicines Agency (EMA) and/or PMDA until 2019, including Crysvita (burosumab), Trogarzo (ibalizumab), Palynziq (pegvaliase), Epidiolex (cannabidiol), Ajovy (fremanezumab), Lorbrena (lorlatinib), Vizimpro (dacomitinib), Libtayo (cemiplimab), Talzenna (talazoparib tosylate), Vitrakvi (larotrectinib sulfate), and Ultomiris (ravulizumab).

	A study of decisions by FDA and EMA for 107 new drug applications during the period 2014–2016 found that the two agencies were in agreement on the majority (more than 90%) of approval decisions (7). Differences were largely due to different conclusions regarding efficacy, sometimes based on the same data and sometimes on differing data.

	In a separate study, it was found that FDA approved 170 new therapeutic agents between 2011 and 2015, while EMA granted marketing authorization to 144 (8). In addition, the median total review time at FDA was much lower, on average 60 days shorter. FDA also approved more orphan drugs than EMA (43.5% vs. 25.0% of the total).

	Many of the new therapies approved by FDA in 2019 were developed to treat rare diseases, and most of these drugs are the first treatments ever approved for these diseases. Examples include Celgene’s Reblozyl (luspatercept–aamt), which reduces the need for blood transfusions in anemic patients with beta thalassemia (9); Ablynx’s Cablivi (caplacizumab-yhdp) injection, the first therapy specifically indicated, in combination with plasma exchange and immunosuppressive therapy, for the treatment of adult patients with acquired thrombotic thrombocytopenic purpura, a rare and life-threatening blood clotting disorder (10); and Pfizer subsidiary FoldRx’s oral therapies Vyndaqel (tafamidis meglumine) and Vyndamax (tafamidis), the first FDA-approved treatments for heart disease caused by transthyretin mediated amyloidosis (11).

	Other therapies for rare diseases for which the first new drugs were approved by FDA in 2019 include Givlaari (givosiran) from Alnylam Pharmaceuticals for acute hepatic porphyria, a genetic disorder resulting in the buildup of toxic porphyrin molecules (12); Turalio (pexidartinib) from Daiichi Sankyo for symptomatic tenosynovial giant cell tumor, a non-malignant tumor that causes the synovium and tendon sheaths to thicken and overgrow (13); targeted therapy Adakveo (crizanlizumab-tmca) from Novartis for vaso-occlusive crisis, a common and painful complication of sickle cell disease (14); and Scenesse (afamelanotide) from Clinuvel for pain due to phototoxic reactions upon exposure to light (15).

	Several of the approved drugs are designed to treat cancer, including the first PIK3 inhibitor Piqray (alpelisib) from Novartis (16). The drug is used in combination with the FDA-approved endocrine therapy fulvestrant, to treat postmenopausal women, and men, with hormone receptor-positive, human epidermal growth factor receptor 2-negative, PIK3CA-mutated, advanced or metastatic breast cancer. The disease must be detected with companion diagnostic test therascreen PIK3CA RGQ PCR Kit from QIAGEN Manchester, which also received FDA approval in 2019.

	Of note, Piqray was the first new drug application for a new molecular entity approved under the Real-Time Oncology (RTOR) pilot program, which permits FDA to begin analyzing key efficacy and safety datasets prior to the official submission of an application, allowing the review team to begin their review and communicate with the applicant earlier. Piqray also used the updated Assessment Aid, a multidisciplinary review template intended to focus FDA’s written review on critical thinking and consistency and reduce time spent on administrative tasks. With these two pilot programs, Piqray was approved approximately three months ahead of the PDUFA VI deadline.

	Two of the new drugs approved by FDA in 2019 address medical conditions suffered by women. Zulresso (brexanolone) injection from Sage Therapeutics is the first drug approved by FDA specifically to treat postpartum depression (17). Vyleesi (bremelanotide) from AMAG Pharmaceuticals treats acquired, generalized hypoactive sexual desire disorder in premenopausal women (18). FDA identified female sexual dysfunction as one of 20 disease areas of high priority and focused attention in 2012.

	Some of the drugs approved by FDA are interesting due to their route of delivery. Novo Nordisk’s Rybelsus (semaglutide), oral tablets, is the first glucagon-like peptide receptor protein approved by the agency to improve control of blood sugar in adult patients with type 2 diabetes that does not need to be injected (19). Baqsimi nasal powder, from Eli Lilly and Company, meanwhile, is the first glucagon therapy approved by FDA for the emergency treatment of severe hypoglycemia that can be administered without an injection (20).

	Also of note was the agency’s approval of Janssen Pharmaceuticals’ Spravato (esketamine) nasal spray, in conjunction with an oral antidepressant, for the treatment of treatment-resistant depression (21). Esketamine is the s-enantiomer of ketamine, which was approved under the brand name Ketalar in 1970.

	New antibacterial, antiviral and antifungal meds

	Developing new antibiotics and antiviral agents is a priority of FDA as concerns over drug resistance continue to rise; three 2019 approvals fall under this category. The Global Alliance for TB Drug Development received approval for Pretomanid tablets in combination with bedaquiline and linezolid for the treatment of a specific type of highly treatment-resistant tuberculosis (TB) of the lungs (22). This drug was approved under the Limited Population Pathway for Antibacterial and Antifungal Drugs pathway and also received FDA’s Qualified Infectious Disease Product (QIDP) designation and a Tropical Disease Priority Review Voucher.

	Nabriva Therapeutics’ Xenleta (lefamulin) was approved for the treatment of with community-acquired bacterial pneumonia and also received QIDP designation (23). Fetroja (cefiderocol), an antibacterial drug from Shionogi & Co. for treatment of complicated urinary tract infections also received the QIDP designation (24).

	FDA also approved Sentosa SQ HIV Genotyping Assay (Vela Diagnostics USA), the first HIV drug resistance assay that uses next-generation sequencing (NGS) technology (25).

