Adeline Siew is editor for Pharmaceutical Technology Europe. She is also science editor for Pharmaceutical Technology.

Adeline Siew, PhD

	Every now and then, you will hear about the rising costs of R&D, which in turn, translate into high prices for new drugs entering the market. Of course, with the blockbuster model becoming obsolete and the pharmaceutical industry shifting focus to personalized medicines, one can only expect drug prices to hit the roof.

	Cost is always a controversial matter that sparks questions regarding affordability and sustainability. Now if you’ve ever wondered how drugmakers come up with a price tag for their product, there is a possibility that the veil may be lifted. The United States could be leading the way in a push for greater transparency in drug pricing as assembly member and San Francisco democrat David Chiu introduced a new bill (AB 463) requiring pharmaceutical companies to disclose information on how they set the price tags for new drugs (1). If this law is passed, the manufacturer of any course of treatment costing $10,000 or more per year must report the production costs for the drug, which include R&D costs paid by the manufacturer or by grants; clinical trial and other regulatory costs; financial assistance offered to patients through various programmes; manufacturing costs; acquisitions costs such as licensing fees or the purchase of patents; total spend on marketing and advertising; and profits attributed to the drug.

	A recent report by the Tufts Centre for the Study of Drug Development estimates that, on average, it costs $2.6 billion to bring a drug to market (2). Whether or not it is a myth or fact, this figure has caused disputes with critics accusing the industry of using R&D cost as an excuse to mark up drug prices. Perhaps the new transparency act on drug pricing will clear up some of the confusion and serve as an eye opener as to why our medicines cost so much. And it would be interesting to see if Europe will follow suit.

AB 463 Pharmaceutical Cost Transparency Act of 2015,

 accessed 2 Mar. 2015.
	2. Tufts CSDD, Cost to Develop and Win Marketing Approval for a New Drug is $2.6 Billion, Press Release, 18 Nov. 2015.

Articles

Every now and then, you will hear about the rising costs of R&D, which in turn, translate into high prices for new drugs entering the market.

Lifting the Veil on Drug Pricing

	While new technologies, such as continuous manufacturing, quality by design (QbD), and process analytical technology (PAT), continue to expand within the pharmaceutical industry, the supply chain will remain an ongoing challenge, commented Chris Kilbee, group director pharma at UBM, in a press release on 2015 pharma trends by CPhI Worldwide’s panel of experts. 

	The report highlighted that the pharmaceutical supply chain does not have a positive outlook. According to Hendrik Baumann, CEO of Arevipharma GmbH, marginal pressures caused by a greater profit-driven approach to sourcing and reduced stockpiling by pharmaceutical producers and wholesalers will have a major effect on Western drug markets. Baumann warned that there is a danger that this will result in drug shortages for popular treatments.

	Outsourcing is set to increase, especially with India and China emerging as manufacturing forces. Prabir Basu, consultant at Pharma Manufacturing, stated that this trend brings along its related quality issues, which are not likely to reduce in the near future.

	Hedley Rees, managing consultant at PharmaFlow, was in the opinion that Big Pharma may start to repatriate some of the drug development value chain activities to regain control over quality, lead times, and costs. “Could a Big Pharma buy a CMO?” was the question posed by Rees. Rees also projected that there will be a strain on the industry as drug developers come under pressure to meet increasing GMP/GDP demands as well as exceed expectations of potential pharma partners with respect to the robustness of their CMC packages.

The pharmaceutical supply chain continues to be a challenge for the industry despite advances in QbD and PAT.

Pharma’s Ongoing Supply-Chain Woes

	CPhI Worldwide has released its expert panel projections for the pharmaceutical industry in 2015. With quality constantly being the main emphasis in this highly regulated industry, it comes as no surprise that companies are widely adopting quality by design (QbD) and process analytical technology (PAT) in their processes. CMOs and generic drug companies, in particular, are actively implementing these approaches as a means of providing a competitive edge.

	“Smart operation of multipurpose plants, continuous improvement of the current processes, permanent investments in technology, people, and cGMP compliance without compromise will guarantee the existence of the European API producers and key for their success,” notes Hendrik Baumann, CEO of Arevipharma GmbH.

	There is a strong move towards continuous manufacturing and FDA’s role is evolving from being merely an “investigator” of products to an “enabler of better quality processes and cultures within pharma manufacturing. According Prabir Basu, consultant at Pharma Manufacturing, regulators will be expected to provide some guidance on the regulation of continuous manufacturing.  

	FDA will continue to emphasize on quality metrics. Girish Malhotra, president of EPCOT International, however, points out that the industry is still in it’s “regulation-centric” mode despite the benefits of “process-centric” methodology. API manufacturers, for example, will need to review its current model and practices and start embracing QbD and PAT. Brian Carlin, director, Open Innovation, FMC, adds that more attention should be given to the impact of raw material variability on the quality of finished pharmaceutical products.  

