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

Cynthia A. Challener

Established vaccine manufacturing technologies are complex, often highly customized, and typically require dedicated facilities. Development and approval times can be lengthy, presenting hurdles for medical professionals seeking rapid response to pandemic diseases.

	Standardized vaccine manufacturing platforms that can be deployed across different vaccines and vaccine types, could speed the delivery of vaccines and enable manufacturing in emerging economies. Technical and funding challenges must be overcome to bring the advanced manufacturing potential to reality.

Pharmaceutical Technology and BioPharm International

Vaccine Development and Manufacturing 2017

	When referring to this article, please cite it as C Challener, " Accelerating Vaccine Development and Manufacturing," 

Pharmaceutical Technology and BioPharm International Vaccine Development and Manufacturing 2017 eBook

Articles

The use of approved platform technologies can reduce the time and cost required to generate new vaccines.

Accelerating Vaccine Development and Manufacturing

Taste masking is a method used to improve not only the taste of bitter APIs in formulated drug products, but also mouth feel (smooth vs. gritty) and the overall acceptability of medications to patients. It can be achieved in various ways. Common methods include changing the API to a different form (e.g., drug freebase, salt, or pro-drug) that is less bitter; using sweeteners, buffers, taste modifiers, or aromas to reduce aversive sensory attributes; and complexing the API to prevent its interaction with taste receptors, such as with cyclodextrins and ion exchange resins. The API can be physically encapsulated through application of a coating (or coatings) to a drug particle or tablet, the use of barrier membranes, or the formation of solid dispersions via spray drying or hot-melt extrusion. Another potential method involves blocking of taste receptors directly or along the signaling pathway.

	Taste masking is important because poor patient adherence often leads to worsening health and poor outcomes, along with increased costs across the healthcare system. A broader range of taste-masking technologies is required for the growing number of different dosage forms and drug-delivery approaches in use and under development today to meet the unique and specialized needs of different patient populations. Pediatric patients consist of subgroups from babies to teenagers, each with its own requirements. Geriatric patients may have difficulty swallowing. Patients with neurological conditions may resist taking medications if the drug product is not easy to take. Excipient manufacturers and service providers focused on taste masking are overcoming a range of challenges--from regulatory hurdles to an ability to accurately predict and measure taste-masking performance--to develop technologies suitable for the growing range of dosage forms and delivery systems.

	The use of taste-masking technologies is driven by a number of factors that can be classified as regulatory, economic, demographic, and philanthropic, according to Jeff Worthington, president and founder of Senopsys. Regulations in the United States and European Union both require the development of pediatric medicines and the submission of detailed plans that outline the studies to be conducted as part of the pharmaceutical and clinical development programs. Unless a waiver is granted, pharmaceutical companies are required to develop pediatric formulations suitable for patients from birth to adolescence requiring age-appropriate dosage forms, such as liquids, orally dissolving films, fast dissolving tablets, granules (sprinkles), and mini-tablets, Worthington reports. 

	Tablets and capsules effectively bypass the taste receptors in the oral cavity, but most of the pediatric (and geriatric) dosage forms have residence times in the oral cavity, meaning the drug active can be perceived. Palatability, however, is essential to dosing compliance and adherence; therefore, regulatory agencies require that companies demonstrate the palatability of drug products in pediatric patient populations as part of the pharmaceutical and clinical development program. “Because many drug actives are bitter or have other aversive attributes including trigeminal irritation, malodor, and texture (e.g., grittiness), taste masking is required to ensure successful dose administration,” Worthington explains.

	Non-governmental organizations also support the development of pediatric dosage forms for use in developing countries. Solid-dosage forms that permit flexible dosing (e.g., granules and mini-tablets) are preferred due to a variety of factors, including lack of potable water, complex logistics, and lack of refrigeration, according to Worthington.

	Geriatric patients present additional adherence challenges. “This demographic group is often taking multiple medications and dosages provided by their caregivers; they also have a greater degree of challenges with swallowing and handling. Taste-masking is just one element that has to be considered,” according to Ali Rajabi-Siahboomi, vice-president and chief scientific officer of Colorcon.

