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

Cynthia A. Challener

Editor’s Note: This article was published in the September 2019 issue of Pharmaceutical Technology Europe.

Modeling and simulation are recognized to provide assistance in predicting bioreactor performance, both for initial laboratory runs and when scaling to the pilot plant and commercial manufacturing. “Bioreactor performance has consistently improved in biomanufacturing due to a greater understanding of the engineering and biology of cellular systems. To continue to drive efficiency and reduce cost of manufacturing while maintaining or improving quality, reducing bioreactor variation will be required, and predictive models can enable these variation reductions,” asserts Tom Mistretta, director of data sciences at Amgen.

	The continual development of higher-producing cell lines, the switch to chemically defined media, and other advances in cell-culture technology are, however, challenging developers of modeling and simulation software.

	Typically, bench-top bioreactors are operated in the performance design space of larger bioreactors to model scale-up performance, according to Parrish M. Galliher, chief technical officer for upstream and founder of Xcellerex, a GE Healthcare Life Sciences business. “Successful modeling of bioreactors can reduce costs associated with engineering testing and engineering runs, accelerating pathways through preclinical and early clinical phases,” adds Mark T. Smith, staff engineer for single-use technologies in the BioProduction Division of Thermo Fisher Scientific.

	In particular, the inexpensive computing power and excellent computational talent entering the biopharma industry, combined with improvements in computational fluid dynamics (CFD) and metabolic modeling software, are promising to open new doors for more complete cell culture models, according to Smith. “I predict that as modeling becomes more robust, it may be possible to entirely eliminate traditionally performed engineering runs, which in fact is already being done to some extent because in many cases companies and engineers simply ‘get to know’ their bioreactor networks empirically and heuristically, without the need for complex physical modeling in silico,” he asserts.

	The key to successful modeling for the prediction of bioreactor performance, stresses Galliher, is to first calculate the performance design space of the bioreactor at all relevant scales. “Once these design spaces are understood, the scientist can operate any scale bioreactor within the design space of any other bioreactor scale and model any bioreactor of any scale using a bioreactor of any other scale,” Galliher says. He adds that this approach has been used effectively for decades.

	The process of modeling has, in fact, advanced due to the increased capability to aggregate various sources of data (sensor measurements, batch record entries, product quality measurements, maintenance records, etc.) needed to model bioreactor performance, according to Mistretta. “The industry has become more efficient due to the significant improvements in technology for centralized access to various data sources via data lakes,” he observes. In addition, the information systems technology used has been validated and demonstrated to be cost effective, secure, compliant, and scalable with the volume of data generated from manufacturing processes.

	In the past, continuous processing in perfusion mode was generally limited to cell-culture processes involving sensitive products that degrade under conventional batch conditions. Today, there is increasing use of perfusion cell culture to realize other cost, efficiency, and productivity advantages.

	Some of the challenges with perfusion processes include sensor instability and fouling over the course of the culture, which impacts the ability to effectively monitor and control these processes, according to Galliher. Perfusion mode can also create additional risks of contamination due to the continuous removal of depleted media and introduction of fresh media, and these processes involve a significant increase in the media volumes required, which has logistical and cost implications for a manufacturing facility, adds Mistretta.

	Overall, perfusion mode generally places higher performance demands on the reactor, according to Smith. He points to the higher cell densities in perfusion mode, which result in increased oxygen demand in a higher viscosity solution with potentially more cell-free biopolymers and lipids than a fed-batch culture. “Viscosity and solution property changes can fundamentally change mass-transfer mechanisms by impacting bubble size, bubble coalescence, mixing times, shear rates, and other parameters,” he explains.

	As a result, in-silico modeling of systems must be adjusted for such parameters in a meaningful way. There is, however, a limited understanding in how to model such high cell densities due to the relative newness of these operating regimes, according to Smith. Computational models of perfusion processes additionally require consideration of filter effects such as fouling and sieving, Mistretta notes.

	The shift to single-use technologies has also introduced a wider diversity of bioreactor designs, particularly with respect to spargers, baffles, and impeller configurations. “Whereas stainless-steel reactors could be made with relatively similar configurations within and across bioreactor networks, having multiple vendors represented in single-use bioreactor networks increases the level of data and modeling required to successfully predict behavior,” Smith explains.

	In addition, Smith notes there is a need for improved mass transfer performance with reduced shear in single-use bioreactors. “As cultures become more intensified, the need for oxygen transfer increases and is generally accommodated by increasing the oxygen gas passing through the micro-sparger. In many cases, the increased flow through the micro-sparger leads to shear-associated cell damage, likely related to increasing shear at the gas-liquid interface,” he explains.

	To address this problem, oxygen delivery must be achieved through efficient spargers with lower gas-liquid shear rates. “While some vendors already offer such sparger technologies, the industry has been slow to update to these sparger technologies, likely due to the revalidation burden of changes to established bioprocesses,” Smith comments.

	Mistretta agrees that variability of single-use systems does impact cell culture, but Amgen has been able to rapidly detect and determine the root cause for this cell culture variation through the accessibility of process data, material lot data, and process performance models. “These models also support data-driven decision making for impacted lots, and we continue to develop monitoring strategies through predictive models, near-real-time monitoring, and material-control strategies to mitigate unintended variation in performance,” he states.

	More advanced use of hybrid models such as CFD combined with first principles kinetic models are also effective to assess the impact of bioreactor design on performance, according to Mistretta. “Use of CFD models to determine kLa [mass transfer coefficients], mixing time, and shear effect have become pretty standard applications for technology transfer, scale-up, process optimization, and troubleshooting activities at Amgen,” he says.

	In addition, Amgen has demonstrated the utility of finite element models for studying operational risk factors for single-use bioreactor systems. For instance, models were developed to predict how material science aspects of the single-use bioreactors impact pressurization and other operational risks, according to Mistretta. Amgen has also observed more collaboration with single-use bioreactor suppliers with respect to exchanging data and developing models that enable more reliable operations.

	Ultimate Goal: Modeling complete bioreactor performance

	Modeling of the performance of the whole bioreactor including predicting full cell-culture performance seems to be the holy grail, but according to Smith, broad application of complete models has yet to be achieved due to to the complexities of the variables required for accuracy, including the oxygen transfer rate, oxygen uptake rate, carbon dioxide evolution, carbon dioxide stripping, metabolism, cell-line traits, etc.

	In addition, Smith notes that improvements in understanding of cell biology have led to dramatic changes in what a “typical” cell line is capable of producing, with improvements coming from cell-line traits such as gene promoter strength, gene integration site, integration stability, modified metabolic pathways, etc. Furthermore, aspects of bioprocess engineering have shifted, for example from natural to chemically defined media and from batch to fed-batch (and toward continuous) modes.

	“In short,” Smith concludes, “modeling the ‘complete’ bioreactor cell-culture performance for universal application may be not yet be in sight.” He does comment that there appears to be a push among large biopharma toward the use of complete models for the prediction of bioreactor performance in their given networks. However, in any published literature regarding these efforts, there is rarely sufficient detail provided to enable easy reproduction of the models used, and almost never is source code made public for direct application. “In this sense, trade secrecy and intellectual property concerns may be stymying the potential rapid industry-wide advances in performance prediction,” Smith observes.

	 “Even simpler aspects of bioreactor performance are generally still poorly modeled.” He points to the modeling of mass transfer coefficients, which dictate whether a reactor can provide sufficient oxygen to maintain viable cell mass, as an example. “In the past three years, there have been several hundred publications on CFD-based prediction of kLa in bioreactors. While this quantity suggests great interest in the area, they frequently describe the resulting models as ‘adequate,’ ‘sufficient,’ or similarly hesitant verbiage, suggesting in fact there is still space to improve in our performance prediction as an industry,” Smith explains.