	Hedley Rees, managing consultant at PharmaFlow, commends FDA’s launch of the new Office of Pharmaceutical Quality, “which will provide the much needed focus and integration in the US regulatory process and therefore drive drug developers and manufacturers to design quality into their products at a much earlier stage.”

CPhI experts make predictions on pharma trends for 2015.

Quality is Key

	Oral formulations, which form the largest category of pharmaceutical dosage forms, tend to require more excipients than other preparations. As a result, trends in oral solid-dosage forms have a significant impact on excipient demand (1).


	While the high number of poorly soluble APIs continues to be a challenge in the formulation of tablets and capsules, there is now a wider range of options available for these drugs, observes Paul Titley, business development director, Aesica. “Approaches such as spray drying, hot-melt extrusion, lipid-based drug delivery, and multiparticulate systems are increasingly being used in both drug development and the manufacture of solid dosage forms,” notes Anil Kane, PhD, global head of formulations at Patheon.

	Randy Wald, senior research fellow at Bend Research, part of Capsugel Dosage Form Solutions, adds that the demand for specialized dosage forms, such as pediatric/geriatric, abuse-deterrent, and controlled-release formulations, has also grown over the past decade.

	“More than ever before, pharmaceutical companies are looking to take a holistic approach that places the patient at the center of everything they do,” says Verena Garsuch, PhD, pharmacist and senior manager of formulation development, Hermes Pharma. “A recent survey (2) highlighted that more than 50% of people find it hard to swallow traditional solid tablets. This presents exciting new opportunities to better meet consumer needs, for example, by making pharmaceuticals more user-friendly.”

	“Dosage forms such as orally disintegrating granules (ODGs), effervescent tablets, lozenges, instant drinks, and chewable tablets are specifically designed to be easier to swallow and offer a more pleasant experience,” explains Martin Köberle, PhD, senior manager of analytical development, Hermes Pharma. “By creating medicines that people ‘want to take’ rather than ‘have to take,’ we have the opportunity to improve treatment compliance while boosting product differentiation and brand recognition.”


	Wald observes that another major trend has been towards continuous processing, where multiple continuous unit operations are coupled into an integrated system. “The genre includes the primary processes in oral solid-dosage form manufacture such as direct blend, wet and dry granulation, tableting/encapsulation, and film coating. Drivers include streamlined development, lower and more flexible manufacturing, higher quality product, and lower net costs,” says Wald. “Moreover, with quality by design (QbD) and process analytical technology (PAT) becoming more mainstream, manufacturers are placing increasing emphasis on raw materials and process controls.”

	The adoption of QbD continues to improve pharmaceutical development. Garsuch notes that more companies are moving away from traditional, empirical methods towards a rational, systematic approach. “QbD enables a more robust approach and more accurate decision making, as it is based on data and facts rather than relying on trial and error or instinct,” Köberle comments. “The adoption of QbD across the industry is still in progress, but with regulatory authorities demanding that more processes meet QbD requirements, this trend is expected to continue.”

	“Excipient manufacturers are implementing the concepts of QbD in their manufacturing processes to improve the quality and consistency of the excipients. There is also an improvement in the quality from the perspective of higher purity and lower impurities in the excipients,” Kane points out.


	Excipients make the bulk of a solid-dosage form and they play a crucial role in the properties and performance of the finished formulation, such as its stability, drug release, bioavailability, taste, and texture. Garsuch and Köberle emphasize that excipient quality is not only essential for meeting the requirements set out in the pharmacopeias, but it is also necessary for creating reliable products and enabling the production process to proceed unhindered. “It is, therefore, essential to characterize and understand your excipients as comprehensively as possible, including particle size and shape,” they remark.

	“Primarily, pharmaceutical excipients need to be GRAS-listed (i.e., generally regarded as safe), have a bovine spongiform encephalopathy (BSE)/transmissible spongiform encephalopathies (TSEs) free certification, well-established specification sheets and control test procedures, and quality control,” explains Kane. “The key consideration in selecting excipients in solid oral-dosage forms is its global acceptance. Most of the products developed are intended for global markets and seldom do we see any products focused on a selected market. Hence, raw materials, excipients, packaging formats, and stability requirements are considered for global filing. Beyond its global regulatory acceptance is the functionality of the excipients. The functionality of an excipient and its lot-to-lot consistency in meeting the quality specifications are critical in vendor selection and qualification.”

	Wald adds that excipient selection needs to take into account the API chemical and physical properties (e.g., solubility, permeability, chemical stability, particle properties, and physical form); dosage form; and manufacturing process preferences or constraints. “It is also important to consider the required pharmacokinetic performance (especially the required absorption profile and plasma-time targets, as predicted from pharmacokinetics-pharmacodynamics modeling) and functional excipients used for enhanced absorption, controlled or modified release, chemical stability, taste masking, or commercial manufacturing process viability” continues Wald.

