Compared with small-molecule therapies, which have been in use for more than a century, the field of biological therapies is reasonably nascent. Since the approval of the first recombinant protein in the 1980s, numerous biological therapies have been developed for a variety of indications (1).

“The field of biological therapies has indeed experienced a significant expansion and transformation, largely driven by advancements in molecular and cellular biology,” says Anupreet Kaur, post-doctoral associate in the Brinson Research Group, Institute for Bioscience and Biotechnology Research, National Institute for Standards and Technology, University of Maryland. “One of the key breakthroughs has been the widespread adoption of recombinant DNA technology, which has revolutionized the production of protein therapeutics.”

“[Going back to] the end of the last century, when there were mainly small-molecule drugs on the market, [patients were typically] suffering from major side effects,” notes Peter Timmerman, head of Peptide Science at Biosynth. “[These unwanted effects] paved the way for a new generation of drugs that were more specific and more biologically based.”

A major benefit of biological therapies is their ability to reach targets that have previously been thought of as ‘undruggable’ with more traditional small-molecule therapies (2). Despite being much larger than small-molecule drugs, biological therapies feature a greater affinity and selectivity to a specific target, making them more effective with less off-target toxicity, Timmerman explains.

Specificity to molecular targets is a key attribute of monoclonal antibodies (mAbs), remarks Kaur. “This specificity allows [mAbs] to distinguish between subtle differences, such as single amino acid variation and conformational changes, enabling precise targeting of disease-related molecules,” she says. “Additionally, the bivalency of each antibody molecule contributes to the higher binding strength, which enables mAbs to bind with exceptional affinities, typically ranging from picomolar to nanomolar levels.”

This high binding strength is important for differentiating between the binding partners and for enhancing the therapeutic efficacy of the mAbs, continues Kaur. “Moreover, the Fc [fragment, crystallizable] domains of mAbs play a crucial role in [biological] properties,” she adds. “This region can engage with various components of the immune system leading to functions such as antibody-dependent cellular cytotoxicity, or complete complement dependent cytotoxicity. These effector function-enhanced antibodies have the ability to eliminate the target cells and modulate the immune response, further improving therapeutic potential.”

“As targets for mAb therapies are identified, fewer are agonist targets and more are antagonistic targets,” remarks Scott Rudge, founder of SynerG Biopharma Group. “When an agonist target is found, the antibody can be designed to stimulate a cell or an organ to produce a certain outcome.For example, insulin is a protein agonist, because it causes cells to take up glucose and store it as glucagon.”

However, antagonists, such as check point inhibitors, actually block something from happening, Rudge emphasizes. “If every receptor in the body needs to be inhibited or down-regulated in some way, antagonists are going to require ever increasing doses, which means higher concentration, to be administered subcutaneously or some other way less invasive than intravenous administration,” he adds.

“Subcutaneous and intramuscular delivery, in particular, require highly concentrated formulations, as the maximum volume these routes of administration can accommodate is about 1 mL,” Rudge explains. “Alternatively, other means for delivering high doses of mAbs could be found that deliver more than 1 mL. Research in these two areas is very active.”

Additionally, the development of bispecific antibodies represents a significant milestone for the biopharma industry, Kaur adds. “This platform holds promise for enhancing the efficacy, increasing the half-life, and improving the stability and specificity of biologic therapies, while minimizing the side effects,” she says.

Looking beyond antibody therapeutics, Kaur highlights RNA vaccines as a promising platform that will enable the development of novel vaccines to tackle infectious and other diseases. “Ongoing research aims to further optimise RNA vaccine platforms and expand their application beyond infectious diseases,” she says.

“Also, there are short oligonucleotide therapies, which will present a versatile and promising approach for the treatment of orphan rare diseases by targeting specific genes and other regulatory elements,” Kaur specifies. “Continued research and development efforts are focused on improving the delivery strategies, enhancing the specificity and efficacy, and expanding the clinical application of oligonucleotide-based therapeutics.”

Focusing on potential future trends for the biologics field, Hans de Backer, head of the Peptide Division, Biosynth, emphasizes that there are several key trends that will be ongoing for a long period of time. Some are more industry generalized trends, such as accelerating time-to-market and moving towards more green solutions in manufacturing; however, a more specific trend for biologics will be the drive for increasingly patient-specific solutions instead of generic ‘one-size-fits-all’ type therapies.

A further trend that is shaping the biologics sector is the development of combination therapies, whereby companies are developing these products as combination options from the outset rather than considering a potential combination later on, De Backer specifies. “The expanded range of biological and small-molecule drugs already approved can be being used during development to act on multiple pathways or the same pathways through differing mechanisms. It is therefore increasingly important for researchers to be able to access reagents (as biosimilars or generics) early in development to understand modes and mechanism of action, and routes for improving efficacy,” he says.

“All of the aforementioned trends will be mainly boosted by the introduction of artificial intelligence (AI), which is being seen in all areas playing a key role” De Backer says. In particular, he points out that AI is helping to accelerate time-to-market for drug development, all the way from discovery to production. AI is helping companies to deliver results that in turn are fitting in with funding cycles and ultimately helping them commercialize their drug products.