	Potentially because of this remaining gap in the predictive power of CFD, many of the product development/pilot-scale models used today, according to Smith, are driven primarily by empirical data leveraging fairly traditional correlations. “This approach seems to be able to provide a similar predictive power to that of CFD without the additional computational effort and resources,” he comments.

	Useful models must be robustly validated with real data. The greater the amount of good data incorporated into a model, the more insight the model can provide. Traditional off-line sampling once or twice daily of key parameters (e.g., viable cell density, glucose, key amino acids, titer, etc.) will not provide the robust data set needed to robustly predict performance on a moment-by-moment basis, according to Smith.

	That is where process analytical technology (PAT) comes in. “Relatively recent application of PAT, such as Raman spectroscopy and biomass probes for inline sensing, are now providing more complete data on cell-culture performance and enabling better, more timely control of reactor automation,” he observes. Advances in sensor technologies have also contributed to enhanced real-time performance predictions of bioreactor performance and enable approaches such as model predictive control-based models, according to Mistretta. It is, in fact, improvement of automation control strategies within the existing capabilities of the bioreactor-not sampling and sensing alone-that is essential to improving bioreactor performance, stresses Smith.

	Further advances in PAT are still needed, however, to enable them to really contribute to model development, according to Galliher. As an example, he notes that while infrared sensors have been touted as being able to measure relevant bioreactor parameters and generate predictive models, they require large computing power and algorithms to cancel out interference from cells, gas bubbles, and media components, all of which change over the course of the run.

	The availability of data (particularly genomic and proteomic data on cellular systems) and the capability to efficiently capture, store, and contextualize the process data needed for modeling have been the most notable advances in predictive bioreactor performance, according to Mistretta. The decreasing cost of the infrastructure needed to run computational models has, he adds, also made it faster to prototype and deploy predictive models for bioreactors.

	One area of rapid development involves the potential application of artificial intelligence (AI) and machine learning (ML) to bioreactor performance modeling. Bioengineers are beginning to discuss how to apply machine learning and digital-twin algorithms to the modeling of bioreactor performance, coupled with CFD modeling of multiphase (liquid and gas) solutions, according to Galliher. He does note, though, that these approaches are in conceptual stage.

	In fact, the capability to design, develop, and implement machine learning algorithms more efficiently has also allowed modeling to be done in a more systematic way, according to Mistretta. “Machine learning models based on the ability to leverage larger datasets have enabled automated data-driven models of bioreactor performance and offer the potential to help determine fitted parameters in first principles models of bioreactor performance,” he remarks.

	One possible challenge to leveraging AI/ML could be the need for massive data sets in order to avoid overfitting issues. “Considering that biopharma companies are generally trying to minimize the number of culture runs for any given product and that even processes for blockbuster products may only be run 12–20 times per year, there is a relative dearth of information for most cell-line cultures with respect to typical ML data sets,” Smith explains. While each run produces thousands of data points (e.g., second-by-second pH, dissolved oxygen, viable cell density, etc.), these variables are inextricably linked to the chronology of the culture and could lead to further bias, he adds.

	Smith stresses, though, that although AI and ML pose unique challenges to bioengineers, it is possible that experts well-versed in how and when to apply AI may develop creative solutions for deconvoluting and dealing with these types of challenges in the near future, to successfully enable the utilization of these technologies within the bioprocess industry.

	Today, most of the measures of bioreactor performance are at a macro level in commercial facilities, according to Mistretta. To get to highly accurate predictions of bioreactor outputs, additional measurements and a framework for extensible modeling that describes the physiology, chemistry, and mechanics of a bioprocess must be developed, he says. “Genomics and metabolomics-level understanding of bioreactor performance supplemented by real-time advanced sensors to measure post-translational modifications are necessary, and we predict to see these types of advances in the next three to five years,” Mistretta asserts.

Vol. 31, No. 9
	September 2019
	Pages:42–44

	When referring to this article, please cite it as C. Challener, “Cell-Culture Advances Test Bioreactor Performance Models,"

Articles

The evolution of cell-culture technology is driving the need for improvements in modeling solutions.

Cell-Culture Advances Test Bioreactor Performance Models

Fixed-dose combination drugs formulated with two or more APIs in a single product to improve compliance through simplification of medication needs has been a popular and beneficial lifecycle management strategy. Controlled-release formulations are also a subject of continued interest in the pharmaceutical industry.

	“There is a particular focus on creating smarter dosage forms that primarily meet the therapeutic index by maintaining the desired plasma concentrations for extended time periods,” states Deep Patel, a senior formulation scientist with Cambrex. “Reducing the frequency of administration to prolong the duration of effective blood levels increases patient compliance, particularly when API combinations are involved,” he adds. Combining multiple APIs in a single product can create a complex situation, if the actives have varying and/or conflicting properties. Selection of the right excipients can be crucial to the successful development of controlled-release products containing multiple APIs.

	Achieving the desired drug-release profile

	There are many different types of controlled-release formulations, and one of the first steps must be to establish whether the drug should be formulated as delayed-release, sustained-release, controlled-release, etc., according to Ronak Savla, global scientific affairs manager at Catalent. The next step is to determine the optimal release kinetics (zero order, first order, or pseudo-zero order), after which point the formulator must design a formulation that meets the dissolution (release) profile. Catalent uses physiologically-based pharmacokinetic modeling (PBPK) to determine the optimal desired dissolution rate via construction of a plasma concentration profile.

o correlation is still one of the biggest challenges, according to Patel, along with risk management of dose dumping under different gastrointestinal (GI) tract conditions. Designing controlled-release delivery systems for biologic APIs that deliver abuse resistance and meet specific requirements for pediatric dosage forms are also key areas of focus.

	Each modified-release dosage form faces both common and unique challenges and requires prototyping to fine-tune the drug release rate and achieve the target pharmacokinetics. “Because modified-release dosage forms contain higher drug loads than immediate-release dosage forms, the potential for dose dumping is a greater concern and must be addressed. API compatibility and stability with excipients must also be assured,” Savla notes. Manufacturing challenges unique to each dosage form include the coating level and drying requirements for coated tablets and multi-layered drug-containing beads and ensuring a homogeneous viscosity and uniform filling of semi-sold fill material incorporated into soft capsules.

	Different target patient populations also present different challenges. “Controlled-release formulations are typically solid dosage forms that can be rather large. Patient populations prone to swallowability issues, such as geriatric and pediatric patients, may not be able to swallow the intact dosage forms,” says Savla. For these patient populations, coated beads (or ‘sprinkles’) filled in two-piece capsules are a popular option. The capsule can be opened, and the contents can be mixed with food or certain liquids. Mini-tablets are another attractive option for toddlers and older children. Another consideration for pediatric patients is whether the excipients are acceptable for use in the population.

	In addition, because controlled-release formulations typically aim to provide once-daily dosing, the drug must have some colonic absorption, which can be an issue for poorly-soluble APIs. With the increasing prevalence of poorly-soluble drug candidates, the use of solubility-enhancing technologies such as amorphous solid dispersions and lipid-based drug delivery systems are necessary. “Coupling solubility enhancement and modified-release requires the use of a significant quantity of excipients and presents great difficulty to achieve target drug load while retaining a patient-friendly dosage form size,” Savla observes.

	Added challenges with multi-API formulations

	The critical properties of controlled-release drugs containing two or more APIs include the potential dose differential and varying release profiles for the different drug substances, the stability and compatibility of the APIs with one another and other ingredients in the formulation, and content uniformity, according to Anil Kane, executive director and global head of technology and scientific affairs at Thermo Fisher Scientific. “If the APIs reside within a monolithic tablet or capsule, additional steps may be required to ensure dose uniformity and compatibility and to maintain the target release rate,” agrees Savla.