	The role of the excipient is a key consideration, according to Titley. “For example, you would ask, what do we expect this excipient to achieve? Do we understand the differences between the various grades? Are we using it in the correct manner? What weight will we need per dosage form-is it too high for the target size?” However, familiarity is also one of the factors in excipient selection, Titley points out. “The drug developer or formulation scientist will consider, have we used this excipient before; is it on our database; has the quality assurance manager approved the source?”

	“Other factors, such as batch-to-batch variation, stock limitations, and price fluctuations, can also have an impact on the supply of trustworthy, well-characterized excipients,” notes Köberle. “In these cases, it is essential to know if the excipients that you select will perform as expected, will continue to be reliably available, and whether they can be easily replaced should the need arise.”

	Garsuch explains that taking a QbD approach to selecting excipients involves a rational and systematic process that evaluates these risk factors. Often this approach includes conducting compatibility studies or applying design-of-experiment (DoE) approaches to collect the data needed to better understand how the excipients will perform during formulation development and manufacture. “If pharmaceutical companies fail to pay close attention to excipient quality, they run the risk of creating products that are outside of specifications. This can ultimately lead to manufacturing downtime, supply shortages, increased costs, and damage established business relationships,” says Garsuch.

Impact of excipients on drug product performance


	“QbD has always been used to understand the variation of the properties of an individual excipient,” observes Titley. “Excipient properties can affect critical quality attributes (CQAs) of the drug product, such as flow, compaction, and content uniformity. The most obvious one is the variation in particle size of a particular excipient, especially in solid-dosage forms. Excipient manufacturers and formulators have used QbD to show that the particle size variance is not an issue from batch to batch. If there is variation from batch to batch, then the critical process parameters (CPPs) can be investigated to make the product more robust.”

	“Although the selection of the excipients with the proper functionality and their corresponding levels in the drug product formulation are critical to drug product performance, a deeper understanding of how variability in the excipients can affect drug product performance and the proposed control strategy has also been identified as an important component of improved drug product development,” comments Kane, citing a study by Kushner et al. (3). “A number of drug product recalls identified excipient variability, and, therefore, a lack of an adequate control strategy, as a contributor to failure of the drug product, further underscoring the need for improved excipients variability understanding.” Evaluating the impact of excipient variability on drug product performance, however, has presented a greater challenge to date than evaluating API and process impacts on drug product performance, according to Kane. “This is partially because of the pharmaceutical manufacturer having more internal capability to manipulate the API and the manufacturing process for experimental study. For excipients, the observed lot-to-lot variability for an individual grade is a function of the control strategy put in place by the excipient supplier. Because of the scale of excipients manufacture and the broader industrial application of many pharmaceutical excipients, it can be difficult for pharmaceutical manufacturers to easily obtain an ideal set of samples to adequately investigate the impact of excipient material properties on drug product performance,” he explains.

	“The science- and risk-based approach to regulating pharmaceutical manufacturing, developed in 2004 by the Office of New Drug Chemistry in the United States Food and Drug Administration, focuses on the impact of chemistry, pharmaceutical formulation, and manufacturing processes on drug product CQAs and their impact on safety and efficacy. In 2012, FDA released a guidance for abbreviated new drug applications (ANDA) that further affirmed the role of excipient material property understanding (along with drug substance and manufacturing process understanding) as a significant aspect of QbD drug product development. As a direct result of these regulatory expectations, the impact of excipients on the manufacturability and performance of new drug products has recently received increased scrutiny in the pharmaceutical industry,” Kane elaborates.


	Excipient choices for a formulation are typically driven by functionality requirements and compatibility with the API, observes Ali Rajabi-Siahboomi, PhD, chief scientific officer, Colorcon. “Excipient grade choices are most important when deciding which manufacturing process will be used,” he stresses, and further explains that direct compression (DC) may not always be suitable for formulations due to several reasons.

	The primary reasons typically revolve around the API and its particular properties, according to Rajabi-Siahboomi. “Many times, APIs are very fine powders, sometimes below 10 microns in size. Small particles do not flow well and good flow is essential in the tableting process to achieve low tablet weight consistency. Both wet and dry granulation techniques can be used to agglomerate fine powders and improve flow,” says Rajabi-Siahboomi. “Another main reason DC may not be suitable is the compactibility of the API; as poor compactibility results in low tablet hardness. Wet granulation techniques are typically used to resolve this issue.”

	DC grades of excipients facilitate both direct compression and roller compaction processes, producing robust tablets that are also suitable for subsequent film coating, notes Rajabi-Siahboomi. “Some of the factors to consider when choosing grades of excipients are particle size, flow properties, density, and compactibility.”