	Dosage forms other than conventional tablets and capsules-which may be difficult for older patients to swallow-need to be developed, adds Don Barbieri, technical product manager at SPI Pharma. He points to chewable tablets, dispersible powders, and orally disintegrating tablets as alternate dosage forms that can be administered to address swallowing difficulties. With orally dispersible products, however, there is a need for taste masking so that these dosage forms have acceptable palatability with little or no bitterness.

	On the economic front, many of the growing number of drug candidates being developed to target orphan diseases, including many pediatric medicines, are often financially attractive due to premium pricing opportunities, market exclusivity, and priority regulatory reviews among other benefits; however, the formulations require taste masking, according to Worthington. 

	A number of hurdles must be overcome when developing taste-masking technologies that are effective. Top among them is achieving the necessary balance between preventing release of the API while the drug product is in the mouth but allowing for release once the medicine is in the stomach, according to Rajabi-Siahboomi. “The pharmacodynamics and pharmacokinetics (PK) cannot be effected by the taste-masking technology,” he says. The taste-masking technology must be appropriate for the extent of bitterness of the API, its solubility, and dosage level, as well as the dosage form (coated tablet, liquid, or granules to be distributed on food, etc.). In some cases, adding a sweetener or flavor is sufficient; in others, some type of barrier membrane must be used. Taste maskers that sequester the API from taste receptors (e.g., particle coating, adsorption, complexation), however, may adversely affect PK, leading to effective taste-masking but reduced bioavailability, according to Worthington.

	Developers of taste-masking technologies also often work with APIs that have been incompletely or inaccurately characterized with respect to their aversive taste attributes because many APIs have multiple aversive taste attributes--such as bitter and sour basic tastes with trigeminal irritation--each of which may require a different mitigation approach, according to Worthington. In addition, he notes that the absence of a robust measurement system to provide quantitative data on the intensity and duration of aversive taste attributes makes optimization of taste-masking technologies difficult.

 taste models (e.g., the electronic tongue with human data) often results in sub-optimal taste-masking and palatability issues, according to Worthington. There is also increasing regulatory pressure to reduce the number of excipients used in pediatric formulations, leading to the availability of fewer tools and lack of regulatory clarity on how specifically to approach pediatric formulation development. “For the most part, agencies have adopted a ‘propose-and-justify’ approach, rather than ascribing a defined process,” Worthington comments.

	Modifying existing APIs through complexation to ionic exchange resins, while effective for taste masking, can present regulatory challenges, according to Barbieri. “In the United States, over-the-counter (OTC) monographed drugs can be marketed without further FDA approval. When an OTC monographed drug is attached to an ion-exchange resin, however, FDA considers the complexed drug to be a new compound that requires regulatory approval,” he explains. 

	Getting novel excipients of any kind approved for use in prescription medicines is also challenging because there is no separate approval pathway for excipients. A new excipient must be approved along with a formulated drug product, and often drug developers are hesitant to take on the added risk of using an excipient that has not already been proven acceptable to regulators.

	On the production side, Barbieri notes that the use of solvents for the application of taste-masked polymer systems is an issue. “Aqueous systems are definitely preferred to avoid the additional testing that is required to quantify residual solvent levels and the added cost of handling and disposing of solvents,” he says.

	Recent innovations in established taste-masking approaches have been incremental rather than breakthrough or novel technologies, according to Worthington. As an example, he notes that in particle coating there have been advances in barrier coatings used for taste masking that are better at balancing the need to maintain the target PK profile while minimizing particle size to minimize grittiness. 

	Senopsys applies expertise in sensory and formulation science garnered from the food industry to the development of palatable, patient-accepted drug products for its clients. These efforts include advances in the company’s quantitative FlavorMetrics Taste Assessment and FlavorOpt Taste Masking platforms and the development of methods for assessing the ease-of swallowing multi-particulate dosage forms (granules and mini-tablets), and a science-based approach to screening age-appropriate dosing vehicles for multi-particulate products. The company also is focused on the development of novel dosage forms to address unique dosing challenges, such as bars, chews, gummies, and ready-to-use puddings for high drug loading applications, according to Worthington.