	One of the critical considerations during design of controlled-release formulations containing multiple APIs is the half-lives of each API, which may be different and generally require careful selection of excipients to control each drug release, according to Kane. The dose differential is also important. “Combining a very low dose API (e.g., 1–5 mg) with an API with a high dose (450–550 mg) could be another challenge to controlling the release of each in a specific target site in the GI tract,” he states. The site of absorption of each API may also be different, making it difficult to design a controlled-release strategy for the best clinical efficacy.

	The size of fixed-dose combinations (FDCs) formulated as controlled-release products must be considered where swallowability may be an issue, because these dosage forms cannot be split or crushed due to the modified-release functionality. It is prudent, according to Savla, to not create FDCs of two or more APIs with doses in the hundreds of milligrams.

	“A promising approach to creating modified-release FDCs in tablet form would be to borrow and learn from formulations containing both immediate-release and modified-release functionalities,” Savla says. He points to multi-layered drugs containing beads, which allow the creation of multiple-release profiles within a single dosage form.

	Designing a strategy for development of a controlled-release target product profile is important for many reasons. To achieve this goal, according Kane, a thorough understanding of the pharmacokinetic and pharmacodynamic profile of the small molecule related to its clinical efficacy is needed.

	A proper design strategy can have many benefits, including reduction of the frequency of dosing (once-a-day or twice-a-day preparation) and increased safety through avoidance of the possibility of dose dumping. The target patient population may better comply to the medication regimen when consideration is given to the size of the pill to swallow and the time of administration. Another potential benefit is the ability to formulate small-molecule APIs into controlled-release oral solid dosage formats (e.g., tablets, capsules, etc.) or as long-acting sterile injectables.

	The primary role of functional excipients in controlled-release formulations is to modify the release of the drug from the dosage form. There are numerous mechanisms by which these excipients function to release drug, according to Savla: time; pH responsiveness; erosion; osmotic concentration, etc. Controlled-release characteristics can be achieved by the use of polymeric coatings over solid dosage forms such as tablets, capsules, granules, sugar spheres, or ion exchange resins, or alternatively by incorporating polymer matrix systems within solid dosage forms or systems that respond to changes in physical conditions within the formulation, adds Patel.

	Excipients used in controlled-release formulations can, according to Patel, also help to overcome the bitter taste of active ingredients or to protect the gastric mucosa. In some cases, these excipients can be used to deter abuse or to help avoid alcohol-induced dumping. In addition, excipients can be used wisely to target the release of the active drug at a specific site of the gastrointestinal tract, behavior that is triggered by pH of the specific region of the GI tract.

	“The choice of the right excipient in terms of its properties as well as the right quantity and quality, directly impact the release of the drug in the gut for an oral solid dose controlled-release preparation or in the plasma concentration in the systemic circulation for an injectable form,” says Kane. The level of excipients is critical from a safety aspect as well. Patel adds that proper selection of excipients can help drug product manufacturing through improved flowability, enhanced compressibility, improved bioavailability, and particle size distribution specifically when using different combinations of APIs.

	The following categories of excipients and the grade selection are critical in developing an optimal controlled-release formulation, he notes:

		Solubility/permeability enhancers/modifiers

		pH modifiers (to improve stability or release at a targeted region of the gastro intestinal tract)

		Polymers that retard the release the drug based on diffusion, erosion, swelling, etc.

		Excipients/materials that can act as physical barriers.

	Savla provides two examples. pH-responsive polymers are a good choice to protect acid-labile APIs from gastric acid or for those APIs that need to reach the colon. Matrix tablets and semi-solid lipids within soft capsules work via erosion and are options for sustained-release delivery.

	Pulsatile release polymeric excipients, meanwhile, control the selective delivery (when and where) the API is desired, according to Patel. “These excipients provide a unique advantage due to the timely release that is controlled by coating the drug with selective polymers having sustained or enteric modified-release characteristics,” he observes.

	“Overall, the quality and consistency of performance of the excipients, 

, are critical to the success of controlled-release dosage forms,” Kane concludes.

	While the selection of the excipients with the proper functionality at appropriate levels in the drug product formulation are crucial to drug product performance, a deeper understanding of how variability in the excipients can affect drug product performance is also essential. “Maintaining variability of critical material attributes within an acceptable range is important for a given drug product as excipient concentration and characteristics greatly influence drug product performance with respect to stability, bioavailability, and manufacturability,” Patel notes. In addition, says Kane, development of a control strategy is an important component of improved drug product development.

	“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 excipient variability understanding,” he observes. To date, however, Kane notes that evaluating the impact of excipient variability on drug product performance has presented a greater challenge than evaluating API and process impacts.

	The overall variability in a particular critical quality attribute is a combination of the variability of the API, the excipients, the manufacturing process, and interactions of any of these individual factors, he notes.

	“At Thermo Fisher Scientific, we have found that identification of the most impactful material properties is critical to evaluating the effects of excipient variability on drug product performance. This excipient variability understanding can then be combined with the knowledge of each of the API properties and the process parameters used to manufacture the drug product to develop an appropriate control strategy that ensures consistent supply of safe and efficacious drug product,” Kane says.

	In the end, says Kane, the experience of the formulation scientists is the key to developing robust optimized controlled-release formulations that perform consistently in a predictable manner. For controlled-release formulations with multiple APIs, a systematic and well-characterized data set for each of the APIs, along with a scientifically designed strategy for the dosage form and the selection of the right quantity and quality of excipients, are key to successful formulation development. The experience and expertise of the formulator will help to avoid any ‘trial and error’ experimentation and can bring speed to these projects, he concludes.

	For Patel, new excipient development is also essential. “The development of excipients that are capable of fulfilling multifunctional roles such as enhancing drug bioavailability and drug stability, as well as controlling the release of the drug according to the therapeutic need, is one of the most important prerequisites for further progress in the design of novel drug delivery systems,” he asserts.

Vol. 43, No. 8
	August 2019
	Pages: 20–22, 45

	When referring to this article, please cite it as C. Challener, “Combo Drugs Require a Complex Design Approach," 

A design strategy can ensure conflicting properties are managed appropriately for multi-API controlled-release formulations.

Combo Drugs Require a Complex Design Approach

Selection of a sterilization strategy for a drug product that requires aseptic manufacturing is generally determined by the stability of the drug substance. For stable products, terminal sterilization, including heat sterilization or exposure to radiation or chemicals such as ethylene oxide or vaporous hydrogen peroxide, is the preferred strategy. These methods are advantageous because the processes can be monitored and validated and they tend to be less prone to error.

	Most biologic drug substances, however, are unstable when exposed to heat, radiation, or chemicals and generally require aseptic manufacturing using sterile filtration. Successful sterile filtration requires a drug product formulation with an appropriate viscosity and compatibility with the contact surfaces and shear stresses involved in pumping the fluid. Single-use technology is widely used in sterile fill/finish operations today, reducing the turnover time and cross-contamination risk.

	The main factors that must be considered from the outset when developing formulations for biologic drug substances that require sterile filtration include, according to Yunsong (Frank) Li, director of process development at Catalent Biologics, the product’s chemical and physical stability under various stress conditions (e.g., thermal, mechanical, photostability) during product manufacturing, storage, shipping, and administration; compatibility with the materials used during sterile fill/finish processing; and compatibility with the final container closure system and administration device.