	Methocel (The Dow Chemical Company) DC2 is a polymeric hydroxypropyl methylcellulose (HPMC) excipient that provides an alternative to wet granulation in matrix tablet production. “Methocel DC2 has been specially engineered, by The Dow Chemical Company, with particle morphology to facilitate the flow of the material, as compared to the controlled-release (CR) grade of Methocel,” explains Rajabi-Siahboomi. “Improved flow properties may allow for simpler and less costly manufacturing techniques, such as DC. These changes must be balanced with the other properties and functionalities of the polymer, compactibility of the blend, and controlled-release performance of the final tablet.”

	Rajabi-Siahboomi highlights that several case studies showing the improvement in flow properties and the impact on tablet properties have been published (4–6). “These studies have ranged in scale and looked at API concentration and solubility, and process technique. The outcome of these studies has demonstrated tighter tablet weight and hardness, as well as more consistent drug uniformity when Methocel DC2 is utilized as the controlling polymer,” adds Rajabi-Siahboomi.

	According to Rajabi-Siahboomi, Colorcon and Dow have generated a cost-comparison spreadsheet to facilitate analysis of tablet manufacturing costs. “When comparing a wet granulation process to a DC process, the model shows a potential reduction in costs of around 60%, when utilizing a direct compression process enabled by Methocel DC2,” says Rajabi-Siahboomi.


	“Affinisol (The Dow Chemical Company) HPMCAS (hydroxypropyl methylcellulose acetate succinate) has a number of properties that make it ideal for formulating poorly soluble drugs as solid dispersions,” says Robert Schmitt, PhD, fellow, R&D, Dow Pharma & Food Solutions. “The polymer is soluble in a range of organic solvents, which provides a wide latitude for developing spray-dried dispersions from solvents most compatible with a specific API. The glass transition temperature (Tg) of the polymer is high enough to produce a stable dispersion for many APIs and the water uptake of the solid dispersion is low, which leads to a solid dispersion with good shelf stability. Finally, the polymer can be tailored via control of both the succinate and acetate groups as well as the molecular weight in order to optimize the performance of the dispersion both in the solid state and upon dissolution,” explains Schmitt. “This high level of control allows for the development of a stable dispersion that has the optimum dissolution and super-saturation performance 

	According to Schmitt, there are many factors to consider when selecting the best formulation for a specific API. “The choice of polymer family is typically driven by the drug class and the desired release profile,” he asserts. “We currently combine a review of the API physical properties along with high throughput screening to define which polymer is most effective for a particular API.” Schmitt observes that HPMCAS is a suitable choice for highly crystalline compounds, “provided enteric performance is consistent with the delivery goals,” he says.

	“If HPMCAS is selected as the lead polymer, we then evaluate a series of custom produced polymer grades, which allow for the optimization of the API performance. This effort is primarily focused on optimizing the drug-release rate and super-saturation performance as the solid-state properties of the polymer do not vary much over the composition range available,” continues Schmitt. “The API to polymer ratio is driven by the dose requirements as well as the stability of the API in the polymer. We find stable formulations in the range of 10–50% API are typical. One of our goals is to provide solubility-enhancement polymers that don’t require additional excipients for optimum performance. This leads to more predictable performance while providing stable formulations.”

	Dow has also designed a novel cellulosic polymer with improved thermal properties for hot-melt extrusion (HME) applications. Affinisol HPMC HME can be readily extruded over a wide range of temperatures without the need to add plasticisers. The standard pharma grades of HPMC have high Tg’s and very low melt flow, observes Schmitt. “To process these polymers via HME, it is critical to add high levels of plasticizers. However, the plasticizers tend to increase the mobility of the API within the solid matrix leading to lower API stability.”

	According to Schmitt, the Affinisol HPMC HME grades have been tailored to reduce the Tg of the material and significantly improve the melt flow of the product. “With Affinisol HPMC HME, it is possible to extrude the neat polymer, which represents a breakthrough because it is now possible to use this excipient in the highly efficient extrusion process,” he explains. “By eliminating the need for plasticizer in the formulation, a simple binary system can be utilized, providing good shelf stability. While greatly improving the processability of HPMC within the HME processing space, the Affinisol HPMC HME maintains good sustainment properties already known for HPMC.”

Eudragit polymers for controlled-release formulations


	Evonik’s poly(meth)acrylates, better known as Eudragit, represent a versatile toolbox for controlled-release formulations, remarks Ann Gray, market segment manager oral excipients, Evonik Pharma Polymers & Services.

	“Eudragit L 30 D-55 and L 100-55 are used for enteric coatings that dissolve quickly after stomach transit. For drug release in lower sections of the small intestine, Eudragit L and S can be used in variable mixtures to target specific dissolution pH values. Eudragit S or FS grades with a dissolution pH of 7 are recommended for pharmaceutical forms that are intended for colonic drug delivery,” explains Gray.