	SPI Pharma has developed a gelatin coacervation process as a new approach to taste masking of APIs. Originally introduced to manufacture carbonless copy paper, the process is performed in water without the need for solvents. The gelatin coating provides an excellent taste-masking barrier, and the resulting hydrogel film around the particle provides great mouth feel with smooth sensation and improved swallowability, according to Barbieri. “Interest in acetaminophen and ibuprofen taste masked with coacervated gelatin has increased significantly and, worldwide, more products are planned for introduction in the next one to two years,” he observes. Given the continuing increase in both interest and sales, SPI is actively evaluating other APIs that would be good candidates for taste masking via a coacervation process, according to Barbieri. 

	In addition to offering its established barrier membrane technology (Surelease/Opadry combination) and ion exchange resin (using DowDuPont AMBERLITE and DUOLITE) taste-masking solutions, Colorcon is working with BASF on the application of that company’s Kollicoat Smartseal, a novel, reverse enteric polymer that is highly stable in saliva and specifically soluble in gastric juice. It is being evaluated by Colorcon for inclusion in a film-coating system to simplify and accelerate coating operations and provide new options for taste masking of drug particles and granules. Colorcon also recognizes the industry need for a pigmented taste-masking solution to address the issues of differentiation and patient recognition. “However, because the polymer is a novel excipient, there is a real challenge to get it approved. We are working with some branded pharmaceutical companies who are evaluating its use in their formulations,” Rajabi-Siahboomi says.

	As demand for taste masking increases further, the industry will look for ways to improve coating efficiency, reduce processing time, reduce waste, and maximize product yields, according to Barbieri. “Excipient manufacturers can be of benefit here by providing to pharmaceutical manufacturers a selection of different substrates for bead coating in a range of different particle sizes. The various core beads can be made to target specific particle size specifications and narrow particle size ranges. In this way, manufacturers can select the most appropriate seed product for their process without having to perform additional screening steps to sort out the inappropriate size beads, thereby facilitating manufacturing and reducing waste,” he comments.

	To better support the application of its new taste-masking technologies and offerings, Colorcon is developing new methods for in-vitro testing using artificial saliva that will be truer tests and resemble what occurs within the body. They will be intended for use in conjunction with conventional dissolution testing. “There needs to be a mechanism for confirming that formulated products are indeed taste masked. Current dissolution testing methods don’t accurately model what is in the mouth,” says Rajabi-Siahboomi.

	Worthington expects to see advances in barrier coatings in which these systems are combined with smaller particles to yield further improvements in palatability and swallowability. He also notes that further advances in 3D-printing may provide additional opportunities for taste masking as well as enable on-demand production of custom drug products.

	Receptor disruption as a long-term solution?

	Humans have approximately 30-40 known bitter taste receptors (many more than for sweet, sour, or salty); different compounds activate different sets of receptors. Efforts are underway to identify compounds that block bitter perception. Because individual compounds activate different sets of receptors, the prospect would appear greater for interrupting the common signaling pathway rather than a multiplicity of receptors, according to Worthington. “Regulatory agencies might consider these to be biologically active components at either the receptor or signaling pathway level, however, meaning that the bitter blockers would be regulated as drugs--or at best novel excipients--either way impeding adoption by industry,” he states. It is possible though, that basic research in taste perception may lead to future advances in taste-masking technology.

	When referring to this article, please cite it as C. Challener, “Growing Demand for Taste-Masking Technologies," 

Taste masking increases drug acceptability and medication adherence in pediatric, geriatric, and other special patient populations.

Growing Demand for Taste-Masking Technologies

The global market for highly potent APIs (HPAPIs) is growing at a healthy rate. According to market research firm Markets and Markets, demand for HPAPIs will expand at a compound annual growth rate of 8.5% from $16.02 billion in 2016 to $24.09 billion in 2021 (1). Rapid growth in the markets for oncology treatments (including generics) and antibody-drug conjugates is driving this expansion. 

	The manufacture of HPAPIs requires specialized facilities, equipment, and skills to ensure the safety of operators and the environment. As such, the bulk of HPAPIs are produced by contract development and manufacturing organizations (CDMOs) with these specialized capabilities. One of the challenges for CDMOs when accepting new projects, particularly those involving new chemical entities (NCEs), is identifying the level of potency, or biological activity, in order to establish appropriate safety and handling procedures.