	Factors related specifically to sterile filtration that can impact the final formulation include the contact surfaces, shear forces involved, and the potential to induce aggregation or particle formation, notes Margaret Faul, vice-president of drug product for Amgen. The method used to sterilize the fill/finish equipment or primary packaging must also be considered because any residual chemicals may interact with the biologic. For instance, antibodies containing methionine residues in the related binding regions of an antibody can become ineffective (lose efficacy) if (accidentally) oxidized (e.g., by vaporous hydrogen peroxide during manufacture), according to Hanns-Christian Mahler, head of drug product services at Lonza Pharma & Biotech.

	In addition, Faul observes that the concentrations of excipients or the biologic drug substance may be reduced by the filter. “The material of construction may affect material loss, while the filter system design may affect the flow properties, and the size may affect the overall process throughput,” she explains.

	To address these issues, certain excipients may be added to minimize process loss to the filter, negative interactions between the biologic and the filter, or residual sterilization chemicals that might lead to particle formation, aggregation, or decreased rates of filtration, according to Faul.

	To ensure that sterile filtration will be effective and that contaminants are excluded, it is therefore necessary to test the process for each batch, adds Andrew Bulpin, head of process solutions at MilliporeSigma. “The adsorption of components of the final formulations and the biologic drug substance to the sterile filter must be analyzed to ensure that the formulation and final concentrations remain within the desired values,” he says.

	In addition, Bulpin notes that because biologic drug substances may be sensitive to shear stress, the mixing and pumping during formulation must be gentle to prevent aggregation and/or conformational changes of the biologic. Fill/finish processes must also be performed under controlled temperature conditions to avoid thermal degradation. Many biologics are in fact lyophilized during final fill due to their thermal instability.

	“In general,” concludes Mahler, product development should carefully consider the intended method of manufacturing. Less obvious and less regulated, but equally important, is evaluation of whether each critical process unit operation (process step) of the manufacturing process within and beyond normal operating parameters would impact critical quality attributes.

	As an example, Mahler points to the type of filling pump chosen, which is not only relevant from a good manufacturing practice and fill precision perspective but can also significantly and adversely impact product quality by causing aggregation and/or precipitation when operating within or beyond its parameters. He also observes that the choice of disposable tubing, filters, and primary packaging components, as well as the capping and 100% visual inspection processes, also play a significant role in impacting product quality and yield.

	There are significant advantages with single-use technology in both drug substance and drug product manufacturing. “Its use eliminates the cleaning step, which saves time with batch turn-over, saves costs in facility construction, and improves the process flexibility,” states Li. Using sterile, disposable materials also mitigates the risk of microbial ingress during processing, according to Christy Eatmon, SME for sterile drug products at Thermo Fisher Scientific. “Using disposable technologies makes it possible to complete sterile connections in a non-sterile compounding area. The product can be almost completely processed using closed systems for product flow from a manufacturing area to the aseptic filling core,” she comments.

	Li stresses, though, that with disposable systems, it is also necessary to understand and ensure compatibility between the single-use materials employed and the final drug product as part of the formulation development efforts. “It is necessary to ensure that there are no adverse impacts, such as aggregation, denaturation, or adsorption to the product by the materials used in the process. The extractable and leachable profile also needs to be fully understood to ensure that it is compatible with the product formulation,” he explains.

	Other issues to consider with the application of single-use Â­technologies during sterile fill/finish operations include particle shedding, permeation of water or oxygen out of or into the product (possibly leading to degradation), or loss of critical excipients such as polysorbate or preservatives, according to Mahler. “The latter components have been shown to possibly be adsorbed or be able to permeate across disposables, with the potential to lead to significant quality issues in the final product,” he remarks.

	Therefore, while there are time savings during production, Faul observes that the advent of single-use technology for sterile fill/finish has impacted the formulation development process by increasing the need to assess and prevent degradations due to interactions with the contact surfaces of the disposable systems.

	Generally, many biologic drugs require high doses for injection via the subcutaneous route of administration, with the volume of injection typically less than two milliliters. “This delivery method requires the formulation to be at a very high concentration, and it becomes highly viscous, posing difficulties in both manufacturing and administration,” notes Li. Due to the need of high protein concentrations in many biologic formulations, high viscosities still pose difficulties during sterile fill/finish, adds Bulpin.

	Different approaches are under development to address this issue. Viscosity reduction in high-concentration solutions can be achieved through the modification of the formulation, Bulpin notes. One potential solution, according to Faul, is the use of excipients to reduce viscosity. The excipients can bind to certain regions of a protein to reduce viscosity and enable delivery of high-concentration formulations. “The method of interaction can be determined using various analytical methods to support understanding of the binding interactions in formulation development,” she comments. However, according to Li, currently there is no general strategy for using these excipients, and their application must be explored on a case-by-case basis.

	Conducting fill/finish processes at higher temperatures is also being investigated, according to Faul. This approach is, of course, only relevant for more thermally stable biologic drug products.

	An alternative strategy, according to Bulpin, involves enablement of lower protein concentrations using advanced biologic administration technologies that allow for higher volumes during administration.

	One of the biggest challenges faced when formulating biologic drug substances is trying to improve the stability of the molecules to achieve the target shelf-life of the product. “Formulation studies can only stabilize the target molecule to a limited extent. While there are many new excipients and approaches being attempted today, the molecule’s structure itself still plays a critical role in determining the molecular stability,” says Li.

	The best way to address this issue is to conduct developability/manufacturability studies as early as the molecular screening step in the drug discovery stage, he notes. “By taking this approach, the most stable molecule will be selected from many molecules having the same bioactivities, and this selected molecule will have the best success rate in the subsequent development and manufacturing stages,” Li adds.

	Compatibility of biologic drug substances with various surfaces is another challenge, according to Malgorzata Tracka, senior staff scientist at Thermo Fisher Scientific. “Compatibility studies should be conducted to ensure suitability of the formulation against product contact surfaces, including the primary packaging (IV bags, auto injectors, Â­pre-filled syringes, etc.) and manufacturing design (tubing, connectors, filters, etc.),” she observes.

	Formulations can be optimized with surfactants using a design-of-experiment approach, and contact surfaces can be selected carefully to minimize their negative impact. “Very often formulations advanced to the sterile fill/finish stage require optimization of surfactants to protect the biomolecules from effects that can lead to aggregation and subvisible particle or visible particle formation,” notes Tracka.

	Controlling the fluid viscosity, product sensitivity to shear stress, and thermal stability are particularly important for biologics subjected to sterile fill/finish processing, says Bulpin. “Optimal formulation might reduce product viscosity with the addition of salts and other stabilizers or through pH adjustments, while temperature control during sterile fill/finish will prevent thermal degradation and adequate filter sizing, and process design will reduce pumping requirements and, therefore, shear stress,” he comments.

	The key component of successful formulation strategy for biologics that will be subjected to sterile fill/finish processing is early identification of potential degradation factors. It is important to determine if the active will be damaged by the mechanical forces involved during processing or fill/finish activities, according to Eatmon. “The formulation development process involves identifying degradation pathways for the biologic, assessing which may be impacted by sterile fill/finish processing, identifying excipients that minimize potential degradation of the biologic and maintain potency, and empirical testing/verification of assumptions at appropriate milestones,” adds Faul. The potential for excipients to foam during processing or adhere to filter membrane during aseptic filtration must also be evaluated, Eatmon notes.

	That is where process, flush, shear-stress, and compatibility studies come in, to ensure the correct mechanical parameters, determine the quantity of material required to saturate the product contact surfaces, identify the appropriate fill speed and pressure, and assure the suitability of the materials in contact with the drug substance.

	The final filtration process is often developed on a small scale in parallel to formulation development to determine a chemically compatible filter membrane, according to Bulpin. Extractables in the final formulation, protein adsorption, and the adequate filter size are then factored in.