	One of FDA’s 2019 approvals making significant news for its price and questions about data integrity was for Zolgensma (onasemnogene abeparvovec-xioi), the first gene therapy approved to treat children less than two years of age with spinal muscular atrophy (SMA), the most severe form of SMA and a leading genetic cause of infant mortality (26). AveXis, which is a division of Novartis, also received a rare pediatric disease priority review voucher.

	The oral drug Ruzurgi (amifampridine) from Jacobus Pharmaceutical Company was granted the first FDA approval of a treatment specifically for pediatric patients with Lambert-Eaton myasthenic syndrome (27). Pfizer’s Fragmin (dalteparin sodium) injection, initially approved for adults in 1994, received approval for reducing the recurrence of symptomatic venous thromboembolism in pediatric patients (28).

	AbbVie’s oral drug Mavyret (glecaprevir and pibrentasvir), approved to treat hepatitis C virus (HCV) in adults in 2017, received approval for the treatment of children in 2019 (29). Glaxo-
	SmithKline also won approval for Benlysta (belimumab) intravenous (IV) infusion (initially approved for adults in 2011) for treatment of children with systemic lupus erythematosus (SLE) (30).

	Two new vaccines received approval from FDA in 2019: Jynneos Smallpox and Monkeypox Vaccine, Live, Non-Replicating from Bavarian Nordic, the only current FDA-approved vaccine for the prevention of monkeypox disease (31), and Dengvaxia from Sanofi Pasteur, the first vaccine approved by FDA for the prevention of dengue disease caused by all dengue virus serotypes in people ages 9 through 16 (32).

, accessed Dec. 18, 2019.
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New Drug Approvals in Japan: Drugs with New Active Ingredients

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European Public Assessment Reports (EPAR) Authorised Medicine

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	6. Kyoto Encyclopedia of Genes and Genomes Drug Database, “

New Drug Approvals in the USA, Europe and Japan

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	7. M. Kashoki, et. al., 

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FDA Approves First Therapy to Treat Patients with Rare Blood Disorder

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	10. FDA, “

FDA Approves First Therapy for the Treatment of Adult Patients with a Rare Blood Clotting Disorder

,” Press Release, Feb. 6, 2019.
	11. FDA, “

FDA Approves New Treatments for Heart Disease Caused by a Serious Rare Disease, Transthyretin Mediated Amyloidosis

,” Press Release, May 6, 2019.
	12. FDA, “

FDA Approves First Treatment for Inherited Rare Disease

,” Press Release, Nov. 20, 2019.
	13. FDA, “

FDA Approves First Therapy for Rare Joint Tumor

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	14. FDA, “

FDA Approves First Targeted Therapy to Treat Patients with Painful Complication of Sickle Cell Disease

,” Press Release, Nov. 15, 2019.
	15.       FDA, “

FDA Approves First Treatment to Increase Pain-Free Light Exposure in Patients with a Rare Disorder

,” Press Release, Oct. 8, 2019.
	16. FDA, “

FDA Approves First PI3K Inhibitor for Breast Cancer

,” Press Release, May 24, 2019.
	17. FDA, “

FDA Approves First Treatment for Post-Partum Depression

,” Press Release, March 19, 2019.
	18. FDA, “

FDA Approves New Treatment for Hypoactive Sexual Desire Disorder in Premenopausal Women

,” Press Release, June 21, 2019.
	19. FDA, “

FDA Approves First Oral GLP-1 Treatment for Type 2 Diabetes

,” Press Release, Sept. 20, 2019.
	20. FDA, “

FDA Approves First Treatment for Severe Hypoglycemia that Can Be Administered without an Injection

,” Press Release, July 24, 2019.
	21. FDA, “

FDA Approves New Nasal Spray Medication for Treatment-Resistant Depression; Available Only at a Certified Doctor’s Office or Clinic

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	22. FDA, “

FDA Approves New Drug for Treatment-Resistant Forms of Tuberculosis that Affects the Lungs

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	23. FDA, “

FDA Approves New Antibiotic to Treat Community-Acquired Bacterial Pneumonia

,” Press Release, Aug. 19, 2019.
	24. FDA, “

FDA Approves New Antibacterial Drug to Treat Complicated Urinary Tract Infections as Part of Ongoing Efforts to Address Antimicrobial Resistance

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	25. FDA, “

FDA Authorizes Marketing of First Next-Generation Sequencing Test for Detecting HIV-1 Drug Resistance Mutations

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	26. FDA, “

FDA Approves Innovative Gene Therapy to Treat Pediatric Patients with Spinal Muscular Atrophy, s Rare Disease and Leading Genetic Cause of Infant Mortalit

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FDA Approves First Treatment for Children with Lambert-Eaton Myasthenic Syndrome, a Rare Autoimmune Disorder

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	28. FDA, “

FDA Approves First Anticoagulant (Blood Thinner) for Pediatric Patients to Treat Potentially Life-Threatening Blood Clots

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FDA Approves First Treatment for All Genotypes of Hepatitis C in Pediatric Patients

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	30. FDA, “

FDA Approves First Treatment for Pediatric Patients with Lupus

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	31. FDA, “

FDA Approves First Live, Non-Replicating Vaccine to Prevent Smallpox and Monkeypox

,” Press Release, Sept. 24, 2019.
	32. FDA, “

First FDA-Approved Vaccine for the Prevention of Dengue Disease in Endemic Regions

Vol. 44, No. 1
	January 2020
	Pages: 28–32

	When referring to this article, please cite it as C. Challener, “US Maintains Lead in Drug Development Despite Fewer Approvals in 2019,"

Articles

FDA’s approval rate slowed, but the US agency is still ahead of its international counterparts in green-lighting new drugs for market.