	There are two parts to an assessment of an NCE with respect to potency or biological activity. The first part, according to Simon Edwards, vice-president of sales and business development at Cambrex, is a risk assessment that produces an exposure “limit” or an exposure “range” (i.e., occupational exposure limit [OEL] or occupational exposure band [OEB], respectively). The limit or range is in units of mass per volume air (micrograms per cubic meter, mg/m

). The second part of the assessment is risk management, which involves establishing actions or preventive steps to keep exposures below the limit or range determined by the risk assessment. This work is undertaken through a collaborative effort between toxicology, safety, and engineering departments.

	Standardized approach, but no standard system

	This general approach to risk assessment is reasonably well-standardized, but the way the results are displayed varies greatly because the application of uncertainty factors differs depending on the individual risk assessor. The standard approach, according to Cambrex, is incorporated into the International Council for Harmonization (ICH) guidance on impurity assessment (2), which is used by the American Conference of Governmental Industrial Hygienists (ACGIH) in a manner similar to the way FDA and the European Food Safety Authority (EFSA) conduct food ingredient risk assessments and EPA undertakes environmental risk assessments.

	There is no standard system, however, because there are no regulatory compliance requirements for occupational exposure limits. “Having discussed this in depth with expert consultants, we feel that it is very unlikely that there will ever be a collaborative effort to establish a common system unless a regulatory mandate is introduced,” says Edwards. 

	This lack of a standard classification system is not a hindrance for CDMOs, however, according to Ramesh Subramanian, vice-president of strategic marketing with Piramal Pharma Solutions. “The actual nomenclature is not as relevant as the OELs themselves. So despite using various classifications, the industry is able to move forward with minimal issues, as the OELs provide the clarity that is needed to establish containment solutions,” he observes. 

	Because there are many possible pharmacophores and toxicity endpoints, Edwards stresses that risk assessment/risk management of NCEs takes a great deal of experience and chemical intuition. “Managing the potential exposure in manufacturing is not prescriptive either, as it is facility- and equipment-specific, and requires experienced engineers and safety people to implement correctly in order to protect employees, the facility, and the surrounding environment,” he states.

	The key, according to Vivek Sharma, CEO of Piramal Pharma Solutions, is having an established potent compound category policy that defines the level of containment required depending on OELs or other toxicity data (e.g., LD50) and having predefined default criteria for containment in the absence of such data. “Quality CDMOs tend to default to a higher OEL when in doubt to ensure operator and staff safety,” he says. “Achieving a strong track record of safe HPAPI manufacturing also requires an understanding of the systems that are required to minimize the risks associated with potent compounds.” Sharma notes that proper facility design and engineering, including the heating, ventilation, and air-conditioning (HVAC) system, barrier isolation, and gowning/degowning areas are key considerations. “Appropriate facilities, engineering controls, and safety protocols are, in fact, increasingly imperative as newer HPAPIs under development have ever declining OELs,” he comments.

	During early development stages, many drug sponsor companies do not have the toxicity data needed to ensure that their compounds are handled safely. The challenge for CDMOs then becomes ensuring that the right level of safety measures is implemented and followed when handling the molecule and the associated chemistry, according to Vince Ammoscato, site head-Riverview at Piramal Pharma Solutions. In the absence of any data, many companies default to a potent designation Category 3 (10µg/m

	“The responsibility for containment rests with the CDMO since they are responsible for the safety of their employees. Having environmental, health, and safety (EHS) best practices with an excellent chemical hygiene program, which includes a potent compound handling policy that has been vetted through testing of the engineering controls and procedures, is paramount to ensuring NCEs are handled safely regardless of whether the potency levels have been established,” Ammoscato asserts.

	There is the additional challenge, however, of the tendency by chemists and operators to make “dread”-based decisions on handling and how to respond to incidents, according to Edwards. “This type of response is really not necessary if the appropriate risk assessment is performed,” he notes.

	The most common strategy when a CDMO is presented with a project involving an NCE, according to Edwards, is to initially ask the drug sponsor for available data, as this information should be available because the results-probably 

-have led them to move forward in the development of a NCE. 