In terms of personalized medicine, AI will enable further personalized drug manufacture options—but also personalized prescription. AI can be used by clinicians to assess the sort of response they can expect from a therapy, which is allowing them to choose the best therapeutic option for their patients (3). “AI will definitely bring new opportunities and allow for better forecasts [of therapy outcomes], driving personalized medicine to the next level,” De Backer asserts.

Additionally, Timmerman states that he has high expectations of AI as it has the potential to provide information that hasn’t been seen before, particularly for drug discovery. Currently, when trying to look at the drug space, industry is limited to only a fraction of what is available, he emphasizes, whereas AI has the potential to be used to map out the complete drug space, enabling the identification of a greater number of novel chemical entities (4).

There are other areas where AI can provide new information, Timmerman notes. “For example, there is a new modeling program called AlphaFold that basically can predict the structure of all proteins from their primary amino acid sequences,” he says. “[So, now it is not necessary to have] any crystal structure or any other type of 3D structure to know how the protein looks and how to develop a drug against it.”

Recent research has also demonstrated the ability for AI and machine learning to speed up protein engineering (5,6). “Advancements in protein engineering have facilitated the development of novel molecular scaffolds, optimized molecular functions, and improved drug properties,” Kaur adds. “These molecule-level innovations have opened up new possibilities for creating biologics with enhanced therapeutic effects.”

“Gaining an in depth understanding of accepted and multiple antibody interactions will undoubtedly revolutionize the future of biological formulation, as this knowledge can lead to the design of excipients that have beneficial impacts on the protein structure, thereby improving quality and efficacy [of antibody therapeutics] in the future,” summarizes Kaur. “Moreover, understanding the dynamics of antibody and antigen binding properties at the molecule level will help in designing the effective next-level antibodies. Manipulating these binding properties can optimize antibody performance and specificity and affinity for specific application.”

1. Andrews, L; Ralston, S; Blomme, E.; Barnhart, K. A Snapshot of Biologic Drug Development: Challenges and Opportunities. 

, 34 (12), 1279–1285.
2. Leach, M.W.; Rottman, J.B.; Hock, B.; et al. Immunogenicity/Hypersensitivity of Biologics. 

, 42 (1), 293–300.
3. Johnson, K.B.; Wei, W.-Q.; Weeraratne, D.; et al. Precision Medicin, AI, and the Future of Personalized Health Care. 

, 14 (1), 86–93.
4. Duncan, K.K.; Rudnicki, D.D.; Austin, C.P.; Tagle, D.A. Exploring Novel Biologically-Relevant Chemical Space Through Artificial Intelligence: The NCATS ASPIRE Program. 

, 6, 1–6. DOI: 10.3389/frobt.2019.00143
5. Wait, S.J.; Expòsit, M.; Lin, S. Machine Learning-Guided Engineering of Genetically Encoded Fluorescent Calcium Indicators. 

, 4, 224–236.
6. Case, M.; Smith, M.; Vinh, J.; Thurber, G. Machine Learning to Predict Continuous Protein Properties from Binary Cell Sorting Data and Map Unseen Sequence Space. 

Felicity Thomas is the European/senior editor for Pharmaceutical Technology Group.

When referring to this article, please cite it as Thomas, F. Biologics on Target for the Future. 

Articles

Cover Story

A greater understanding of molecular and cellular biology and technological advancements are revolutionizing the field of biologics. 

Biologics on Target for the Future

Submitted: January 9, 2024.
Accepted: February 6, 2024.

This study reviews changes in in-country testing (registration testing, import testing) requirements and analyzes current trends. In the context of international harmonization of good practices and standards as well as the improved information exchange between national regulatory authorities, in-country testing is considered outdated and redundant in many cases. More and more countries acknowledge the limited value of in-country testing compared to the benefit of full or partial testing waivers. Analyzing the changing testing requirements, the authors find that a predominant trend is waiver schemes based on unilateral reliance on reference countries with a mature regulatory system.

In 2015, an overview of in-country quality control (QC) testing requirements for pharmaceutical products—the term “pharmaceutical product”, or simply “product”, will be used in this article to refer to small molecules, biologicals, and/or vaccines—and data on the efficiency of this practice were published by Garbe 

 (1). Findings from this study on import testing, based on data collected from multinational pharmaceutical companies, suggested that import testing does not add significant benefits to the quality or safety of drugs, provided that the products are uninterruptedly controlled according to the globally harmonized manufacturing and distribution standards. The authors, therefore, recommended establishing more exemptions from import testing, subject to well-defined conditions, and that waiver procedures are applied where a company has demonstrated appropriate controls of manufacturing and distribution processes.

This article reviews changes in in-country testing requirements since this key publication in 2015, summarizes the evolution in the field of in-country testing, and analyzes current trends.

Two categories of in-country testing form part of the considerations of the review:

 which is performed in conjunction with the product registration.

 which is the repeated QC testing of pharmaceutical products entering a country, including lot release testing of vaccines and blood products.