US Maintains Lead in Drug Development Despite Fewer Approvals in 2019

Excipients are essential components of drug formulations. While physiologically inert, they have functionalities that achieve many formulation goals, such as API stabilization, taste masking, and extended release. “Functional excipients play an integral role in achieving the desired biological performance of many drug formulations,” asserts Robert Lee, president of the CDMO division of Lubrizol Life Science Health. Many excipients, however-depending on the API structure, dosage form, packaging configuration, and storage, handling, and use conditions-may also interact via mechanisms that negatively impact efficacy and safety. The challenge is to identify the right excipients for each formulation.

	Interactions between excipients and APIs can occur via both chemical and physical mechanisms. “Many small-molecule APIs are inherently complex and have diverse functional groups that can undergo multiple reactions either simultaneously or in sequence,” says Anil Kane, executive director and global head of technical and scientific affairs with Thermo Fisher Scientific. Thus, the degradation of pharmaceuticals is an area that is complex.

	The major mechanisms for chemical decomposition of pharmaceuticals with other excipients include hydrolysis, dehydration, oxidation, isomerization/epimerization, decarboxylation, dimerization, polymerization, and photolysis, according to Kane. Complexation with excipients to form salts is also possible.

	In some cases, the excipient itself doesn’t initially interact in a negative manner with the API. For that to occur, it must first undergo a chemical reaction to generate a new species that can affect the API. For instance, Lee notes that some unsaturated polymers can in the presence of oxygen form peroxides that will degrade oxidatively labile APIs.

	“While many pathways of degradation are obvious from basic organic chemistry principles, it is not uncommon to find surprising degradation chemistry leading to unexpected degradation products and pathways,” Kane observes.

	Physical interactions include hydrophobic and hydrophilic media interaction and interactions that affect particle size and shape. For instance, in nanoparticulate suspensions, excipients physically adsorb to the surfaces of the API nanoparticles via steric or charge-charge interactions.

	In fact, the dosage form plays a role in determining the potential interactions that can occur. Molecular mobility in solids is far less than in the liquid state, hence chemical reactivity in the solid state generally occurs under severe conditions of temperature and/or humidity or in presence of a catalyst, according to Kane. Considerations for chemical degradation are more critical for non-sterile oral liquids and sterile parenteral products because the excipients and APIs are more mobile and there are more opportunities for chemical reactions. For lyophilized products, interactions can occur in the initial buffered solution as well as in the lyophilized cake.

	Interactions between excipients can be either beneficial or harmful. Hydrophilic and hydrophobic media interactions are generally beneficial, according to Richard Shook, director of drug product technical services and business integration at Cambrex. “Through these mechanisms, polymers can delay the release of the drug substance to better target the site of absorption and protect the API from degradation in the gastrointestinal tract. Polymers can also be used to enable prolonged duration of API release, increasing the time of drug absorption and decreasing patient dosing frequency, thereby assisting treatment compliance,” he explains.

	For example, lower-molecular-weight polymers assist in “bridging” of particles during wet granulation processing and can be used to coat API particles to increase their interface with surrounding media during dissolution. Higher-molecular-weight polymers, meanwhile, can form hydrogels that restrict the release of API, enabling extended release. Preferential polymer bridging between smaller and larger particles during wet granulation, meanwhile, can affect particle size and shape selection, leading to desirable granule formation. Saturated fat-based lubricants such as magnesium stearate and stearic acid can create a hydrophobic barrier within the dosage form to ensure proper delivery.

	For nanoparticulate suspensions, which offer increased dissolution rates and potential higher bioavailability due to the high surface area of the particles, the high-energy milling process used to create the nanoparticles generates crystals with high surface energies. Use of excipients to lower the surface energy and stabilize the nanoparticles is therefore necessary, according to Lee. Polymeric stabilizers such as polyvinyl pyrrolidone provide steric stabilization, while charged excipients such as sodium lauryl sulfate participate in electrostatic interactions.

	APIs containing aromatic groups can, in some cases, benefit from interactions with polymeric excipients that also possess aromatic functionality, according to Lee. In this case, there is the potential for the aromatic rings to stack together (π-π stacking), which may lead to stabilization of the API.

	Some chemical reactions are also desirable. For instance, Shook points to the use of pH-enhancing excipients such as citric acid, sodium hydroxide, etc., which can stabilize pH-sensitive APIs by creating microenvironments within dosage forms. Alternatively, certain excipients can be added to participate in competitive reactions as a means of protecting the API. One example is butylated hydroxytoluene, which in some formulations is preferentially oxidized over the API.

	When interactions of the API with functional excipients lead to degradation and the formation of known or unknown impurities, however, the results are generally not beneficial. In these cases, the interactions can have a number of consequences, such as physical changes including discoloration, agglomeration, plug formation, precipitation, cloudiness in liquids, etc., according to Kane.

	One of the most important undesirable reactions is the Maillard reaction between amines and alcohol functionalities, the latter of which are common in sugar excipients such as lactose. The result is formation of high-molecular-weight brown polymeric material with a strong aroma, leading to formulations that are discolored and have an unpleasant odor, according to Shook. Another common issue is the degradation of pH-sensitive APIs due to exposure to acidic or basic environments. In some cases, encapsulation can protect APIs that are hydrolytically or enzymatically labile, Lee notes.

	“The main impact of the interactions of APIs and excipients, however, is not only loss of potency and the intended therapeutic efficacy, but also formation of degradation products that may or may not be toxic,” he observes. “Degradation of the API will result in a drug product not meeting specifications for its assay (or potency) and content uniformity. The dissolution performance of the active drug substance may also decrease. Thus, the drug product will not meet its expected critical quality attributes,” he continues.

	Chemical functionality must be understood

	With efforts to reduce time and cost to market, the potential for stability issues increases dramatically, according to Kane. The ability to rapidly predict and assess the potential for stability and safety concerns is, therefore, an important part of speeding the development of innovative drug therapies. “Degradation prediction enables understanding of labile functionalities critical in designing less reactive, more stable analogs, and degradation studies conducted by a chemistry-guided, predictive, stability approach enable analysts to deliver stability-indicating methodology more efficiently,” he asserts.