	According to Gray, the insoluble Eudragit polymers enable a variety of formulation strategies for controlled-release dosage forms. “Eudragit RL/RS and Eudragit NE/NM 30 D are insoluble, independent of pH, and swell in physiological media, thus releasing the API in a diffusion-controlled manner,” she continues. “Eudragit polymers of different functionalities can be combined to achieve release profiles tailored to the API and therapeutic requirements, for example zero-order, circadian, and pulsatile release. They may be applied in one layer or also as multiple layer coatings.”

	“Eudragit polymers form inert matrix structures from which the active is released through (pore) diffusion. Depending on the physicochemical properties of the active and the desired release profile, both pH-independent and pH-dependent Eudragit grades can be used to develop extended-release matrix formulations,” says Gray, adding that conventional granulation technologies can be used, as well as direct compression of the polymer powders. She also highlights that the added advantage of these matrix systems is their higher resistance to the influence of alcohol.

	Gray also mentioned that the company’s Eudragit E PO and its customized ready-to-use variant, Eudragit E PO ReadyMix, form protective coatings of low water vapor permeability that dissolve quickly in acidic media leaving disintegration and release behavior in the stomach unaffected, even when used in thicker polymer layers.

	When asked about Evonik’s QbD approach, Gray said that in general, as fully-synthetic polymers, Eudragit does not have the high variability that is inherent in excipients manufactured from natural substances. She emphasizes that quality is planned into the manufacturing processes of Eudragit polymers by means of well-established chemistry, highly controlled processes, and tightly specified raw materials from backward vertical integration. “Evonik offers in-depth knowledge of polymer properties to customers so they can achieve robust formulations that meet QbD requirements,” adds Gray.

Other innovative excipients on the market


	One of the key advances in excipients is the development of coprocessed products for solid-dosage forms, observes Titley. “These products are a combination of two or more standard excipients, which speeds up the formulation development process. Typically these coprocessed excipients are already standard ingredients used in the pharmaceutical market, hence, the acceptability of the product is a key advantage for formulators,” Titley adds. “An example of a co-processed excipient is Prosolv Easytab from JRS Pharma, which combines four standard excipients into one product. All the formulator has to do is add the API.” Prosolv Easytab is an all-in-one, ready-to-use, high functionality, excipient composite that combines binder/filler, glidant, superdisintegrant, and lubricant for rapid formulation development and convenient tablet manufacture.

	GalenIQ, a multifunctional excipient from Beneo-Palatinit for oral solid dosage forms, is available in a variety of particle sizes and morphologies. This non-hygroscopic, physically and chemically stable excipient serves as an anti-caking agent, anti-humectant agent, stabilizer, and taste-masking agent among its various functions.

	Roquette has developed Pearlitol Flash, a mannitol–starch compound with unique disintegrative properties for the formulation of orodispersible tablets. The company recently introduced a new range of directly compressible polyols-maltitol, xylitol, and sorbitol-that can be used to create a variety of tastes and textures for all types of tablets. Kleptose Linecaps (maltodextrin) has been developed as a taste-masking agent, particularly for pediatric formulations. It masks the bitter taste of the API by decreasing the overall amount of drug particles exposed to the taste buds. Roquette’s Lycatab C-LM is a low-moisture partially pregelatinized maize starch, used as a hard capsule filler for moisture-sensitive APIs to add stability to the formulation.

Advances in spectroscopic and imaging technologies


	“Advances in spectroscopic and imaging technologies allow us to better analyze excipients in a way that is not possible using chemical analysis alone,” observe Garsuch and Köberle. “For example, we can now photograph 60 to 70,000 particles in a single, rapid analysis that provides deep insights into particle size distribution and shape, all key characteristics that influence excipient behavior during formulation development and manufacture,” remarks Köberle. “Modern DoE approaches also utilize powerful, multivariate data analysis methods that allow us to systematically screen and select excipients in a way that is faster and more informative than traditional, univariate methods.”

	“New PAT systems are also enabling us to improve the formulation process in real time, both during development and later during manufacture. In this case, spectroscopic methods can be used to monitor the process inline and in real-time without the need for sampling, allowing us to rapidly fine-tune and troubleshoot the process. Some of these technologies can even provide data without needing to be in contact with the product, reducing the chances of contamination,” adds Garsuch.

	1. Freedonia Group, “US Pharma Excipients Demand to Reach $2 Billion in 2018,” press release (March 28, 2014).
	2. Hermes Pharma, “Conventional Tablets May No Longer Be the Go-To-Solution,” press release (Sept. 2, 2014).
	3. J. Kushner et al., 

 103 (2) 527-538 (2014).
	4. G. LaBella, J. Theuerkauf, and T.L. Rogers, “Pilot Scale Evaluation of Methocel DC2 for Robust Matrix Tablet Manufacture by Direct Compression,” poster at AAPS (San Diego, Nov. 2014).
	5. Colorcon with Dow Pharma & Food Solutions, “Reduce Production Time and Cost Using a New Directly Compressible HPMC, Methocel DC2,” webinar (broadcasted Sept. 23, 2014).
	6. A. Faham, “A Novel Hypromellose Excipient for Direct Compression Controlled Release Applications,” oral presentation at Modified Release Forum (Barcelona, Spain, Oct. 2014).