	Other questions must also be addressed, Bulpin adds, such as whether the bulk drug substance will be frozen before final fill and whether the drug product will be lyophilized, because these processing steps require excipients such as sugars to stabilize protein conformation. In addition, based on the hydrophobicity of the biologic, an optimal solvent must be identified (mostly aqueous), the optimal pH conditions under final concentration must be determined, and the need for the addition of antioxidants evaluated. “In essence,” Bulpin remarks, “the entire formulation needs to be tweaked to ensure maximum stability of the product.”

	Exploring new technologies in sterile fill/finish

	A new form of sterile fill/finish being explored through the development of several new technologies involves the production of sterile biologics in a powdered state, which are then used to produce sterile suspensions for injection. “All of these technologies are relatively new and in various stages of clinical studies,” Li observes.

	The sterile filling of powders is also relatively new to the industry and poses additional challenges. Li adds, however, that these technologies are attractive because they can achieve an extremely high “concentration” of biologics even beyond the protein’s solubility.

Vol. 43, No. 7
	July 2019
	Pages: 30–32

	When referring to this article, please cite it as C. Challener, “Considerations for Sterile Filtration of Biologic Drugs," 

Sterile filtration is often required for biologics but presents degradation and compatibility challenges.

Considerations for Sterile Filtration of Biologic Drugs

Many of the drug substances under development today pose formulation challenges, from poor solubility and bioavailability to limited stability under physiological conditions. The application of nanotechnology in formulation development has the potential to address many of these issues, while also enabling targeted delivery to reduce off-target effects. Nanoparticles have the advantage of allowing chemical modifications, such as addition of ligands to target cell receptors and surface coatings (e.g., PEGylation) to prevent recognition by the body’s immune system, allowing them to carry drug compounds to the site of the disease without being captured and eliminated. The result: rapid development of the emerging field of nanomedicine.

	Indeed, the value of the global market for nanopharmaceutical drugs is estimated by market research firm BIS Research to be expanding at a compound annual growth rate of 8.1% and predicted to reach $79.3 billion by 2026 (1). Polymer and liposome drug carrier systems are the two sub-segments driving this growth.

	Pharmaceutical companies are always looking for opportunities to improve medicines for patients. Applying the use of nanoformulations to existing drugs based on previous learnings is one avenue for leveraging nanotechnology, but Pfizer is focused on developing nanoformulations and nanotechnology solutions that will ultimately enable the next generation of nanotherapeutics to be brought to cancer and other patients, according to Young-Ho Song, a research fellow at Pfizer Oncology. “We complete this work as early as the discovery phase in collaboration with our medicinal chemistry colleagues,” she notes.

	Nanomedicine research is focused in two main areas, agrees Stephan T. Stern, acting deputy director of the Nanotechnology Characterization Lab, which is part of the Frederick National Laboratory for Cancer Research. One area involves reformulation of existing drugs or drug combinations to decrease toxicity and/or increase efficacy, or to develop competing formulations of existing marketed products (e.g., nanosimilars). The second research area involves the formulation of novel drugs, such as difficult-to-formulate new chemical entities, oligonucleotides, and vaccines. “Most pharmaceutical companies have nanotechnology formulation research groups that are involved in resurrecting drugs that are failing due to toxicity/efficacy/formulation issues,” he adds.

	The most common dosage forms of nanomedicines on the market are intravenous liposomes (e.g., Doxil [Janssen], Onivyde [Ipsen Biopharm], Vyxeos [Jazz Pharmaceuticals]), intravenous polymerics (e.g., Genexol [Samyang Biopharm]), intravenous nanoemulsions (e.g., Diprivan, Intralipid), and oral nanocrystals (e.g., Rapamune [Pfizer], Tricor [AbbVie]), according to Stern. Generally, he observes that the formulation type is first chosen based on API liabilities. “The issue may be solubility, toxicity to off-target tissues, rapid metabolism, or poor distribution to target tissues. Once the liability is recognized, a series of formulations that could overcome the liability are identified and the optimum formulation and preparation method are chosen based on chemical compatibility of the API,” says Stern.

	Choice of appropriate formulation type is dictated by the properties of the API. “Certain APIs are better suited to certain formulation, due to loading efficiency and stability,” Stern explains. “Amphiphilic weakly basic/acidic drugs, for instance, are suitable for active loading of nanoliposomes, while highly hydrophobic drugs are better suited to nanoemulsions, polymeric nanoparticles, and micelles,” he says. High-temperature techniques, such as microfluidizer homogenizing, are not ideal for thermally labile APIs.

	The formulation type will then determine the formulation method. Liposomes and emulsions are produced by solvent evaporation or thin-film hydration followed by extrusion/homogenization (microfluidizer), according to Stern. Polymeric nanoparticles and micelle formulations are generated by solvent evaporation, single/double emulsion, or precipitation techniques. Nanocrystals are generated by top-down techniques, such as milling and homogenization. Stern notes that bottom-up techniques (i.e., precipitation) are not currently used in the manufacture of nanocrystalline APIs.

	Pfizer has been working with biodegradable polymeric nanoparticles for drug delivery. The most widely used processes for the production of these nanoparticles are nanoemulsion, in which the organic phase consists of water-immiscible solvents such as ethyl acetate or dichloromethane, and nanoprecipitation, in which the organic polymer-drug solution contains water-miscible solvents such as dimethyl sulfoxide, tetrahydrofuran, or acetonitrile, according to Song.

	The polymeric nanoparticles developed by Pfizer are based on a biodegradable polyester polymer commonly used in dissolving stitches. Loaded nanoparticles are prepared by dissolving the polyester polymer, API, solvents, and excipients in an oil to form an emulsion. Thousands of pounds of pressure are applied to the emulsion, leading to the formation of nanosized droplets. The solvents are removed, and the droplets are hardened into polymeric nanoparticles encasing up to 10,000 API molecules. The surfaces of the nanoparticles are modified with antibodies that will bind to a specific cell type, such as a specific tumor cell. Once bound, the drug will diffuse out from the nanoparticles, slowly releasing and accumulating the API at the desirable target sites.

	To date, the Pfizer researchers have investigated the application of their nanopolymeric technology for the development of drugs to treat various cancers and cardiovascular, inflammatory, and infectious diseases. “Our state-of-the-art nanoemulsion process and formulation approach have expanded the chemical space of compounds that can be formulated into our nanoformulations,” Song asserts.

	Despite their advantages, nanomedicines do face several challenges, including the lack of predictive preclinical models for safety/efficacy, problems with scale up, and an uncertain regulatory path, according to Stern. Song agrees that there are multiple hurdles that must be overcome before nanomedicines can become commercial products. “An individual’s perspective often determines what is the biggest challenge, and each medicine can present its own set of challenges. It takes a team effort, from discovery through approval, to address each challenge as it is presented,” she observes.

	Advances in technology are helping, too. “Recent advances in nanodelivery formulation around oligonucleotides (siRNA, mRNA, and genetic materials) using lipid nanoparticle (LNP) nanosystems are breakthrough technologies,” according to Song. “These new approaches can help to convert a very difficult drug formulation challenge ultimately into a drug product,” she states.

	Stern points to a gradual refinement in established platforms regarding improved stability, safety, and delivery as important evolutionary advances in nanomedicine. One example he highlights is the approval of Onpattro, a treatment of transthyretin-mediated amyloidosis and the first gene-delivery nanoliposome, which has been decades in the making. “With market approvals like this one, more venture funding will flow into nanotech, allowing for more research and advancement in the field,” Stern asserts.

	Another way these challenges are being overcome, says Stern, is through the participation of contract research, development, and manufacturing organizations that are developing and expanding specialized capabilities at the lab to commercial scale and can assist with the scaleup of complex drug formulations. He also notes that as more nanomedicines reach the clinic and eventually the marketplace, a better understanding of which preclinical studies are useful and important is being realized. In addition, FDA and the European Medicines Agency are in the process of streamlining the regulatory approval process for complex drugs, including their generic/follow-on versions, which should expedite future development.