	“In instances where the OEL of the molecule is unknown, there is no one common strategy for NCEs, but most CDMOs use some form of standard operation procedure,” Edwards says. “For either an NCE or synthetic intermediate, qualitative, or quantitative structure activity- and reactivity assessment-related data coupled with read-across and 

 data are used at Cambrex. These data are all important and come from many sources. The amount of data available depends on novelty of the pharmacophore,” he explains. Cambrex also calculates an OEL even when the sponsor has provided one.

	Ramesh from Piramal adds that firms may use substructure- or chemotype-based OEL estimations that include consideration of the therapeutic class for guidance. “One approach involves comparison of the API’s properties to those of similar substances with known toxicities and assumption of a similar level of risk,” he notes. Some CDMOs have in-house categorization systems for determining the required safety measures. 

	“Regardless of the method, if there is any uncertainty, it is best to assume a compound is potent, despite the potential higher costs incurred,” Ammoscato states. “Frequent communication between the sponsor and the CDMO is then essential to ensure that necessary data can be generated and an appropriate decision on the potency of the compound be reached as early as possible,” he adds. The CDMO should also be prepared to respond as needed if additional toxicity data are developed indicating that reclassification is warranted. Furthermore, CDMOs should perform additional risk assessments as an HPAPI project moves through different stages of development and commercialization to ensure the implementation of optimal procedures and practices that both protect operators and the environment but are also practical and cost effective, according to Ammoscato.

	1. Markets and Markets, “High Potency API /HPAPI Market by Type (Innovative, Generic), Synthesis (Synthetic, Biotech (Biologic, Biosimilar), Manufacturer (Captive, Merchant), Therapy (Oncology, Glaucoma, Hormonal Imbalance)-Global Forecast to 2021,” January 2017.
	2. ICH, 

M7 Guideline on Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk

CDMOs sharpen skills for identifying API potency risks and establishing safety and handling procedures.

Setting Safety Processes for Potentially Potent APIs

Although drug repurposing has been receiving growing attention in recent years, drug companies have taken this approach, at least serendipitously, for decades. Viagra, originally developed as an angina treatment, is one of the most well-known examples. Today many groups are focused on identifying new indications for existing drugs including academic researchers; nonprofits such as Cures Within Reach; and government institutions including the National Center for Advancing Translational Sciences in the United States and the UK Medical Research Council. Other drug repurposing research ventures include public-private partnerships like the Center for Drug Repurposing (operated jointly by Ariel University [Israel] and Drug Rediscovery Ltd) and the Drug Repurposing Hub (a collaboration between the Broad Institute Cancer Program, the Center for the Development of Therapeutics, and the Connectivity Map group); and companies such as Biovista, GVK Bio, and NuMedii. In 2012, it was estimated that drug repurposing, repositioning, and rescue (DRPx) efforts accounted for 30% of FDA-approved new drug products (1). In addition, in 2015, Biovista President and Co-founder Aris Persidis reported that these drugs accounted for approximately 25% of pharmaceutical industry revenues (2).

	The known safety of existing APIs is one of the biggest benefits of using them for new indications discovery, according to Gini Deshpande, founder and CEO of NuMedii, a drug-discovery company that applies its proprietary big data platform based on integrative genomics, network-based methods, large-scale machine learning, and chemoinformatics to improve efficacy. One application of NuMedii’s technology is to identify new indications for existing APIs. “Safety data for these APIs can be leveraged to expedite the development process via the 505(b) 2 approach in the United States or the hybrid approach in the European Union,” she explains.

	For instance, a new chemical entity would normally take 10-15 years to get to the market, whereas an existing API being developed for a new indication can reach the market in five to eight years depending on the indication for which it is being developed. “This reduction in development time results in a significant cost and time savings for companies,” Deshpande says.

	Success rates in Phase III trials are also much higher for products developed via 505(b) 2-type approaches compared to traditional approaches. Evaluating new indications for existing APIs can also expedite the discovery and development of new therapies for rare diseases and for other conditions where existing APIs can be used to validate novel biology and thus speed up the discovery process, according to Deshpande. She notes that there are approximately 2800 drugs and more than 4000 compounds that have been discontinued at Phase II development, so there is a rich pool of existing APIs with which to work. 