	The first step is to examine the possible mechanisms of chemical decomposition for the API in the context of the common functional groups that are present in the molecule. APIs containing functional groups, such as ionizable moieties, amines, and carboxylic acids, present the greatest risk for interacting with excipients, according to Lee. Excipients that can enhance these mechanisms should be avoided.

	In addition, it is important, according to Kane, to consider the excipient impurity profile, which is generally lot specific. Impurities of concern include water, solvents, metals, acidic/basic compounds, and reactive molecules (e.g., peroxides, aldehydes, organic acids). The amorphous content is an additional factor, as is the equilibrium moisture content and hygroscopicity profile, effective pH in water, and the thermal and thermal/humidity solid-state stability (chemical and physical).

	It is worth noting, adds Lee, that in some cases, excipient suppliers offer special, extremely high-purity versions of certain products. When an API is identified as being susceptible to oxidation, for instance, “cleaner” excipients with very low limits of oxidizing substances would be preferred as a means for reducing the risk of undesirable interactions.

	The choice of excipients is usually based on forced degradation data for the API. “Forced degradation data provide information about the potential degradation pathways for the API and for the formation of impurities in acidic and basic environments, when exposed to light or oxidative conditions, and under high temperature and high humidity conditions,” he says.

	Proper characterization of excipient/drug substance interactions using physical and chemical testing is paramount, Shook agrees. “No stone should be left unturned when identifying critical material attributes of a target drug substance. For example, various excipient compatibility studies, such as binary blends, formulated prototypes, compressed versus loose powder, etc., can be designed to align with the available API and timeline,” he observes. The studies, he adds, are best employed in a phase-appropriate manner.

	Prior to initiation of any compatibility studies, a thorough review of relevant drug substance information available at the time of the compatibility studies is extremely important, according to Kane. This evaluation should include a review of the structural understanding of the molecular scaffold and the sites of known reactivity, a detailed review of the synthetic route, a review of the API solution state stability data (pH, thermal, and photostability challenges), any and all forced degradation data, and metabolite formation information and output from predictive models of degradation.

	“A risk-assessment approach should be used that involves combining the knowledge of reactive impurities in excipients along with an understanding of the potential degradation pathways for the API. Other factors such as the drug-to-excipient ratio, crystal form of the API, environmental conditions, surface acidity, and microenvironmental pH must also be considered during the assessment and mitigation of risk. Finally, mitigation strategies should include ‘designing out’ the incompatibilities through formulation design, packaging configurations, or through establishment of a control strategy,” Kane comments.

	There are a number of strategies that can be employed to minimize undesirable excipient-API interactions ranging from avoidance of problematic excipients to the use of excipients that stabilize or protect APIs, devices that physically separate formulation ingredients until the time of administration, and packaging that incorporates chemical agents that inhibit certain chemical reactions.

	For oral solid-dosage forms, there are several different options for protecting APIs that are sensitive to moisture or oxidative degradation. In the former case, Kane notes that excipients with low moisture content should be used to reduce the probability of chemical interaction due to hydrolysis and silica should be added to adsorb any moisture picked up in the formulation. Moisture-barrier film coatings can also be employed, according to Shook. Inclusion of desiccant packs in the drug product bottle pack and/or flushing an inert gas such as nitrogen or dry air during packaging are also recommended.

	For APIs that can react with oxygen, excipients that contain or can generate peroxides should be avoided, while antioxidants should be added to the formulation in the optimal quantity, according to Kane. Selection of the right packaging components and reducing the headspace of the pack may help prevent exposure of the product to an oxidative environment. Inclusion of oxygen scavengers in the bottle pack and blistering and/or bottling the drug product under inert atmosphere (dry air or nitrogen) is also recommended to improve the stability of a packaged product.

	For fat-based lubricants such as magnesium stearate and stearic acid, proper characterization of material blending is important, according to Shook. “Blending times must be optimized to balance proper introduction of the lubricant with minimal coating of the drug substance particles,” he says.

	For non-sterile oral liquids and liquid injectables, the addition of acidic or basic excipients or buffering agents as appropriate can help maintain the desired pH and prevent degradation of the API, according to Kane. If an excipient is necessary to achieve proper delivery but cannot be in contact with the API for extended periods of time, the use of dual-chamber syringes or packaging in separate vials are options for keeping the ingredients physically separate until the time of administration, according to Lee. “The key for these solutions is to have a good understanding of the length of time during which the combined ingredients will be stable in order to ensure that patients are being administered the correct formulation,” he comments.

	Whichever solutions are chosen, they are selected based on extensive pre-formulation and analytical work and intended to provide design control that will lead to acceptable products, according to Lee.

	Most importantly, Lee stresses that it is essential to make certain that each excipient used in a formulation has a well-defined function. “It is critical to question every excipient that is added to a formulation. Formulators must be able to justify the use of each and every excipient in their formulations and ensure that they are using the minimal quantity for each that will afford the target product profile,” he asserts.

	To achieve that goal requires experience and expertise in formulation development and the potential physical and chemical mechanisms of interaction between excipients and APIs with a wide range of functionalities.

	“The best strategy to prevent future drug substance and excipient interaction headaches is to partner with an experienced, proactive formulation team,” says Shook. “Strong collaboration between a formulation team and an analytical team conducting forced degradation on the drug substance can also prove highly beneficial,” he concludes.

Vol. 43, No. 12
	December 2019
	Pages: 20–23

	When referring to this article, please cite it as C. Challener, “Managing Excipient Interactions," 

The key is to ensure that excipients only interact with APIs via desired mechanisms.

Managing Excipient Interactions

Cell lines determine the performance of bioprocesses and the quality of the biologic drug substances they produce. As such, selection of the optimum cell line for each specific biologic is essential. Given the number of potential factors that can influence cell-line quality, the increasing complexity of biologic drug substances and growing regulatory requirements, identifying the optimum cell line can be challenging.