The different categories of in-country testing and their impact are further described by Garbe 

Both categories of re-testing raise comparable challenges with regards to technical/supply/environmental issues, efficiency, and costs. One of the main impacts of in-country testing is delayed supply of products to patients. Import testing can cause drug shortage situations (1–5) while registration testing (initial market application) can cause long delays in patient access to new medicines (6), which can damage public health.

Several articles on in-country testing were published since 2015 (1–6). Therein, import testing was analyzed regarding its efficiency, the associated risks, challenges, issues, and costs. Supported by real data (1), a limited value of the procedure was concluded.

, is global quality manager and policy lead, F. Hoffmann‑La Roche Ltd., Pharma Global Technical Operations, Basel, Switzerland; 

is quality unit lead and technical director, Bristol Myers Squibb, São Paulo, Brazil; 

is director CMC Technical Advocacy and Policy, Merck Sharp and Dohme, LLC, Horsham, PA, USA;

 is director, Regulatory Intelligence, Pfizer Global Supply, New York, US; 

is chief pharmaceutical officer, Sanofi Vaccines, Lyon, France; 

is quality assurance director, Merck & Co. Inc., Rahway, NJ, USA; 

is scientific director, Johnson & Johnson Innovative Medicine, Leiden, The Netherlands; 

is head of External Affairs Europe, Amgen (Europe) GmbH, Rotkreuz, Switzerland; 

is global SME Quality Advocacy, Vaccines Global Quality, GSK, Wavre, Belgium.

*To whom all correspondence should be addressed

When referring to this article, please cite it as Garbe, J.H.O.; Conti, T.; McGoldrick, M.; et al. Reliance-Based Waivers Become the Predominant Trend in In-Country Testing of Pharmaceutical Products. 

Peer-Reviewed Research

This study reviews changes in in-country testing (registration testing, import testing) requirements and analyzes current trends. In the context of international harmonization of good practices and standards as well as the improved information exchange between national regulatory authorities, in-country testing is considered outdated and redundant in many cases.

Reliance-Based Waivers Become the Predominant Trend in In-Country Testing of Pharmaceutical Products

Taking patient needs and expectations into consideration when designing and developing new drugs has become increasingly important as evidence mounts that more convenient and easier-to-take products increase medication compliance. To be effective, drug therapies require patient adherence. For oral dosage drugs, particularly liquids, dispersible powders (granules), orally disintegrating tablets or films, chewable tablets, and minitablets, taste-masking can be critical for increasing patient compliance, as these dosage forms result in exposure of taste buds to the APIs, which can be an unpleasant experience, according to Liliana Miinea, technology manager, Pharmaceutical Excipients, at Lubrizol Life Science.

Leveraging taste-masking technologies and solutions that impart an attractive appearance help overcome such issues. It is a complex task, however, that requires consideration of numerous variables including patient profiles, drug product characteristics, and regulatory rules, among others, observes Nasrin Mahmoudi, Sr., lead applications scientist at IFF Pharma Solutions. As such, expertise in these formulation technologies is essential to implementing a patient-centered approach to drug development and ultimately improved therapy outcomes.

Important factors influencing patient compliance

With respect to factors influencing patient compliance, most experts agree that taste and appearance are two of the most important. For oral administration, they may only fall below ease of swallowing and dosing frequency, says Steve Amoussou-Guenou, technical development manager Europe at Roquette.

In addition to palatability and swallowability, other attributes that help ensure patients follow medication regimens as recommended and without delays, skips, or increasing/lowering the dose include acceptable aroma/smell and product packaging, adds Mahmoudi. “In 2006, the [European Medicines Agency] (EMA) reported that taste, smell, and texture were all integral factors in determining a medication’s acceptability for patients,” she says (1).

“The importance of taste masking can be attributed to the fact that many APIs can be unpleasant or even impossible to consume without an effective taste-masking strategy—literally the spoonful of sugar that helps the medicine go down,” Amoussou-Guenou says.

Appearance, meanwhile, can be viewed by patients as an indication of quality. In certain markets such as Japan, Amoussou-Guenou notes, even a slight visual defect such as black specs or color heterogeneity will immediately cause concern. A consistent appearance is therefore vital for securing patient trust, as well as their attention. Some drug companies also use a unique visual appearance as a marketing tool and/or to help consumers identify their medications, limit cross contamination, and avoid potential toxicity.

Taking a patient-centric approach to formulation design

The most effective strategy for developing patient-centric formulations and achieving higher levels of medication adherence is to adopt a patient-centric design approach from the outset, Mahmoudi contends.

“Understanding how patient needs and preferences are influenced by variables such as age, overall health, disease states, and physical condition can help to design a patient-centric drug product suitable for a target patient population,” she explains.

For oral formulations, Mahmoudi says, tablet size, shape, smell, taste, and mouthfeel—or, for liquid dosages forms, rheology and volume as well as smell and taste—all impact a drug’s overall acceptability because they all impact swallowability.

To identify the needs of the target population, formulators rely on experience, sensory analysis, and marketing studies, according to Amoussou-Guenou. “[These] data are used alongside other factors like the dosage form type and advice of marketing teams to inform decisions around the drug product’s appearance and desired properties, as well as the most effective taste-masking technique to employ,” she comments.