	One of the biggest challenges to repurposing of existing drugs is in finding that novel indication. NuMedii’s big-data, artificial intelligence (AI)-driven methods provide a distinct advantage with such an effort, according to Deshpande. “Because our technology uses both data- and knowledge-driven approaches to find the broadest possible landscape of new indications, we often identify out-of-the-box new indications for existing APIs that have previously been undiscovered,” she explains. 

	Ensuring market exclusivity and reimbursement level are two other key challenges with this approach. These issues are further compounded by geographic-specific issues, because rules differ in the EU, the US, and the rest of the world. 

	Four major factors need to be considered when looking for new indications for existing APIs, according to Desphande: scientific rationale, clinical viability, commercial viability, and regulatory path. 

	Scientific rationale involves ensuring that the biology being modulated by an existing API is relevant and assessing whether formulation or delivery for the new indication is feasible. Clinical viability relates to whether there is a specific unmet need being solved by the API, the fit within a treatment paradigm, and the risk/benefit equation for using an existing API for the new indication. Commercial viability factors include the nature of the competitive landscape, reimbursement issues, and target pricing. Questions regarding the regulatory path are focused around the type and amount of available existing data that can be used for approval, and what additional studies need to be conducted and at what cost for the various phases of development.

	In the past, drug repurposing relied on serendipity, according to Deshpande. “Case reports of off-label uses and side effects of drugs reported (either approved or those in development) were often a good starting point to look for new indications/uses for these APIs,” she notes. Recent approaches have now made the process more systematic. She points to i

 approaches that span a multitude of both computational biology approaches such as the one NuMedii uses and computational chemistry methods, screening approaches using both cell lines and animal models and literature-based searches, although the latter can have potential issues due to prior art with regards to intellectual property.

	NuMedii began using genomic-data driven methods, but now leverages multiple types of ‘omic’ information, as well as information from the literature, clinical information, and other data types to ensure that the company captures the best possible indications for existing APIs, according to Deshpande.

	“Since 2010, NuMedii has demonstrated that its discovery technology and machine learning algorithms can dramatically impact the time, cost, and success rate of drug development in the fields of inflammation and oncology, thus revolutionizing the drug discovery process as we know it,” she attests. “We have demonstrated the predictive capability of NuMedii’s technology in the areas of inflammation and oncology with many pharma partnerships and projects,” Deshpande adds. One such example is NM-001, an undisclosed cardiovascular drug that has been in use for more than 30 years. NuMedii’s software uncovered wide-spectrum anti-inflammatory activity that was validated in an ulcerative colitis animal model. 

	Even with the successes being achieved with the information and approaches in use today, Deshpande believes that more data needs to be incorporated to ensure that the 

 approaches become more predictive. Policy changes are also necessary, in addition to advancements in the technology to identify new indications for existing APIs, to ensure appropriate incentives are in place to speed the use of existing drugs for new indications. “For instance,” says Deshpande, “the [Orphan Product Extensions Now] OPEN Act--a key part of the 21st Century Cures Act before it was removed from that bill just before being signed into law in December 2016--would have provided an additional six months of market exclusivity to drugs that are repurposed and approved by FDA to treat rare diseases,” she observes. 

	There remains significant pressure on the pharmaceutical industry both from payers and patients to identify new therapies for diseases. “Given the total cost and time to develop new therapies from scratch, and the high failure rates involved, drug repurposing will provide an important avenue for the discovery of new treatments, particularly for rare diseases,” asserts Deshpande. She does not expect such an approach to completely replace traditional approaches, but rather supplement them. 

	“If one looks at developing countries like China and India, where the costs for new drug development will seem prohibitive, drug repurposing, repositioning, and rescue can rapidly create new treatments in these areas. That is precisely why we at NuMedii are proud to pioneer the use of big data and AI with systems biology to disrupt the traditional drug discovery paradigm and create a faster, highly predictive drug discovery engine to help more rapidly bring life-saving medicines to the people who need them most,” she states.