	Shift in focus to next-generation therapies

	The future for nanomedicines certainly appears to be exciting. “Complex drug formulation will always have a place in modern drug development for the simple reason that new drugs and drug classes are getting ever more difficult to deliver as a result of poor stability, solubility, and permeability,” Stern observes. There will still be reformulation and repurposing of existing drugs as nanoformulations, but Song agrees that research will move toward a focus on developing new drugs in nanoformulations starting from the early drug discovery phase.

	Research in nanomedicine has, according to Stern, already begun shifting toward the use of nanoformulation for immunomodulatory drugs and gene therapies. “Nanotech is well suited to overcoming the lack of selectivity and poor delivery, respectively, of immunotherapies and oligonucleotide-based drugs,” he says. One example is the use of gold nanoparticles rather than an inactivated virus to deliver clustered regularly interspaced short palindromic repeats (CRISPR) gene-editing tools to cells (2).

	1. BIS Research, “Global Nanopharmaceutical Drugs Market is Expected to Reach $79.29 Billion by 2026,” Press Release, Dec 11, 2018.
	2. Fred Hutchinson Cancer Research Center, “Could Gold Be the Key to Making Gene Therapy for HIV, Blood Disorders More Accessible?” News Release, May 27, 2019. 


	Vol. 43, No. 7
	July 2019
	Pages: 22–23

	When referring to this article, please cite it as C. Challener, “Facilitating API Delivery with Nanoscale Solutions,” 

Nanotechnology is enabling enhanced bioavailability, improved stability, and targeted delivery of challenging APIs.

Facilitating API Delivery with Nanoscale Solutions

Continuous bioprocessing provides numerous benefits, from high product quality and consistency to smaller physical footprints, increased flexibility, and potentially lower capital and operating expenses. It also provides the greatest benefits if real-time monitoring of process parameters and product quality, including detection and quantitation of residual impurities, is performed. While advances in analytical methods and automation are occurring, practical implementation of real-time monitoring of product- and process-related impurities has yet to be achieved.

	“In an ideal world,” asserts Byron Kneller, senior director for analytical and formulation development with AGC Biologics, “we would have simple, rapid, physicochemical tests for residual impurities.” 

	Unfortunately, there are different types of residual impurities. They can be classified as product- and process-related, and as chemical impurities (typically small-molecule process additives such as isopropyl β-D-1-thiogalactopyranoside, antifoams, antibiotics, etc.) or biological impurities from the host cell or processing steps (host-cell proteins [HCP], host-cell DNA, residual Protein-A, etc.).

	“Technologies that utilize mass spectrometry (MS) capabilities are, at this point, the seemingly best choice for residual impurity detection, characterization, and/or quantitation when coupled with liquid chromatography (LC) systems,” observes Amit Katiyar, director of analytical and formulation sciences for Thermo Fisher Scientific. Multi-attribute methods (MAM) capable of detecting, quantifying, and characterizing multiple product quality attributes are currently being developed. 

	A triple-quadrupole system would be best suited for quantitative analysis of small molecules/peptides, with quadrupole time-of-flight systems more appropriate for the characterization and semi-quantitative analysis of larger molecules, according to Charles Heise, senior staff scientist for bioprocess strategy and development at Fujifilm Diosynth Biotechnologies.

	An amalgamation of both technologies for a single quantitative measurement covering a wide mass range would be an ultimate solution, he adds. A series of standards could be used to generate standard curves for target impurities and the software needed for identification and quantification of peaks. Aspects of high-throughput proteomics analysis may also be applicable to the continuous manufacturing environment.

	Other analytical techniques Heise highlights include in-line spectroscopic measurements such as Raman, Fourier-transform infrared (FTIR), ultraviolet/visible (UV/Vis), and refractive index methods that can give quantitative as well as qualitative, real-time analysis. “The particular technology used will depend on the impurity, its expected concentration range, and whether monitoring is performed during steady-state conditions,” he observes. New methods or advances in current technologies may also become available that allow in-line measurements using nuclear magnetic resonance (NMR) imaging, sensor arrays, or LC systems.

	The final option, Heise says, is to do no process monitoring and rely solely on the quality-by-design data generated during process development to define the operating ranges and edges of failure.

	“In practice, the choice of method may be constrained by the availability of the instrumentation, turn-around time, analyst training, and potential interference from the complex matrices that may be involved in the downstream purification,” comments Ian Parsons, director of analytical development for biologics at Charles River Laboratories. His company has focused primarily on the use of high-performance LC (HPLC) and the enzyme-linked immunosorbent assay (ELISA), with MS and gas chromatography (GC) also used frequently. 

	“The pharmaceutical industry is still heavily utilizing traditional separation techniques such as size-exclusion, ion-exchange, and reversed-phase chromatography in addition to immunoassays such as ELISA and electrophoresis as the primary means for quantifying product- and process-related impurities,” notes Katiyar. 

	Often tests for process-related impurities require specialized reagents such as anti-HCP antibodies or natural products like limulus amebocyte lysate and can be more complex, adds Kneller. In addition, many impurities are heterogeneous (e.g., HCP) and thus not amenable to detection and quantitation using simple tests.

	In practice for continuous processes, in-line confounded (e.g., spectroscopy) or secondary parameter (e.g., off-gas, pH, basic physical property) measurements are most user friendly, but accuracy of residual impurity detection is sacrificed for real-time control, as a result of the heterogeneous nature of the signal, or to allow for detection of a secondary response, according to Heise. 

	He also notes that off-line LC–MS, where mass windows can be limited by buffer solution and scanning rates, or LC–evaporative light scattering detection (ELSD), is typically used for small molecules, but these are not sensitive enough and can take too long to run for large-molecule manufacture.

	These approaches could potentially be substituted for sensor technologies with equivalent modalities, according to Heise, such as the Octet system from Fortebio, which relies on biolayer interferometry. Similarly, he says that ELISAs for sensitive quantitation of biological impurities that have slow response times (material quarantining needed) could also be replaced with equivalent ‘dip-and-read’ methods.

	The new technologies (e.g., MAM, MS for HCP analysis, automation for DNA, HCP, and Protein A residuals) are just starting to be introduced in the process development space, according to Katiyar. “The transition from exploratory to fully validated methods will be slower for such methods in a cGMP environment where method performance, equipment validation, and data-integrity measures need to be at the highest level. There will also be a significant cost aspect of not only reconfiguring current laboratories to accommodate the equipment, but also for training and/or hiring new personnel with expertise in these new technologies,” he says. 

	At the process level, identifying the residual impurities present and the appropriate analytical assays, with the required detection limits and sensitivity ranges, to monitor them across the process is critical, stresses Dan Pettit, senior staff scientist for analytical development at Fujifilm Diosynth Biotechnologies. At the operations level, he notes that identifying suitably equipped labs to develop and validate methods for GMP analysis is vital because of the heterogeneity of process impurities and the consequent analytical issues they pose. 

	Heterogeneous process-related impurities, specifically HCPs, are often quantified in early-phase projects using generic kits that may result in key impurities going undetected during purification process development. “These impurities have the potential to cause problems at production scale,” Kneller says. Current ELISA techniques lack the ability to identify specific immunogenic proteins, thereby making the process development aspect less strategic in design, agrees Michael Farris, scientific manager of analytical and formulation sciences with Thermo Fisher Scientific. “Building toxicology and immunogenicity databases for various HCPs and other process impurities would help fine-tune process development and, in itself, provide a deeper insight into process performance and robustness,” he asserts.