The dosage form can also be an important dictator of taste-masking requirements. Orally disintegrating tablets (ODTs) and films (ODFs) are good examples, according to Amoussou-Guenou. “These dosage forms are specifically designed to dissolve in the mouth, and their buccal dissolution profile makes taste masking an especially important consideration,” he explains. In these applications, Amoussou-Guenou says producers typically opt for the functional excipient route, rather than coatings, though complexing the API is a viable secondary measure. Multipurpose, co-processed excipients that impart multiple attributes to ODT/ODF formulations are often preferred, as they can support rapid disintegration as well as a pleasant mouthfeel and flavor.

Several different approaches to taste-masking provide formulators with options depending on the various factors in play. The main determinant is the extent and type of the unpleasant taste or smell of the drug, whether it is bitter, sour, or an irritant, according to Mahmoudi. An API’s aqueous solubility also plays an important role in selecting a taste-masking technique because it can be more challenging to cover the poor taste of highly water-soluble drug substances. It is also often easier to taste-mask low dose formulations. In addition, the particle size and shape and the texture of the API, as well as the target release profile, should be considered when selecting a taste-masking method, Mahmoudi says.

The API can be chemically modified into a salt or pro-drug form to reduce its bitterness, according to Amoussou-Guenou. This technique is limited, however, to drug substances with suitable functional properties. More commonly, excipients with a sweetening effect are added to hide the bitter/unpleasant taste of the API. Molecular complexation with compounds such as cyclodextrins can also mask the taste of the API while the drug resides in the mouth. Finally, application of a barrier coating that remains intact while in the mouth but degrades once exposed to the acidic conditions in the stomach is another widely used approach.

“If an API is only bland or mildly off-putting, formulators can simply add acids, flavorants, or sweeteners to combat the bitterness. Excipients with a naturally sweet flavor, like mannitol, are common here, as they combine good tabletability with effective taste-masking characteristics,” Amoussou-Guenou notes.Coatings are preferred if the API has a strong bitter taste that cannot be sufficiently masked with the addition of excipients to the formulation.

Formulations can also combine taste-masking techniques in the case of particularly unpleasant-tasting APIs. When formulating ODTs for example, Amoussou-Guenou observes that the API can first be complexed with cyclodextrins, hiding the bitter compounds from the patient’s taste buds. Then the complexed API can be combined with a high-performance mannitol to lend reduced friability and a better overall tablet consistency, as well as a cool, almost minty taste that makes the disintegrating tablet a pleasure to take.

With respect to polymer coating technologies used for taste masking, Mahmoudi notes there are several hydrophobic or hydrophilic polymers and lipidic compounds available that can be used as coating materials. She highlights ethyl cellulose as a hydrophobic polymer with a pH-independent release. Mahmoudi also notes that the coating level will be determined by the bitterness level of the API and the dosage form properties and can be adjusted to not only provide taste masking, but also control the API release profile.

Miinea emphasizes how taste-masking technologies are continuously evolving to align with the progress in novel oral dosage forms that address the needs for patients with difficulty in swallowing. She points to polymethacrylates as being increasingly used as barrier coatings to taste-mask bitter APIs. Miinea specifically notes that crosslinked polyacrylic acid polymers (carbomers) having structures similar to weak cation exchange resins may form complexes with weakly basic drugs. “The resultant complexes are insoluble in saliva and mask the bitter taste of the API. When they reach the stomach and are exposed to its low-pH environment, they ionize and release the API without impacting its bioavailability. These polymers may also provide additional functionality, including reduced ‘throat catch,’ or the burning sensation associated with some APIs, including ibuprofen,” she remarks.

Meeting the needs of specific patient populations

Patient-centric formulation requires consideration of the close connections between user needs, dosage form design, and taste-masking technologies, because each patient population has its own discrete preferences and priorities, making some delivery methods more-or-less suitable, Amoussou-Guenou observes. In turn, he notes that just as the choice of delivery method stems from patients’ needs, taste-masking technologies are influenced by a drug’s format and target application.

In particular, Amoussou-Guenou notes that for groups with specialized needs such as pediatric, geriatric, and psychiatric patients, it may be necessary for drug developers to “think outside the pill box” to ensure taste-masking methods remain effective. For instance, some medications prescribed to children are not manufactured in pediatric dosage levels. For tablets, breakage in half or smaller units is therefore required. Barrier coatings used for taste masking will not be effective in this case due to exposure of the tablet core. Formulators therefore need to consider all potential patient populations when selecting a taste-masking technology for a given drug substance.

Taste masking is, in fact, extremely important for pediatric medications because it can significantly improve adherence in this population, according to Mahmoudi. It can also be more challenging, because the specific age, growth level and physiological development, underlying diseases, and regional/geographical preferences must all be considered during formulation development, including selection of the optimum taste-masking approach. As an example, Mahmoudi observes that market research suggests there are ‘favorite’ flavors that vary from country to country and indication to indication.