	A high-quality cell line will demonstrate high stability, scalability, and high titer, so that it can provide reproducible results-with all product quality attributes consistent with the desired profile-with high manufacturing efficiency, according to Ian Collins, senior director of process development at Catalent. “A high-quality cell line should be well-rounded among the critical chemistry, manufacture, and control (CMC) production cell line attributes,” agrees Jill Cai, vice-president of cell and protein sciences with WuXi Biologics.

	Before scaling up to bioreactors, the productivity level for monoclonal antibodies (mAbs) should be 2–7 g/L (fed-batch culture in shake flask) or greater, observes Selexis CEO Igor Fisch. Once scaled up, the mAbs should be 5 to >10 g/L in the bioreactor. “This high level of productivity must also be maintained for more than 60 generations at the high-density and in the defined media conditions of large-scale manufacturing,” he says. In addition, the parent cell line must be characterized and maintain consistency between each cell-line development campaign. Similarly, production cell lines must be well characterized so that scale-up from shake flasks to large bioreactors does not require extensive optimization.

	“Equally important,” stresses Fisch, “is the quality of the recombinant DNA expression and transfection systems. Incorporating recombinant DNA stably into a cell line’s genome in a manner that promotes high-level, stable gene expression while minimizing gene silencing is non-trivial. Therefore, both the cell line and the vectors systems must be optimal in order to achieve optimal results.”

	These aspects are in general desirable for both new biomolecules and biosimilars, although cells expressing biosimilars should be able to produce the target proteins comparable to the cells expressing the original therapeutic proteins. Special attention in this case should be paid to critical quality attributes such as glycosylation, which can add a variable to clonal selection and process development and can lead to a scenario where titer may have to be sacrificed for comparability, Collins notes.

	It is also desirable to leverage a cell-line development technology that has already been utilized in clinical trials or in approved products so that there is a precedent for regulatory filings, he adds.

	Many factors influence the selection of a quality cell line for a given biologic drug substance. First is the product itself, which can affect the choice parental cell line (e.g., Chinese hamster ovary [CHO] or other type). “The advent of more complex molecules rather than monospecific antibodies requires technology that can effectively express multiple genes, as well as titrate the genes as needed; this allows the correct product formation or the expression of other processing proteins to further ensure product quality,” Collins says.

	The capacity of the cell line to cope with the secretory stresses associated with the high level of protein production, particularly with many of the newer, non-natural proteins such as bi-specifics or multi-specifics, is also important, adds Fisch. “It is within the secretory pathway the proteins are properly folded, secondary modifications like glycosylation are added, and protein molecules are paired, when necessary, such as in monoclonal antibodies. If the secretory system cannot handle the secretory throughput, the biotherapeutic proteins can become misfolded or have improper secondary modifications,” he explains.

	This issue is particularly important for biosimilars, because the glycosylation patterns of biosimilars must meet biosimilarity specifications when compared to the innovator drugs, which can only be achieved when they are generated in cell lines with well-functioning and stable secretory machinery, according to Fisch.

	The plasmid ratio and medium are also important, Cai observes. “A balanced plasmid ratio usually has dramatic impact on some formats of bispecific antibody cell line development to minimize impurities,” she explains. Factors such as cell growth and metabolism, as well as product quality attributes like glycosylation distribution, are closely related to the culture medium, and thus many cell-culture media components can impact cell line and product characteristics, she also notes.

	Lastly, comments Fisch, optimized, well-defined, and maintained manufacturing conditions are absolutely critical to achieving the highest level of productivity of a high-quality product. “Feed streams, gas-exchange, metabolic bi-products, and temperature all play critical roles in the amount and quality of the biotherapeutic products. Only the highest quality manufacturing will result in the levels of productivity needed for clinical and commercial manufacturing,” he asserts.

	Challenges with variability, complexity, and regulations

	Selecting the optimum cell line must occur in the face of several Â­challenges. For Fisch, the biggest hurdle to overcome is the variability in the expression and secretion requirements of different biotherapeutic proteins. “Each cell-line development campaign brings its own set of challenges that can affect cell-line productivity and product quality,” he says. “Recombinant biotherapeutic proteins can misfold and stress the secretory pathway, leading to protein degradation or cell death, multi-chain biotherapeutic proteins may not pair properly, or perhaps glycosylation is incomplete. The challenge is to have a cell-line development platform that is robust and flexible enough to allow for troubleshooting across a broad range of production issues,” Fisch concludes.

	Added to this challenge is the strong focus by regulatory authorities on monoclonality, according to Collins. “Whichever method is used for clonal selection should be well validated and ideally utilized in programs that have gained regulatory approval,” he states.

	The sharp increase in the complexity of biomolecules is also increasing the difficulties associated with optimum cell-line selection, adds Cai. “Quickly developing and delivering high-quality cell lines is getting more and more challenging for bispecific antibodies and fusion proteins. It is difficult to rapidly screen more cell types clones to enable the selection of scalable cell lines capable of generating better cell productivity and product quality that are stable over extended cell-line generations and the long production cycle from vial thaw to harvest at large manufacturing scales,” she explains.

	Two specific challenges, Cai continues, include clippings for bispecific antibody and fusion proteins, which may require many constructs screening and extensive mass analysis or peptide mapping to detect the problem early on. Another challenge for bispecific antibody cell line development is the percentage of the desired product vs. byproduct impurities, which requires early downstream involvement to determine the types of impurities that are easy to remove, and consequently influences clone selection.

	The importance of an experienced team in cell-line development should not be underestimated, according to Fisch. “While there can be straightforward cell-line development campaigns with mAbs, there are also numerous examples of difficult-to-express mAbs. In addition, the newer bi-specifics and multi-specifics, which are non-natural proteins, can really challenge a production cell line’s capacity to produce and secrete,” he states. A team that has seen and solved many of the issues associated with difficult-to-express proteins has the know-how to identify issues early and a history of solutions to address them, Fisch asserts. Having an experienced team with access to the right technologies can save months on any cell-line development campaign, he adds.