: When referring to this article, please cite as A. Siew, “Innovative Excipients in Solid-Dosage Manufacturing,” 

Experts review considerations for selecting excipients for solid-dosage drug performance and manufacturability.

New Excipient Options for Oral Solid Dosage Drugs

Oral formulations, which form the largest category of pharmaceutical dosage forms, tend to require more excipients than other preparations. As a result, trends in oral solid-dosage forms have a significant impact on excipient demand (1).
	
	

	“More than ever before, pharmaceutical companies are looking to take a holistic approach that places the patient at the center of everything they do,” says Verena Garsuch, PhD, pharmacist and senior manager of formulation development, Hermes Pharma. “A recent survey (2) highlighted that more than 50% of people find it hard to swallow traditional solid tablets. This presents exciting new opportunities to better meet consumer needs, for example, by making pharmaceuticals more user-friendly.”  

	One of the key advances in excipients is the development of coprocessed products for solid-dosage forms, observes Titley. “These products are a combination of two or more standard excipients, which speeds up the formulation development process. Typically these coprocessed excipients are already standard ingredients used in the pharmaceutical market, hence, the acceptability of the product is a key advantage for formulators,” Titley adds. “An example of a co-processed excipient is Prosolv Easytab from JRS Pharma, which combines four standard excipients into one product. All the formulator has to do is add the API.” Prosolv Easytab is an all-in-one, ready-to-use, high functionality, excipient composite that combines binder/filler, glidant, superdisintegrant, and lubricant for rapid formulation development and convenient tablet manufacture.

	1. Freedonia Group, “US Pharma Excipients Demand to Reach $2 Billion in 2018,” press release (March 28, 2014).

	2. Hermes Pharma, “Conventional Tablets May No Longer Be the Go-To-Solution,” press release (Sept. 2, 2014).

	4. G. LaBella, J. Theuerkauf, and T.L. Rogers, “Pilot Scale Evaluation of Methocel DC2 for Robust Matrix Tablet Manufacture by Direct Compression,” poster at AAPS (San Diego, Nov. 2014). 

	5. Colorcon with Dow Pharma & Food Solutions, “Reduce Production Time and Cost Using a New Directly Compressible HPMC, Methocel DC2,” webinar (broadcasted Sept. 23, 2014).

	6. A. Faham, “A Novel Hypromellose Excipient for Direct Compression Controlled Release Applications,” oral presentation at Modified Release Forum (Barcelona, Spain, Oct. 2014).

Innovative Excipients in Solid-Dosage Manufacturing

	Biopharmaceutical manufacturers are under increasing pressure from regulators to ensure the safety and quality of their products. Protein aggregation presents a key challenge in the development of biologic formulations as it can have an impact on product quality in terms of efficacy and immunogenicity. Matthew Brown, PhD, product technical specialist, Life Sciences, Malvern Instruments spoke to 

about the causes and risks of protein aggregation and discussed the analytical capabilities available to measure and characterize protein aggregates.

What are the causes of protein aggregation in biologic formulations?

Proteins have a natural propensity to aggregate due to the dynamic nature of their structure, which is held together by a combination of Van der Waals forces, hydrogen bonds, disulfide linkages, and hydrophobic interactions. Disruption of this delicate balance can expose internal hydrophobic regions of the polypeptide chain, which may then interact with areas on other proteins to form larger complexes of misfolded proteins. This aggregation can be ‘native,’ in which the protein structure is maintained and the aggregation is largely reversible, or ‘non-native,’ where denaturation and structural changes mean this effect is largely irreversible. Aggregates may continue to grow and form over a wide size range, including up to and beyond the formation of visible particles, and ultimately this leads to precipitation.

	Protein aggregation is a common consequence of many sample treatments and is a major problem facing the biopharmaceutical industry. These treatments include the addition of chemicals, incorrect reconstitution of lyophilized materials, the effect of mechanical stresses encountered during manufacturing, freeze/thaw cycles, and prolonged storage. The presence of contaminating particles is also known to promote aggregation, with materials such as silicone oil, a commonly used lubricant in pre-filled syringes, acting as a nucleation site for aggregation. Other examples of contaminating materials include silicone rubber from container-closure systems, glass particles from vials, and oxidized metal particles from sources such as filling lines. There is the potential for aggregation to occur at almost every stage of a biopharmaceutical process, such as in development, formulation, manufacturing, storage, and at the point of use, and it may have a number of deleterious consequences.