	Identifying/developing suitably sensitive methods is also challenging, according to Pettit. “Typically, ppm or ppb levels of analyte in a complex mixture must be detected, and therefore, isolation/derivatization of the analyte may be required. These activities tend to be prohibitively expensive to outsource,” he observes. 

	In addition, isolation of impurities prior to quantitation may introduce artifacts or cause the generation of various altered states during the isolation process. For example, physical manipulation of samples prior to the detection of multiple analytes may alter the HCP antigen profile and relative abundance of the analytes themselves, according to Farris. Additional studies to understand the stability of isolated product(s) may therefore also be needed. 

	Other process impurities such as Long R3 IGF-1, which is a component of some cell-culture processes, have a tendency to adhere to certain plastics, thereby making accurate quantitation more difficult, Farris adds. Sampling instructions must be clearly defined so as to not artificially deplete the analyte during sampling and/or storage prior to analysis. 

	Parsons agrees that the main challenges are focused around developing methods of sufficient sensitivity that also minimize product and matrix-related interference, and thereby accomplish sufficient recovery of the analytes. In numerous instances, Farris points out that the quantitation and/or characterization of impurities is hindered by the API and/or the matrix composition. 

	“Immunoassays, such as ELISA, used for quantitation of host-cell proteins and leachables like Protein A are sensitive to extreme pH, high salt concentrations, and certain detergents. The typical means for overcoming the signal suppression or interference associated with their presence is to dilute the sample, which acts to decrease the sensitivity of the method to maintain acceptable precision and accuracy,” Farris explains. As a consequence, sensitivity constraints imposed by matrix/API interference must be a point of consideration when demonstrating that a method is fit for use for process characterization and/or process validation activities.

	For products with small total batch volumes, the quantities of material required for analytical method development and sample analysis can also present a challenge, Parsons notes. “If method sensitivity is also an issue, an approach can be taken to spike an aliquot of upstream material with a higher known concentration of analyte and then demonstrate fold-removal of the analyte across the downstream purification step(s),” he comments.

	For continuous processes, the turnaround time of typical assays is a key issue, according to Kneller. “The challenge is to increase our testing throughput, decrease our reliance on heterogeneous reagents, and to find ways to either simplify the method types used or engineer robust, easy-to-use systems for more complicated analyses (e.g., mass spectrometry),” he says. 

	Real-time monitoring of representative material for modeling the residence time distribution in continuous processes is also required for process control, according to Pettit. “The range of appropriate analytical sensors for continuous monitoring of product specific critical quality attributes and impurities is limited. Additionally, sensor drift over the operating time, any on-going sensor calibration needs, and system suitability testing for quality critical attribute monitoring during continuous operation must be resolved,” he adds. “Here again, sensor technology could be the way to go (e.g., online Octet dip-and-read-type systems); however, the technology is not sufficiently developed yet. The final technology offerings need to be diverse, robust, accurate, reliable, and rapid,” he concludes.

	For off-line analysis of residuals from a continuous process, Parsons notes that methods for aliquot/sample withdrawal are needed. In addition, the sampling frequency should be sufficient to provide a statistical sampling of the material flowing through the process in order to account for any transient variation that may occur while avoiding significant depletion of the process flow.

	The analytical testing strategy associated with continuous processes can, however, be more demanding in terms of the number of sampling time points if in-line or on-line monitoring is not possible, according to Farris. “Process analytical technologies have generally been geared more toward product quality, cell-culture viability, and growth than active monitoring of process impurities. If a deeper understanding of any particular impurity is required, more complicated assays will likely be required,” he observes.

	Residuals analysis when moving from batch to continuous

	When moving from batch to continuous processing, it is likely that the same analytical methods for residual impurities can be used, but potentially in higher throughput versions. Offline analysis using LC, GC, ELISA, and MS should be possible as long as an appropriate sampling approach can be implemented, Parsons observes. 

	The throughput capability and time-to-result for the analytical method employed are also important aspects if at some point the impurity is deemed process critical, according to Darshini Shah, senior scientist and group lead for downstream process development at Thermo Fisher Scientific. Automation of methods (e.g., liquid-handling or use of systems such as the Octet for HCP analysis) can provide higher throughput both in terms of overall speed and analyst effort, according to Kneller.

	The analytical target profile must also be evaluated to ensure methods are capable of monitoring fluctuating analyte levels throughout continuous processing. “Retention rates or biomass removal may result in periods of correspondingly lower impurity levels, and the analytical method must be sensitive enough and support a large enough dynamic range to be able to actively monitor the process range without the need to constantly repeat analyses to better target the operating range of the assay,” Shah explains. 

	In addition, the typical approach in batch processing involves analysis of a small discrete sample number, with reference standards bracketing the samples and system suitability tests performed prior to analysis, according to Heise. Rotating through multiple identical analyzers would allow continuous monitoring, but the solution would be costly. “Validation of both system suitability tests and the analytical methods for continuous control/monitoring will need additional work to demonstrate that results do not require bracketing with reference standards and that the detection methods do not drift or get poisoned over time when operating for 90+ days,” he says. Failure mode mitigation strategies will also need to be developed, for example with respect to system suitability failure.

	In addition, Parsons points out that for in-line and on-line analysis, the inherent requirements of sensitivity and specificity for analysis of process residuals suggests that many of the current and potential online methods would have limitations and may perhaps be able to best provide supportive general information, rather than specific quantitation of residuals. “Offline analysis using traditional validateable techniques might still be required for specific and sensitive quantitation of the process residuals. Nevertheless, if a continuous process can be shown (perhaps by an online monitoring method) to be robust, then reduced sampling of the continuous process for quantitation of residuals may be justifiable,” he notes.

	In fact, because continuous processes are expected to operate under steady-state conditions, control through predictive modeling or by exception should be possible, Heise adds. “Multi-variant analysis during development may identify simple in-line monitoring techniques that can control the process to ensure residual impurities do not pass through the whole process. However, the dynamics of the process will be different at the outset until the steady state is reached. A testing strategy defining the frequency of measurement pre- and post-steady state will be required to identify when process equilibrium has been reached and when it begins to fail,” he observes.

	“Ideally,” Heise continues, “we don’t really want to be testing anything real-time and on-line. Instead, we want to have confidence that the process will deliver consistently over a defined time scale.” 

	Potential for on-line/in-line analysis of residual impurities

	Significant development is still needed before on-line/in-line analysis of residual impurities will be widely implemented. Shah is not aware of any on-line/in-line tools capable of monitoring residual impurities for either quantitation or characterization. “Offline analysis is the predominant means of analysis and the only on-line/in-line tools are related to product quality aspects,” he says. 

	Current solutions exist around automated sampling for at-line analytics, where process changes occur over a longer time span than the analysis time, such as in the case of mammalian bioreactor metabolite analysis, according to Pettit. 

	On-line process analytical techniques such as near-infrared spectroscopy (NIR) or mid-infrared spectroscopy (mid-IR) and UV may provide some general information on clearance of residuals but are unlikely to be specific or sensitive enough to enable detailed quantitation of analytes, according to Parsons. 

	Parsons does note that low-field NMR is a potentially promising technique for on-line analysis of process residuals. “The system is simple to operate, potentially applicable to both batch and continuous processes, and could be performed by stop-flow analysis with direct connection of the flow path to the downstream purification process,” he says. Attractive features of NMR in this regard are its inherently quantitative response factor and its relatively high resolving power, and hence higher specificity than a technique such as UV, while drawbacks include barriers to implementation of a conceptually complex analytical technique and limitations on sensitivity. 


	Vol. 32, No. 7
	July 2019
	Pages: 28–31

	When referring to this article, please cite it as C. Challener, “Analysis of Residual Impurities in Continuous Manufacturing," 

Continuous manufacturing poses its own unique challenges to real-time monitoring of product- and process-related impurities.