Drug Repositioning: Bringing New Life to Shelved Assets and Existing Drugs

 (John Wiley & Sons, Hoboken, NJ, 2012).
	2. A Persidis, “Myths and Realities of Repositioning,” Arrowhead 4th Annual Drug Repositioning, Repurposing & Rescue Conference (Chicago, IL, May 27-28, 2015).

Compared to traditional drug discovery approaches, drug repurposing, repositioning, and rescue can be faster and cheaper.

Finding a New Purpose for an Unproven Drug

	When referring to this article, please cite it as C. Challener, “Managing Potentially Potent APIs," 

CDMOs have established strategies for handling new chemical entities with unknown biological activity.

Managing Potentially Potent APIs

Increased competition, the diversification of biologic drug substances, and globalization of manufacturing have accentuated the need for processes that are not only compatible with modular, flexible, smaller plants but also carry reduced total costs. Continuous and intensified processing is an attractive alternative that fits the needs of current and future manufacturing requirements.

	Cell-culture media directly impact the performance of upstream biomanufacturing processes; they dictate the potential productivity and reproducibility of specific processes while also influencing product quality attributes, according to Andrew Bulpin, head of process solutions for MilliporeSigma. “Cell-culture media must be optimized for each process, particularly perfusion processes and fed-batch systems that support higher profile fed-batch processes designed to minimize cost and speed development,” he observes.

	In addition to the cell line and cell-culture process parameters, cell-culture media is a key driver of bioproduction processes. Cell-culture media can have significant impacts on cell growth and viability and also drive protein production and protein quality, according to James Brooks, manager of worldwide media services and media development for BD.

	While significant time and effort are invested in the generation of an optimal cell line--from transfection to isolation and cloning, to full characterization--it is often the case that little thought is given to selection of the optimal medium for the clone, according to Brooks. He also notes that sub-optimal formulations have been shown to have deleterious effects on bioproduction processes and limit the potential of cell lines.

	Cell-culture media provide not only nutrients to the cell, but stimulatory and regulatory effects. “Media can impact cellular signaling pathways and be involved in the regulation of enzymatic activity within the cell,” Brooks explains. For example, metals contained within media formulations, such as manganese, copper, and iron, can serve as co-factors for enzyme activation and play a key role in driving protein titer and protein quality. In addition, sufficient levels of amino acids are required for normal cell growth and function, but also for the production of the correct target protein,” Brooks says. “Limitations in the amino acid compositions in some chemically defined (CD) media formulations have been shown to result in amino acid substitutions and the generation of sequence variants of the target proteins,” he says.

	The inherent diversity and complexity of animal cells limit the applicability of a universal cell culture media that can be used across all different production processes with optimal performance. Each cell line is unique, and to achieve its full potential, it is imperative to match an individual cell line with the optimal cell-culture medium that specifically meets its metabolic requirements.

	From the isolation of Chinese hamster ovary (CHO) cells into culture in the 1950s, Brooks notes, the cell line has diverged significantly, giving rise to a multitude of CHO subtypes and lines used for bioproduction today. “Each CHO subtype and each individual clone has its own specific nutritional and metabolic requirements. Through a medium optimization process, these requirements can be met, allowing the cell line to reach its full potential,” he explains.

	“Customization of a cell-culture medium requires the right tools and a specialized team of experts that can guide clients in the right direction and/or modify an existing formulation or design a new one if necessary in a minimum amount of time,” states Bulpin.

	Facing the challenges of chemically defined media

	Companies have been moving toward the use of chemically defined media to avoid the potential contamination issues associated with animal-derived ingredients. They have been challenged, however, to achieve the increasingly higher volumetric productivity levels now expected for fed-batch systems, according to Bulpin. “Chemically defined media are not necessarily the optimal process solution,” Brooks agrees.

	Nutritional limitations in chemically defined media formulations have been shown to impact protein titer and protein quality. Additionally, chemically defined does not mean chemically pure, according to Brooks. “Contaminants in CD media, such as trace elements present in vitamin stocks and some salts, have been repeatedly shown to impact cell-culture processes, particularly protein quality,” he says. Multiple companies have, in fact, published information where trace element contaminants in their CD formulations have impacted their monocolonal antibody (mAb) titers or glycosylation profiles, according to Brooks. “This issue is particularly pertinent in the development of biosimilar mAbs, where it is critical to match the quality parameters of the originator molecule,” he adds.