	Bioinformatics tools can also be invaluable. “The ability to sequence the host cell-line genome, and more recently the transcriptome, along with the development of the bioinformatics to analyze that data has allowed for significant advances in cell-line development,” Fisch says. Bioinformatics analysis may help in getting genetic/epigenetic information that could offer insights into issues with protein production or cell stability in advance, agrees Cai.

	“Transcriptomics and proteomics can provide a more comprehensive and quantitative understanding of metabolic, signaling, secretory, and other pathways to determine whether a cell line is good for production,” Cai comments. “Additionally, Cai notes that bioinformatics methods can help rationally design a host cell through determination of ideal genome sites for target Â­integration and pathway engineering based on RNA sequencing results,” she notes.

	For instance, Fisch observes that secretory requirements can vary from protein to protein, and the genomic and transcriptomic analyses of the parent manufacturing cell lines helps to determine where there might be deficiencies or inappropriate expression of cellular proteins affecting a given biotherapeutic’s production.

	Detailed genomic data can also be used to assess cell lines for transgene integrity at junctions within the genome, transgene copy number and correct DNA sequence, number and locations of genomic integration loci, and possible adverse effects from transgene integration, according to Fisch. “[These] data, along with the right bioinformatics, can allow for direct assessment of the clonality of the biotherapeutic production cell line and provide traceability, validation, and origin of cells used to generate the production cell line-a vast improvement over the historical, indirect ways of determining clonality that makes it considerably easier to monitor drift and possible contaminations in production cell lines,” he remarks.

	The best instruments for selecting high-quality cell lines ideally enable the simultaneous screening of productivity (i.e., protein amount) and product quality (e.g., aggregation and cleavage), according to Cai. “Unfortunately,” she says, “currently available instruments mainly focus on productivity-based screening. Flow cytometry does, however, provide a relatively easy and economical way of screening high-producing cell lines.”

	New instruments based on microfluidic and nanofluidic technologies such as Cyto-Mine (Sphere Fluidics) and Beacon (Berkeley Lights), which allow the screening of cell-line productivity during the single-cell cloning step, are emerging, according to Cai, but such instruments are generally more expensive than flow cytometers and their reliability, time savings, and acceptance by regulatory agencies have not been widely demonstrated.

	Other new instruments designed for single-cell deposition are also available such as single-cell printers and verified in-situ plate seeding. Cai notes that these instruments are easy to use and may one day replace flow cytometry for single-cell deposition.

	Catalent has focused on the Beacon platform, which the company believes to be an emerging leader in the area of clonal isolation and selection. “The Beacon platform is an automated system that is able to select the highest expressing clones faster than traditional methods and provides sufficient data to allow for a single round of cloning, which can reduce timelines for early cell-line development,” Collins states.

	The best selection tools depend upon the cell-line development platform, adds Fisch. “In some platforms with lower transfection efficiencies, identifying high-expressing cell clones depends upon screening tens of thousands of clones, which is achievable using flow cytometry and high-throughput screening systems. When transfection efficiencies are high, identifying high-expressing and stable cell clones requires far less screening,” he explains. Selexis uses systems such as the Beacon optofluidic platform and ClonePix to provide faster, more efficient, multiparameter screening approaches for clone selection.

	Catalent is leveraging automation to create high-quality cell lines and timelines. The company combines its proprietary GPEx cell-line development technology with the Beacon platform to generate highly stable, high-titer clonal cell lines for even difficult-to-express proteins, while the ambr15 system (Sartorius AG) allows for early evaluation of the individual clones in a bioreactor environment, according to Collins.

	WuXi Biologics carefully considers each and every step involved in cell-line development using its WuXia platform to ensure the generation of high-quality cell lines. The strategy, according to Cai, typically starts with the molecule of interest and a codon optimization step to optimize the sequence of the gene(s) of interest. Proprietary vectors are then specifically engineered to enhance the expression level of the target genes. The company uses a tandem pool development and clone-screening approach to maximize the chances of deriving high-producing and high-quality cell lines. “We work closely with our analytical and process development teams to ensure the developability, monoclonality, and scalability of our cell lines and to achieve the key product quality attributes that our clients and partners desire,” Cai observes.

	With the Selexis SUREtechnology Platform, Selexis scientists apply a modular approach to generate high-quality cell lines that meet the production needs of each recombinant therapeutic protein. At the foundation of each cell-line development program, according to Fisch, are Selexis SGEs (Selexis Genetic Elements), unique epigenetic DNA-based elements that control the dynamic organization of chromatin in all mammalian cells and allow for higher and more stable expression of recombinant proteins.

	A panel of tens to hundreds of high-expressing clones are assessed for production levels, product quality, and cell viability. If any of these parameters are suboptimal, Selexis has a suite of solutions for addressing them. With this approach, Selexis is able to express almost every protein, including difficult-to-express proteins that have failed in other systems, Fisch says.

	Selecting cell lines for CAR-T therapies

	As opposed to a synthetic drug, chimeric antigen receptor (CAR)-T cells are living therapies that are sensitive to manipulation and difficult to manufacture with consistent quality. As a result, the process of preparing them so they are safe and effective is multifaceted and complicated, according to Kris Simonyi, global marketing manager with Bio-Rad Laboratories. “One must confirm that all replication-competent viruses have been removed before injecting CAR-T cells into the patient, that the transgene has been inserted in the right location in the genome with the right dosage, and that the cell line is free from microbial contamination,” he explains.

	As a result, a high-quality CAR-T-cell line should not contain any replication-competent viruses or microbial contaminants, but should contain one to four copies of the CAR gene (more than four copies exposes patients to the toxic effects of T cells, which include organ dysfunction and death).

	One of the biggest challenges in selecting high-quality CAR-T cells, Simonyi notes, is that even if a transfection is technically successful, it does not mean it will provide a clinical benefit. “Transgenes integrate into random sites and may have detrimental effects on a patient,” he says. For example, a CAR gene could integrate into and activate an oncogene and lead to cancer. It could also integrate into a non-coding region of DNA and never be expressed.