In the European Union, for instance, cherry and strawberry flavors are most preferred in pediatric medications for pain and infectious diseases, while lemon, peppermint, and orange are recommended for indigestion remedies (1). In the United States, meanwhile, children prefer grape, bubble gum, and cherry flavors (2).Maintaining a balance is essential

While developing palatable, pleasant-looking dosage forms is the key to improved patient compliance, it is necessary to not make drug products appear to be anything but medicines. “As an industry, we must make sure not to make drugs too appealing,” contends Amoussou-Guenou. “Drugs are just that in the end,” he continues, “and no matter how innocuous they look or taste, overconsumption can lead to serious complications. This consideration is especially important in dosage forms aimed at children,” states Amoussou-Guenou. “Producers must walk a formulation tightrope; not too bitter or bland that kids refuse their medicines, but not too craveable that they start to see potent drugs as candies,” he concludes.

A few opportunities for further development

There are two groups for which advances in taste-masking technologies could benefit. The first is infants and young children that require liquid medications or small particles that can be dosed with milk or soft food. Complicating their development is the need to meet different flavor preferences for different pediatric populations and different types of medications. The best approach, therefore, according to Mahmoudi, is to develop liquid, suspension, or ODT formulations with a neutral flavor or in a variety of flavors to be acceptable by children in different regions. Multiparticulate systems, including those based on pellets and mini-tablets, says Mahmoudi, are one approach that allows the flexibility needed for pediatric formulations.

The second group highlighted by Amoussou-Guenou is surprisingly patients who do not require any special considerations. “It may seem counterintuitive, but this group receives the least consideration when it comes to taste-masking. Of course, strategies for improving medicine accessibility for vulnerable populations are both important and necessary. However, as drug developers rightly become more sensitive to the needs of specific groups, they equally should not forget the fundamentals of making every day medicines more palatable, swallowable, and so on,” he states.

On a positive note, Amoussou-Guenou points to advances in the nutraceuticals segment, such as fun formats like functional gummies with attractive fruity flavors, that potentially have applicability for drug products as well. He also highlights steady improvements to the taste of established delivery methods like effervescent tablets. “These developments underscore the potential gains the pharmaceutical industry could realize by considering a more crowd-pleasing approach to taste masking,” he concludes.

EMA Committee for Medicinal Products for Human Use. Reflection Paper: Formulations of Choice for the Paediatric Population. European Medicines Agency, London, UK, 2006.

Mennella, J. A.; Roberts, K. M.; Mathew, P. S.; and Reed, D. R. Children’s Perceptions about Medicines: Individual Differences and Taste. 

Cynthia A. Challener, PhD, is a contributing editor to 

When referring to this article, please cite it as Challener, C.A. Using Taste-Masking and Appearance to Address Patient-Specific Needs.

Development

The use of appropriate taste-masking and appearance technologies can facilitate patient compliance.

Using Taste-Masking and Appearance to Address Patient-Specific Needs

"Primary engines of value creation”—this is the role that leading market commentators see biologic drugs playing in the evolving biopharmaceuticals market (1). Demand for protein-based, large-molecule therapies has been growing steadily for decades, but few could have predicted the boom which followed in the wake of COVID-19. Significantly accelerated by the pandemic, sales of biologic drugs are predicted to exceed those of traditional small-molecule medications by 2027 (1). Runaway demand, exciting scientific breakthroughs, and robust interest from governments and the public: the outlook is bright for biologics. To fulfill this growing potential, producers will need to make drug stability a priority, and this could mean reconsidering the use of a ubiquitous processing ingredient.

Polysorbates (PS 20 and PS 80) are the surfactants most used in biopharmaceutical formulations to protect proteins from denaturation, aggregation, and surface adsorption; these surfactants are present in as many as 90% of marketed therapeutic antibodies (2). Despite their popularity, polysorbates are prone to a number of issues, with serious implications for drug safety, stability, and efficacy. As pressures mount, could it be time for the industry to adopt a new favorite excipient solution for biologics? This article will explore the findings of two recent studies comparing the performance of polysorbates with hydroxypropyl -cyclodextrin (HPβCD)—an alternative biologic stabilizer.

Reliable, multifunctional, and safe, is HPβCD the answer to the question: how do you solve a problem like polysorbates?

To fully understand the “problem” with polysorbates, the industry must examine the essential role ultrafiltration/diafiltration (UF/DF) and protein stabilization during the final formulation stage play in biologic drug manufacturing, and why processing aids are crucial to their success. As 

 shows, UF/DF comes relatively late in downstream biopharma processing, helping achieve the desired concentration and formulation composition of biologics, such as monoclonal antibodies (mAbs) and recombinant proteins. Though an indispensable processing step, the shear stress and continuous adsorption/desorption interactions with the filtration membrane during UF/DF subject active ingredients to harsh conditions. This typically results in protein aggregation and the generation of visible and subvisible particles, jeopardizing the safety and efficacy of the drug. It is here that surface active molecules, such as polysorbates, enter the frame. During high-stress processes, polysorbates act as a chaperone, allowing aggregation-prone hydrophobic sites on the surface of the biologic to pass more easily through membranes to preserve their folded structure and reduce interface exposure (3). 