	What is fundamentally important is to understand, at an early stage, the pathway of aggregate formation in order to put in place processes that will help to minimize it. The aggregation behavior of two proteins in a similar process may be quite different.

In terms of safety and efficacy, what are the risks of protein aggregation?

While biological molecules can degrade in a number of ways that make their development as therapeutic agents challenging, the presence of aggregates in biopharmaceutical formulations remains one of the major quality and safety concerns. Not all aggregates will lose the functionality of the original protein, but as aggregation proceeds, the activity of the constituent protein molecules may well diminish or be lost from the therapeutic, thereby reducing its efficacy. Of even greater concern is that numerous studies have shown that the presence of protein aggregates in a therapeutic formulation destined for parenteral administration may trigger an unwanted and/or potentially dangerous immune response in the recipient.

	Efficacy can be affected in a number of different ways, ranging from no impact through to rendering a drug completely ineffective. For example, large aggregates developing during the formulation process may well be filtered out at the later stages of production, which will also reduce the concentration of active molecule in the final product. While the most serious safety issue is the potential to trigger a life-threatening immune response that leads to anaphylactic shock, less devastating immune responses also have serious consequences. For example, the efficacy of a biopharmaceutical may be compromised if an immune response in the patient leads to elimination of the therapeutic protein, resulting in ineffective treatment perhaps where no other alternative treatments exist. Equally important is the administration of proteins designed to supplement levels of a naturally occurring endogenous protein. The triggering of an immune response here can lead not only to destruction of the therapeutic itself, but may also induce an immune response against the intrinsic protein, potentially leaving the patient with additional clinical complications. The case of Eprex-associated Pure Red Cell Aplasia (PRCA) is a well-documented example of such an effect.

Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products,

 issued in August 2014 (1), provides useful summary descriptions of the clinical consequences of immune responses to therapeutic proteins. Reduced efficacy and increased immunogenicity of a drug product are both highly undesirable, so understanding and monitoring protein aggregation in drug formulations is crucial.

Measuring and characterizing protein aggregates 

Can you describe the challenges in measuring and characterizing protein aggregates? What guidelines have FDA or EMA provided?

Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products

 (1) is ‘intended to assist manufacturers and clinical investigators involved in the development of therapeutic protein products for human use.’ It is wide-ranging in its scope and sets out FDA’s current thinking and recommendations, but is not a statutory requirement.

	In this guidance, aggregates are defined as any self-associated protein species, with a monomer defined as the smallest naturally occurring and/or functional subunit. The text points to the criticality of minimizing protein aggregation in therapeutic products and the need to develop minimization strategies as early as is feasible in product development. It states that, ‘methods that individually or in combination enhance detection of protein aggregates should be employed to characterize distinct species of aggregates in a product.’ Recommendation is made that the range and levels of subvisible particles (2–10 microns) present in therapeutic protein products, initially and over the course of the shelflife, should be assessed. The guidance goes on to indicate that as more analytical methods become available, there should be a move to characterize particles in smaller (0.1–2 microns) size ranges. Furthermore, there should be risk assessment of the impact of these particles on the clinical performance of the therapeutic protein product, with the development of control and mitigation strategies based on that assessment.

	Subvisible particles are usually defined as particles that are not visible to the naked eye and have a size of <100 µm. They can further be defined into micron (1–100 µm) and sub-micron (1 µm) size ranges. The 

) chapter <788> relating to particulate matter in injections requires the quantification of subvisible particles that are ≥ 10 µm and ≥ 25 µm in size, usually using light obscuration and flow imaging techniques (2). Meanwhile smaller aggregates (<0.1µm), typically caused by oligomerization, are characterized using size exclusion chromatography (SEC). However, for particles in the 0.1 to 10 µm size range cited in the FDA guidance, very few characterization techniques can provide quantitative sizing details, and there is no single instrument that can cover the full measurement range required for sub-visible particles. Consequently, different analytical approaches must be applied, each with their own distinct sizing range. This poses the challenge of relating results from quite different measurement technologies and raising the need to use truly orthogonal systems that deliver independently achieved data on the same parameter. The use of orthogonal approaches to the characterization of biotherapeutics is strongly encouraged by regulatory authorities. This is to ensure a more detailed and rounded view of the product is obtained, and to prevent reliance on single technologies. Each and every technique used to characterize the complex nature of proteins will have its own limitations; therefore, combining multiple technologies improves understanding of products.

What methods do you use to measure and characterize protein aggregates and how do they compare to each other?

A variety of techniques is available to characterize protein aggregates, which while helpful individually, collectively can offer even more valuable insight into the behavior of biotherapeutics. A number of widely used technologies are described in the following (see 

 Techniques to characterize protein aggregates. DLS is dynamic light scattering. AUC is analytical ultracentrifugation. NTA is nanoparticle tracking analysis. RMM is resonant mass measurement. SEC/GPC is size exclusion chromatography/gel permeation chromatography. LO is light obscuration.