	To detect low levels of CAR genes and microbial contaminants and precisely quantify CAR genes, Bio-Rad’s Droplet Digital PCR (ddPCR) technology is of increasing interest. The technology selectively amplifies targets of interest including genetic alterations and nucleic acid contaminants, making them easy to identify, according to Simonyi.

	Samples are partitioned into 20,000 nanoliter-sized droplets and a polymerase chain reaction (PCR) takes place in each droplet. A fluorescent probe binds to the target DNA, making it fluoresce when it gets amplified. The nucleic acid fragments that do not contain the target sequence do not get amplified and do not fluoresce. By counting the number of fluorescent droplets versus the total number of droplets, the concentration of the target DNA strand can be determined.

	Custom probes can be used to select for cells containing the CAR gene and bacterial contamination. Because the system performs absolute quantification without the need for a standard curve, it can also measure transfection efficiency and precisely quantify the transgene copy number in CAR-T cells. “Our ddPCR technology can be 100- to 1000-fold more sensitive than quantitative PCR, enabling it to detect down to one CAR gene copy per cell,” Simonyi comments.

	It can also detect and precisely quantifying various types of DNA and RNA alterations that can factor into the quality of genetically engineered T cells, including minimal levels of nucleic acids of interest, such as genetic material from replication-competent viruses and other contaminating microbes. In addition, following next-generation sequencing to map transgene integrations, Bio-Rad’s ddPCR technology can be used to track the cells through the manufacturing process, according to Simonyi.


	Vol. 31, No. 10
	October 2019
	Pages: 18–21

	When referring to this article, please cite it as C. Challener, “Best Practices for Selecting a Top-Quality Cell Line,"

Using automation and taking a step-by-step approach can facilitate the process of selecting a top-quality cell-line source.

How Best to Select a Quality Cell-Line Source

Increasing emphasis on patient centricity is driving the development of different oral dosage forms of APIs used in adult products for specialty patient populations. Poor patient compliance is a significant issue in the pharmaceutical industry that can be addressed to a significant degree through the formulation of dosage forms designed for convenience and ease of use.

	Modification of adult medications for children by dividing tablets or capsules into smaller segments to achieve lower doses or aid in swallowing, however, can lead to incorrect and/or nonuniform doses and compromise the performance of any sustained-release coating in the product. In some cases, excipients suitable for adult formulations can pose safety issues for children.

	Age-appropriate medications are now required by regulatory agencies for children. Elderly patients and patients suffering from dementia and disorders that lead to problems with swallowing also benefit from specialty dosage forms. In all of these cases, the emphasis is placed on patient acceptability, which can be impacted by physiological, physical, and psychological factors.

	“Overall, the practicality of administering an existing drug to a different patient population will determine its acceptability,” says Rob Harris, director, science and technology at Catalent.

	Specifically, there are several factors to consider. The first, according to Jim Jingjun Huang, founder and CEO of Ascendia Pharmaceuticals, is efficacy and safety, which is determined by extrapolating the pharmacokinetics and pharmacodynamics for the existing formulation to the targeted population. The acceptability of the excipients in the existing formulation in the target population-taking their use levels into consideration-must also be established. For many specialty populations, the swallowability and taste of the existing formulation may need to be modified. Finally, dose accuracy, and if appropriate flexibility, must also be assured for the target population.

	“Taste-masking, swallowability, dose flexibility, and excipient acceptability are all important for pediatric formulations, whereas dosing accuracy, swallowability, and the capability for multi-drug administration are important for geriatric formulations,” Huang notes.

	For instance, Harris points to tablets, which are suitable for teenagers, but not patients two years old or younger. The pediatric patient group is unique, adds Anthony Qu, vice-president of scientific affairs for Cambrex. “The pediatric patient population includes infants, children, and adolescents. You cannot simply cut an adult dosage down. Pediatric formulations should vary from adult formulations depending on the patient age, body size, swallowing capability, and pharmacokinetic (PK) and pharmacodynamic (PD) profiles,” he asserts.

	It is also important to note, according to Qu, that pediatric formulations can benefit other patient groups that have swallowing issues, such as the elderly, patients who have had a stroke, or patients with long-term diseases such as Parkinson’s or Alzheimer’s.

	There are many sources of information to assess patient acceptability. “The fastest and least costly method of evaluation is to review relevant publications, the content of which include results of patient focus groups or patient and caregiver surveys,” Harris says.

	There are numerous challenges to reformulating existing adult products for specialty patient populations. For all of these types of projects, accurate extrapolation of the PK/PD can present real difficulties, according to Huang.

	For most specialty populations, swallowability and palatability are key elements in the design of suitable dosage forms. “Most drugs have an unpleasant taste, and therefore due consideration needs to be given to making medicines acceptable from this perspective,” observes Harris. For children and the elderly, the actual dose can also be problematic; while an adult may be able to take a large tablet containing 500 mg of drug, administering a similar-sized tablet to a five-year-old child is much more challenging, he notes.

	The physical differences of age, body weight, swallowing capability, and tolerance within the pediatric population are also challenging and create the need for dose flexibility, according to Qu. He adds that stability, shelf-life, and bioavailability issues can further complicate formulation development. Ensuring excipient acceptability for pediatric formulations adds another layer of complexity, according to Huang.

	The first step in a reformulation project for a specialty population is to consider the age range of the patients and the dose range of the drug to be given. It is then important, observes Harris, to consider the drivers for achieving dose acceptability and compliance for that patient population, and what might the complications be in administering the drug to these patients.

	Questions that should be answered when looking to overcome reformulation challenges, according to Qu, are:

		What measurable dose form is required based on mg/kg body weight?

		Does the API formulation have a bad taste, and if so, can this taste be suppressed or masked?

		Is the formulation stable or not stable?