	Bulpin notes that nutrients that were traditionally supplied with complex solutions such as hydrolysates need to be now supplied as individual chemically defined chemicals. Some of these nutrients, such as cysteine and tyrosine, have a maximum solubility and stability level below the concentration requirements for optimal performance. “In order to overcome this issue, companies choose different strategies; most commonly, feed solutions are divided in multiple parts, often with extreme pHs, and clients need to add them separately to the bioreactor, leading to potential unwanted pH shifts,” says Bulpin.

	MilliporeSigma developed a single-feed concept in which the nutrients are concentrated to more than 130g/L, allowing a reduction of the volume of feed added to the medium and thereby increasing the volumetric productivity. The feed is added at neutral pH using a simple reconstitution protocol and high solubility, according to Bulpin. It also contains cysteine and tyrosine derivatives that have been shown to release free cysteine and free tyrosine slowly throughout the culture. “This time-dependent release avoids tyrosine depletion, which can lead to sequence variants. It also results in free cysteine release while maintaining a reduced redox environment, which has been related to higher cell growth and productivity,” Bulpin says.

	Poloxamer-188, which has traditionally been available from only a single supplier, is a specific cell-culture ingredient that has caused difficulties in biopharmaceutical manufacturing in recent years. “Significant numbers of lots have resulted in toxic or low shear stress-protectant effects for cells,” Bulpin explains. MilliporeSigma developed an analytical test that allows the identification of the desire variety of poloxamer-188 and is employed by the company to validate each lot of poloxamer-188 to be used in cell-culture media. In addition, MilliporeSigma worked intimately with an alternative manufacturer and has validated this producer as a second supplier for poloxamer-188, according to Bulpin.

	The issues with chemically defined formulations are creating a renewed interest in peptone supplementation for cell-culture media across the biopharma industry, according to Brooks. “While chemically defined remains the ultimate desire, peptones continue to be used as cell-culture media supplements to enhance production and protein quality,” he says. “Peptones are well-characterized and long-established cell-culture supplements and feeds that can enhance cell growth and/or protein titer while helping maintain or achieve desired protein quality. The rich composition of peptones enables them to serve as an optimal nutritional source for cell culture. Peptones have also been shown to inhibit apoptosis and positively affect cell cycles,” Brooks adds.

	The continued move toward perfusion-based production has highlighted the need for more optimally designed formulations that can support the perfusion process. “It is a challenge to achieve steady-state, high-density cultures while maintaining consistent product quality. Most media formulations today are not designed for this application,” states Brooks. The overall cost of the base medium for perfusion processes is also a concern. With higher perfusion rates and medium exchange rates as frequent as 1-2 times per day, significant volumes of media can be consumed during a perfusion process. “It is therefore essential to have a medium that can achieve the desired growth, titer, and quality parameters within a cost-effective and manageable process,” Brooks asserts.

	In response to this need, MilliporeSigma developed and recently introduced a medium specifically for use in perfusion processes. It is designed to reduce the medium exchanges per day in a perfusion process while maintaining or even increasing specific productivity and product quality, according to Bulpin.

	MilliporeSigma expects biopharmaceutical industry needs for intensified fed-batch and perfusion processes and for high control over protein quality attributes to continue to increase, according to Bulpin.

	Brooks agrees that protein quality continues to be a major concern and focus in the industry--particularly given the increasing numbers of biosimilars and biobetters in development--and must be considered when developing cell-culture medium formulations. “The best way to make sure the formulation in use provides acceptable protein quality, such as N-glycan or charge-variant profiles, is to optimize the formulation specifically to match the cell line metabolic requirements,” he says.

	Protein quality supplements designed to modulate quality parameters following addition to existing cell-culture base media, are also beginning to emerge in the market, according to Brooks. “The most efficient way to achieve the desired protein quality parameters, however, is through the development and customization of the cell-culture medium to the specific cell line in use,” he stresses.

Media manufacturers improve processes and technologies to reduce risks and improve quality and consistency, while managing costs.