With decades of evidence pointing to their effectiveness in preventing protein adsorption and aggregation, as well as their demonstrated safety, polysorbate 80 (PS 80) and polysorbate 20 (PS 20) are considered the surfactants of choice in more than 90% of approved mAb products (2). Due to their amphiphilic (both hydrophilic and hydrophobic) properties, mAb proteins have a high propensity for surface-mediated unfolding and adsorbing into interfaces (3). Polysorbates compete with proteins at interface points, reducing the interfacial exposure of the proteins. Despite their widespread use, there are several serious questions surrounding the effectiveness of polysorbates in biologic processing. Polysorbate degradation via oxidation and hydrolysis can produce peroxides and fatty acids that could negatively impact the quality and stability of a medication’s therapeutic protein. Polysorbates can also be nonspecifically absorbed onto UF/DF filter membranes, leading to the formation of micelles bigger than the membrane’s molecular weight cut-off, which in turn causes inconsistencies and membrane blockage. As the biopharma industry moves to make efficiency its central priority, researchers begin to wonder, could an alternative excipient help flip the script?

UF/DF is an integral part of downstream biotherapeutic processing, though it’s not without its challenges. A typical UF/DF process involves several passes through a pump and membrane cassette, often taking several hours to complete (4). During this process, active proteins are exposed to a range of physical stresses, including interfacial interaction, which, as discussed previously, can lead to the formation of higher order aggregates and particulates (4). Therefore, to survive the UF/DF process intact, proteins need an accompanying surfactant to smooth their multiple passes through the membrane cassette and protect them from interfacial stresses (4). For the majority of biologic processing steps, polysorbates are the solution of choice; however, their usefulness runs out by the UF/DF stage. Not only do polysorbate molecules tend to bind nonspecifically with filter membranes, but their propensity to form micelles bigger than the membrane’s molecular weight cut-off can cause filters to become blocked during the UF/DF process (4). Researchers at Roquette postulated that HPβCD could offer biopharma producers an alternative excipient solution, better suited to the requirements of the UF/DF process.

The focus of this study into HPβCD’s potential as an alternative to polysorbates was two-fold: assess its permeability through commonly used filtration membrane types and capacity to reduce particle formation during the UF/DF of human plasma immunoglobulin G (IgG) and adalimumab solutions (4). In the first instance, varying concentrations of HPβCD were observed in the retentate and permeate of solutions passed through a regenerated cellulose-type membrane following centrifugation (4). This indicated that HPβCD may be unsuitable for regenerated cellulose membranes, as it tends to be retained during ultrafiltration (4). With another commonly used polyethersulfone (PES) membrane, however, the results were a complete contrast. In all four variations tested, concentrations of HPβCD remained constant, providing convincing evidence that unlike polysorbates, it is able to pass freely through PES membranes (4). What’s more, these results remained largely consistent when the excipient was tested via an alternative tangential flow filtration (TFF) setup, adding credence to HPβCD’s suitability for UF/DF processes (4).

Regarding the goal of mitigating particle formation, results were similarly promising. Using the same TFF setup, researchers conducted UF/DF experiments using the proteins human plasma IgG and adalimumab in the presence of different concentrations of HPβCD. In the diafiltration setup, both the retentate and permeate were recirculated for four hours to mimic the duration of a typical commercial drug production process. The results at the end of the recirculation process suggested that HPβCD effectively modulated the rate of turbidity (measure of clarity in liquids) increase in the human plasma IgG solution (4). In fact, the turbidity of the sample was reduced by more than two times in the presence of just 10 mM of HPβCD, while dynamic light scattering (DLS) data also showed that it had lowered the relative intensity of larger particles (> 0.1 m) compared to the control sample (4). The addition of a larger, 25 mM concentration of HPβCD reduced the rate of turbidity increase in the adalimumab solution yet further, meaning that the final turbidity was more than four times lower in the sample containing HPβCD than in the control group.

Researchers were able to observe HPβCD’s effectiveness in reducing UF/DF-induced particle formation in both the human plasma IgG and adalimumab solutions too. Looking at the human plasma IgG specifically, a 10% protein loss was observed in the control, whereas all samples containing HPβCD achieved almost complete protein recovery at the end of the four-hour recirculation (4). Not only then is HPβCD suitable for UF/DF where polysorbates are not, but its presence could actually boost efficiencies in biologic production lines—no small point in a time of rising demand for the industry.

The potential benefits HPβCD can bring to the biologic manufacturing process are not exclusive to the UF/DF process. Indeed, this multifunctional excipient exhibits continuous functionality into the final stages of formulation, particularly protein stabilization.

Polysorbates vs. HPβCD: Protein stabilization

The presence of protein aggregates and visible/sub-visible particles within a biologic formulation can compromise product quality and long-term stability, as well as lead to an unwanted immune response in the patient. As such, numerous health regulators around the world require biopharmaceutical companies to closely monitor visible and subvisible particle formation within formulations by comprehensive characterization, as outlined in 

 <789> (5). With scrutiny mounting on the issue of particle formation, recent research has sought to investigate the potential of alternative surfactants that could offer a more stable proposition than traditional polysorbates, with the prime candidate being HPβCD.