	“The goal is to determine the formulation strategy that will ensure similar exposure of the API in the new dosage form for new patient population as is achieved with the adult product and address the specific needs of the target population,” Huang explains. The solubility of the API must be considered to determine if a solubilization technology will be needed to achieve this goal. The stability of the API will determine whether a liquid (i.e., solution, syrup, suspension) product is possible, or if a solid dosage form will be necessary, Qu adds.

	Once the various factors have been considered and the relevant questions answered, it is possible to determine the optimum formulation and oral dosage approach. The main options include traditional liquid and solid (i.e., tablets, capsules) formulations, as well as newer dosage forms, such as chewable and fast dissolving tablets, rapidly dissolving and mucoadhesive oral thin films and multi-particulate formulations such as mini-tablets.

	“The traditional approach has been to use solution or suspension formulations because they are easy to swallow and provide dose flexibility. However, the use of sugars, flavors, and preservatives in these formulations can have detrimental effects on children and are consequently becoming less popular,” says Harris. These formulations also typically need to be refrigerated in-between use, have a shorter shelf-life, and can have overall stability issues compared to other forms, according to Qu. Furthermore, accurate dosing can be an issue due to varying spoon sizes.

	Traditional solid-dosage forms are well-established technologies that have stable, long-term shelf-lives, are pre-measured for dosing, and can be coated to mask taste or added to food, according to Qu. “Conversely,” he says, “they can be difficult to swallow for some patients, and certain types of formulations are hydroscopic and require special packaging and storage in a dry place.”

	Chewable tablets are another option often suitable for pediatric patients. “These formulations typically benefit from enhanced bioavailability and are convenient because water is not required for consumption and absorption is typically fast,” Qu says. On the other hand, these formulations typically contain sorbitol, which could have potential side effects in children. Other factors to consider, according to Qu, are the material flow, types of lubricants used, disintegration acceptance, taste, compressibility, and stability.

	Orally dispersible tablets in which the tablet rapidly disintegrates in the mouth have become popular and certainly allow for easy swallowing of the dose, according to Harris. Qu notes, however, that they present their own hurdles as well, such as lower strength levels and half-lives.

	Mini-tablets are the most common type of multi-particulate formulation. These small (typically 2–3 mm in diameter) versions of normal tablets can be readily swallowed by very young children. They also offer great dose flexibility, because the number of minitabs can be varied to achieve the required dose, according to Harris. In addition, unpleasant tastes can be masked using barrier coatings.

	A further benefit of mini-tablets is the ability to combine different types of tablets in one dosage form (within a capsule or compressed into a tablet), according to Qu. “It is relatively easy to combine immediate and sustained release mini-tablets in one product. Similarly, mini-tablets facilitate the formulation of fixed-dose combination products containing two or three different APIs,” he observes.

	One potential solution to the potential solubility, stability, and taste-masking issues is to utilize nanotechnologies, such as nanoparticles or nanoemulsions, which can encapsulate the molecules into a nano-formulation that can mask the taste and protect the compound from degradation, according to Huang. Another approach, he says, could be a change in the route of administration to injectables; a long-acting injectable for chronic disease would address the taste, dose accuracy, safety, and compliance issues associated with oral dosage forms.

	For pediatric formulations, it is essential to ensure that all of the selected excipients are safe for consumption by children. “Excipients play a key role in how a drug product is manufactured, stored, consumed, and delivered in the body. Just like with dosage and formulation, not all excipients that are appropriate for adult formulations will work in pediatric formulations. Children may not be able to metabolize or eliminate the excipient in the same way that an adult can, which can cause issues such as stomach upset (indigestion, flatulence, diarrhea), breathing trouble, intoxication, or worse,” Qu explains.

	In some cases, there may be different safety limits for an excipient that is used in an adult formulation versus a pediatric formulation, and guidance exists on this topic. In other cases, an excipient may need to be replaced completely with a different alternative. Formulators must decide what function each excipient is serving in the adult formulation, and then select and assess alternative, child-acceptable excipients that offer similar functionality, according to Harris. He points to the European Paediatric Formulation Initiative Safety and Toxicity of Excipients for Paediatrics database as a good place to start in selecting suitable excipients for use in children-friendly medicines.

	“Selecting suitable excipients for a pediatric formulation is not normally challenging, but there are restrictions. The key point here,” Harris stresses, “is that excipients are not inactive ingredients.”

	Successful reformulation of adult medicines for specialty patient populations can be successfully achieved by starting with a ‘patient-first’ mind set, which requires consideration of the formulation type that will best suit the needs of the patient, not only for getting the drug into the body, but also for ease of use and patient acceptance, according to Harris.

	The development of pediatric and other specialty formulations requires extensive knowledge and expertise in not only a wide variety of dosage forms and technologies, but awareness of the key determinants for patient acceptability. “As a result, many companies rely on experienced [contract development and manufacturing organizations] CDMOs with specialist expertise in this field,” says Qu. “Such CDMOs will be able to provide guidance and develop the right formulations that consider the age and taste preferences of the target population in the context of different dosage forms, in addition to paying attention to regulatory approval early in the development process,” he concludes.

	1. European Paediatric Formulation Initiative, 

Safety and Toxicity of Excipients for Paediatrics


	Vol. 43, No. 11
	November 2019
	Pages: 22–24

	When referring to this article, please cite it as C. Challener, “Adapting APIs for Specialty Dosage Forms," 

Several factors must be considered when reformulating APIs for pediatric, geriatric, and other specialty patient populations.

Adapting APIs for Specialty Dosage Forms

The stability of clinical trial materials, regardless of the trial phase, must be understood to ensure patient safety. Stability can be affected by the nature of the API, the production process, the choice of excipients, the container and container closure system, and other factors. Stability testing is therefore essential for demonstrating that the formulations administered to patients in any clinical trial will remain unchanged throughout the duration of the trial. The type and length of stability tests typically depend on the phase of development and the nature of the clinical trial (e.g., use of placebo or comparator drug). 

Limited guidance and numerous challenges create confusion about the scope and timing of stability testing for drugs in development.