						Figure 1 is courtesy of Malvern Instruments.

	SEC separates proteins on the basis of size as they pass through chromatography columns. Consequently, SEC allows characterization of a protein’s oligomeric state and is normally employed as a quality control (QC) release assay. The addition of multiple detectors (such as in the Viscotek TDAmax triple detection system [Malvern Instruments]) enables more extensive characterization, including the determination of molecular weight. However, large aggregates, typically above 200 nm, will block the column, and hence, will not be detected. Consequently, SEC will only provide a picture of the smallest aggregates present.

	Dynamic light scattering (DLS) systems (such as the Zetasizer Nano [Malvern Instruments]) are widely used throughout the lifecycle of biopharmaceuticals to measure the size and size distribution of proteins in solution. DLS is an easy-to-use, rapid and non-invasive method with incredibly high sensitivity to the presence of large particles, allowing the detection of aggregates at the earliest stages of onset. Consequently, DLS is often used as a screening tool and in comparability studies to monitor the oligomeric state of the protein of interest. However, DLS provides qualitative data only, and will not provide particle quantification or concentration.

	Nanoparticle tracking analysis (NTA) uses a high-resolution digital camera and specially designed software to track the movement of particles under a microscope, thereby tracking Brownian motion and determining hydrodynamic size. It generates a high-resolution particle size distribution by sizing each particle individually and also measures the concentration of particles present in the sample. Due to the low refractive index of protein, the lower limit of detection in NTA measurement is approximately 30 nm diameter. So while protein monomer units are not measurable by NTA, aggregates comprised of just a few tens of monomers through to many thousands of units can be sized and counted to determine particle concentration.

	A recent addition to the toolkit is resonant mass measurement (RMM) in the form of the Archimedes system (Malvern Instruments). It uses RMM to detect and accurately count particles in the critically important size range 50 nm–5 µm, and to reliably measure their buoyant mass, dry mass, and size. Furthermore, it can also distinguish between proteinaceous material and contaminants, such as silicone oil, by means of comparing their relative resonant frequencies and buoyant masses.

Recent advances in analytical technologies

What recent advances have you seen in analytical technologies for measuring and characterizing protein aggregates? And what area is still lacking?

There is a growing need to be able to quantify aggregates within the sub-micron size range. Some of the latest technology identified in this article now provides the industry with this capability. This ability is becoming more important as companies are required to assess immunogenic risk for parenterals. In addition to detecting and characterizing aggregates, huge importance is now being placed on the identification of particulates and contaminants. Particles detected in a product may not be protein aggregates, but rather contaminants from manufacturing processes and product contact surfaces. New technologies, such as RMM and morphologically-directed Raman spectroscopy (MDRS), are now providing a means by which particulate matter can be distinguished from protein aggregates, thereby greatly facilitating troubleshooting and deviation resolution.

	One of the newest analytical systems for protein characterization (Zetasizer Helix [Malvern Instruments]) combines industry-leading DLS technology for high sensitivity aggregate sizing with Raman spectroscopy, which allows monitoring of changes in secondary and tertiary protein structure. The combination of DLS and Raman spectroscopy enables measurement of protein size and structure from a single small-volume sample, providing unique insights into protein folding, unfolding, aggregation, agglomeration, and oligomerization. Ultimately, this can lead to identification of the degradation pathways that result in the formation of aggregates and identification of high-risk processes and parameters. Such detailed information supports both the effective application of quality by design (QbD) and the efficient development of biosimilars.

	In conclusion, protein aggregation is a consequence, rather than a cause, of degradation. As discussed at the outset, there is a pressing need to understand aggregation pathways and the risk factors that can induce aggregation right from the start of the drug development process in order to devise mitigation strategies and to align manufacturing processes with QbD principles. Given the importance of aggregates and particles to immunogenicity, it is likely that in the future particle quantification and characterization methods will need to be implemented more readily in GMP-compliant environments and in support of QC activities. Indeed, particle counting is often far more sensitive than methods that measure loss of protein monomer, as the protein mass in sub-visible particles is usually very low, relative to the total protein content. However, this understanding must also extend to the manufacturing process and beyond. Not only is it necessary to identify those processing steps most likely to introduce particles and the types of particles involved, but consideration also has to be given to product handling post release, which includes storage and handling in a clinical setting.

Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products 

General Chapter <788>, “Particulate Matter in Injections” (US Pharmacopeial Convention, Rockville, MD, 2012). 

product technical specialist, Life Sciences, Malvern Instruments, Tel: +44 (0)1684 892456, matthew.brown@malvern.com.

When referring to this article, please cite as A. Siew, “Analyzing Protein Aggregation in Biopharmaceuticals,” 

Understanding and preventing protein aggregation is crucial to ensuring product quality and patient safety.