Due to its regulatory status as an approved excipient for oral and parenteral administration, HPβCD has been considered as a functional biologic surfactant for more than a decade (6). There is robust evidence to support HPβCD’s effectiveness in preventing thermal or agitation- and lyophilization-induced protein aggregation, in addition to its capacity to reduce interfacial stress-induced particle formation during UF/DF (5). This stabilizing effect has been attributed to the excipient’s ability to reduce protein–protein hydrophobic interactions and displace proteins from interfaces thanks to its weak surface activity (5). Unique properties such as those listed above formed the foundation of a recent investigation into HPβCD as a potential replacement for conventional polysorbates in biologic formulations.

The resultant study compared the chemical stability of HPβCD against that of PS 20 and PS 80 under various stress conditions. This included heat and chemical stressors, as well as its capacity to stabilize proteins within a formulation. When subjected to heat stress, HPβCD showed little change in product recovery (90.7–100.7% recovery depending on the HPβCD grade), while PS 20 and PS 80 both underwent significant degradation, with only 11.5% and 7.3% undegraded product remaining, respectively (5). Similar results were recorded when the surfactants were subject to autoclave, light, and oxidative stresses, with the HPβCDs remaining almost completely stable, while both polysorbates displayed some level of degradation (95.5%–98.8% usable product remaining for PS 20 and 85.5%–97.4% for PS 80) (5).On closer analysis, researchers found the HPβCDs’ chemical structure remained consistently stable, compared to the significant hydrolytic degradation and oxidation seen with the polysorbate molecules (5).

Finally, attention turned to HPβCD’s performance as a functional surfactant in a test formulation. Researchers evaluated the stability of various model mAbs when subjected to light stresses. Compared with the more traditional PS 80 preparation, the mAb formulated in the presence of HPβCD showed a notable decrease in protein aggregation, a superior monomer, and total protein recovery while reducing both protein aggregation and subvisible particle formation (5). Evidently, HPβCD excipients hold immense potential as biologic stabilizers.

At the end of this compare-and-contrast exercise, it is important to acknowledge the real value polysorbates bring to biologics processing. In a wide array of applications, they act as effective buffers and mediating agents, and their years of evidence as safe and nontoxic excipients cannot be discounted—but neither can the clear potential of HPβCD. In the studies discussed above, it can be seen what a significant impact stabilizing cyclodextrins could have on protein stability, mitigating particle formation, and streamlining vital processes. In some instances, such as UF/DF, substituting traditional polysorbates for HPβCD appears to be the logical choice, but this will not be so clear cut in every scenario. The future of biologics processing then could be one where these two surfactant groups are used interchangeably or even in tandem, to produce the very best outcomes for patients. That is, after all, the ultimate problem every drug producer is seeking to solve.

Biologic Sales to Pass Innovative Small Molecules in Next Five Years, Says GlobalData

, May 10, 2022.
2. Bollenbach, L.; Buske, J.; Mader, K.; Garidel, P. Poloxamer 188 as Surfactant in Biological Formulations—An Alternative for Polysorbate 20/80? 

, 121706.
3. Grabarek, A. D.; Bozic, U.; Rousel, J.; et al. What Makes Polysorbate Functional? Impact of Polysorbate 80 Grade and Quality on IgG Stability During Mechanical Stress. 

 (1), 871–880. DOI: 10.1016/j.xphs.2019.10.015
4. Hong, S.; Peng, T.; Gokhale, R. Mitigating Particle Formation During Ultrafiltration/Diafiltration of Biologics with KLEPTOSE HPβCD (Hydroxypropyl Beta-Cyclodextrin). Whitepaper, 2023.
5. Zhang, H.; Hong, S.; Tan, S. S. K.; et al. Polysorbates versus Hydroxypropyl Beta-Cyclodextrin (HPβCD): Comparative Study on Excipient Stability and Stabilization Benefits on Monoclonal Antibodies. 

, 6497. DOI: 10.3390/molecules27196497
6. Serno, T.; Carpenter, J. F.; Randolph, T. W.; Winter, G. Inhibition of Agitation-Induced Aggregation of an IgG-Antibody by Hydroxypropyl-Beta-Cyclodextrin. 

Tao Peng, PhD, is biopharma research manager at Roquette.

When referring to this article, please cite it as Peng, T. Polysorbates: Part(icle) of the Problem? 

Outsourcing

A comparison between polysorbates and HPβCD determines the better stabilizer for biologics formulation.

Polysorbates: Part(icle) of the Problem?

Research efforts are continuing to expand in the area of biological therapies. These large-molecule drugs are proving to be significant revenue drivers for bio/pharma companies and are providing therapeutic options for disease areas that have previously suffered from unmet medical needs (1).

Additionally, biologics are useful in providing more targeted disease treatment when compared with small-molecule drugs and lessening the risk of side effects for patients. The targeted nature of these large-molecule therapies is explored in this issue’s cover story, featured on pages 10–13, along with the potential that technological advances hold in pushing future boundaries and some of the challenges facing both the development and manufacture of biologic drugs (2).

Advanced therapy medicinal products (ATMPs), which are promising and often curative biologic therapies, face their own specific development and manufacturing challenges. Scalability of manufacturing for these emerging therapies is a particular bottleneck and something that San Francisco-based Cellares, a self-titled integrated development and manufacturing organization (IDMO), has sought to change. In an interview with Pharmaceutical Technology® Group, Fabian Gerlinghaus, CEO and co-founder of Cellares, detailed how the company’s Cell Shuttle platform and integrated consumable cartridge automated platform can improve scalability of ATMP manufacturing (3).

The IDMO has also garnered interest from Bristol Myers Squibb (BMS), which joined the Technology Adoption Partnership (TAP) program in August 2023 (4). Participation in the TAP program has meant that BMS can use Cellares’ automated manufacturing platform for the manufacture of its pipeline products.

Most recently, in April 2024, BMS and Cellares built upon their relationship by entering into a deal worth $380 million, where Cellares will transfer select chimeric antigen receptor T-cell therapies onto its manufacturing platform, with the aim of meeting growing demand and improving turnaround time (5).

1. Bender, M. Pharmaceutical & Life Sciences Trends 2024. 

 (accessed April 24, 2024).
2. Thomas, F. Biologics On Target for the Future. 

, 48 (5), 10–13.
3. Spivey, C. Cellares Innovates with Automated Cell and Gene Therapy Platform. 

, March 7, 2024.
4. Cellares. Cellares Announces Bristol Myers Squibb has Joined Technology Adoption Partnership Program to Evaluate Automated Manufacturing of CAR-T Cell Therapy on the Cell Shuttle Platform. Press Release, Aug. 28, 2024.
5. Cellares. Bristol Myers Squibb and Cellares Announce a $350M Worldwide Capacity Reservation and Supply Agreement for the Manufacture of CAR T Cell Therapies to Bring the Promise of Cell Therapy to More Patients, Faster. Press Release, April 22, 2024.

Mike Hennessy Jr. is the President and CEO of 

When referring to this article, please cite it as Hennessy, M. Tapping into the Potential of Biologics. 

From the CEO

Technological advances are helping advance biologics development and manufacturing and reduce bottlenecks.

Tapping into the Potential of Biologics

Q: We read in an article (1) that there are no specific good manufacturing practice (GMP) regulations for emerging therapies in the United States, yet we wish to bring our emerging therapy products to the US market. How can we make sure our products comply with current regulations?

A: In the absence of specific regulations for your products, the existing generic regulations, such as 21 Code of Federal Regulations (CFR), 600 Biological Products (2), will apply as appropriate. Also, international harmonized guidelines, such as International Council for Harmonisation (ICH) Q9(R1) Quality Risk Management and ICH Q10 Pharmaceutical Quality System (3,4) need to be followed and complied with. However, these are not the only sources of regulatory information available. FDA has published guidance on cellular and gene therapy products (5), which includes guidance and information on “Regenerative Medicine Advanced Therapy Designation” and on “Interactions with Office of Tissues and Advanced Therapies”.

Companies, especially developers of new types of drugs, are encouraged to interact with the regulatory authorities early on in the drug development cycle, and FDA is no exception. There are multiple opportunities for formal as well as informal meetings with FDA, and FDA’s website provides an excellent overview, including links to further information (6).

There is more information out there, free of charge, and most likely relevant to the products your company is currently developing. Though international regulations are only harmonized to some degree, there is generally a broad consensus on the basic principles of GMP, be it for traditional drug products or novel, emerging therapy products. Therefore, companies should look at regulations from other regions (e.g., the European Union [EU]) and guidance from key agencies, such as the European Medicines Agency (EMA). The EU have issued specific regulations for advanced therapy medicinal products (7), recognizing the need to address compliance for these products separately. The GMP requirements detailed in here will broadly be the same that FDA expects. Aspects that require further guidance and clarification are often covered in “Questions & Answers” documents published by EMA. An EMA Q&A document published in 2011 (8) discusses, for example, how to identify the minimum GMP requirements for starting materials, considering quality management, quality risk management, product development, production, and quality control.

These are just a few pieces of information in response to your question. There is much more information available online, from specialist publications, subject matter experts, and not least directly from the regulatory agencies through their consultation mechanisms.

Thomas, F. Optimizing the Manufacture of Emerging Therapies. 

FDA. Interactions with Office of Therapeutic Products. 

EC. Part IV, GMP requirements for Advanced Therapy Medicinal Products. 

Questions and Answers on the Principles of GMP for the Manufacturing of Starting Materials of Biological Origin Used to Transfer Genetic Material for the Manufacturing of ATMPs,

When referring to this article, please cite it as Schmitt, S. GMPs for Emerging Therapies.

Ask the Expert

Guidance documents and interaction with FDA can help manufacturers stay in GMP compliance, says Siegfried Schmitt, VP Technical, at Parexel.

Emerging Therapies and GMPs

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Product Spotlight

Deitz’s Pharmafill TC4 tabletop tablet counter is suitable for counting and filling capsules, gelcaps, pills, softgels, and other opaque, translucent, and transparent oral solid dosage forms.