Continuous Manufacturing Poses Challenge to Analyzing Residual Impurities

New molecular entities have continued to increase in potency over the past decade as pharmaceutical and biotechnology companies seek to improve treatment options for oncology and other chronic and rare disease areas. Approximately 70–80% of drugs in the pharmaceutical pipeline exhibit low solubility and fall into the Biopharmaceutics Classification System (BCS) Class II or IV, according to a 2015 Market Study by Kline & Co., with the majority of these compounds being Class II (poor solubility, high permeability) (1).
	Lonza Pharma Biotech & Nutrition reported to 

 that 20% of the current drug development pipeline is highly potent and/or requires special handling and also has solubility or permeability challenges.

	While solubility and bioavailability challenges are not unique to highly potent APIs (HPAPIs) and in the small-molecule world are highly dependent upon their particular moieties and morphologies, poorly soluble HPAPIs can pose additional challenges with respect to formulation development and manufacturing. Several enabling technologies are helping drug makers and their outsourcing partners overcome these hurdles.

	Any handling of HPAPIs requires care, appropriate equipment, and good practice to ensure the operator is kept safe at all times, according to Alyn McNaughton, technical director with Lonza Pharma Biotech & Nutrition. “Biopharma companies and manufacturers must be dedicated to ensuring that operators are able to do their work safely and without concern for contamination. With the technical challenges of bioavailability enhancement, additional time and dedicated experimental areas can be needed, which can make it challenging to meet development timelines,” he observes.

	As one example, complex molecules designed to provide targeted activity, while minimizing side effects, also often have permeability that is limited due their larger sizes, according to McNaughton. As a consequence, the variability of absorbed dosage can be large if the product is not robustly formulated.

	Even for materials where bioavailability can be readily enabled, the dosage required in the product can be incredibly low, often only a few micrograms or less, McNaughton adds. “This situation presents the secondary challenge of achieving homogeneity within the product, where an individual particle of the HPAPI may be a large portion of, or sometimes even bigger than, the specified dosage,” he says.

	The particle size of APIs can strongly influence the rate of dissolution. With this approach, according to Jessica Mueller-Albers, strategic marketing director for oral drug delivery solutions at Evonik, the concentration gradient between the gut and blood vessels will be increased to facilitate drug transport and consequently absorption. However, micronized particles can be associated with increased health risks.

	“The high potency of these molecules can create potential exposure concerns for workers, even at extremely small amounts. There is a need for specialized processes and expertise in the handling and containment of the drug substance, as well as intermediates resulting from particle engineering and the manufacturing of the finished drug product,” she notes.

	In fact, challenges in containment can limit the technology that can be deployed for solubility enhancement, according to Adam Kujath, global senior director of manufacturing science and technology at Alcami. “Typical API approaches for solid dosage forms to improve solubility like micronization and spray drying are more difficult to outfit with appropriate containment systems. For instance, milling of solids, while not impossible to do in appropriate containment, poses a challenge since it tends to create dust in the breathing airspace for any worker. For parenteral formulations, lyophilization presents a similar challenge,” he says.

	The toxicity of highly potent compounds also drives low allowable carryovers in the manufacturing equipment at change over. “Low solubility means cleaning reactors is inherently more challenging, and analytically may provide poor surface recoveries or impact the achievable cleaning method sensitivity. Furthermore, if they happen to be organometallic compounds, you have to worry about not only the active compound, but related organometallic and inorganic metallic species (such as mercury, arsenic, and platinum) as typical byproducts,” Kujath explains.

	For contract drug-product manufacturers, the first hurdles can occur upon receipt of the HPAPI. Packaging systems can vary widely, and it is rare for the manufacturer of an HPAPI and the product manufacturer to use a common handling system, according to McNaughton. A suitable mechanism needs to be available for accessing the HPAPI in a safe and hygienic fashion.

	An effective containment strategy should include clear, standardized processes for equipment startup, as well as defined cleaning procedures and robust decontamination procedures, according to Mueller-Albers. “It is integral to have operators who are well-trained in the operation of relevant equipment and procedures for the types of substances being handled,” she adds.

	Any handling, introduction, and transfer of powder between processing steps must be conducted in a closed or isolated system, which requires more complex and specialized equipment, according to McNaughton. “Highly diligent processes are required to prevent operators and the surrounding environment from being exposed to dangerous airborne powders,” Mueller-Albers adds. She notes that in addition to closed systems for milling, containment systems for spray drying have also improved considerably over the past decade, which has created opportunities for the spraying of HPAPIs to form amorphous solid dispersions.

	For some technologies, equipment may be specific to the individual process and require specialized handling protocols. “HPAPI product manufacturing is most straightforward for processes that have minimal steps, can be serially conducted, and do not require the material to be transferred to a completely different area,” adds McNaughton. “The resulting process commonly needs to be qualified to ensure the containment is sufficient for the product being manufactured so that any resulting operator contamination is significantly below the level at which the HPAPI could have a therapeutic effect,” he continues.

	Cleaning of processes involving HPAPIs must also be conducted in a closed system and is often the time where there is the greatest risk of exposure for the operator, according to McNaughton. A key challenge for HPAPIs is the fact that cleaning must be conducted to a level at which there is no risk of contamination of subsequent products-a level at which residual HPAPIs are often difficult to detect. When detection cannot be achieved, dedicated equipment or even facilities are often required for a single product to ensure no cross-contamination with other products is possible.

	Even though risks are believed to be controlled or removed, McNaughton stresses that it is important to ensure plans, systems, and training are established so that in a worst-case scenario, such as an accidental release, there is no risk of contaminating the environment and a mechanism is in place to clean the area back to a safe standard without any risk to the operators.

	Excipients that are helpful in any low solubility drug are generally applicable to an HPAPI with low solubility. Solubilizers and disintegrants are two examples, according to Kujath. Excipient selection should be based on a combination of the chemical and physical properties of the HPAPI and the target product profile, according to McNaughton. “Meeting a target product profile for a poorly soluble API requires both the correct technology selection and the appropriate science-led formulation development,” he comments.

	While there are no specific excipients that are suited to highly potent materials, there are certain aspects of some technologies that offer advantages if they are also the correct technology for the product, McNaughton adds. “Under certain permeability-limited situations or biological obstacles, there are specific lipidic excipients that can provide some permeability enhancement, others that inhibit efflux, and others that could avoid first pass metabolism through utilization of the lymphatic system,” he says.

	The use of polymer-based excipients is widely appreciated in the formulation of poorly soluble HPAPIs, according to Mueller-Albers. They play a key role in the formation of solid solutions, stabilizing the amorphous HPAPI in the solid state to prevent recrystallization, and also helping to maintain HPAPI supersaturation in physiological media, she explains. Polymeric excipients are also used in formulations prepared via granulation using high-shear mixing or fluid-bed spraying, which can under certain conditions be applicable for poorly soluble HPAPIs.

	The first HPAPIs were often formulated as liquids and then filled into capsules to reduce safety risks such as dust formation. Liquids, however, present challenges when solubility is an issue, according to Kujath. “While micronization and spray drying of the HPAPI can be helpful for oral solid formulations, these techniques to drive initial dissolution for liquid formulations can present stability challenges, such as crystallization of an initially dissolved amorphous HPAPI from the vehicle or ripening in a micronized dispersion,” he explains. Thermocycling during terminal sterilization can also cause ripening or recrystallization or break complexes formed by solubilizers such as (2-hydroxypropyl) beta-cyclodextrin.

	As the number of HPAPIs in development has increased, however, manufacturers have aggressively examined new processing techniques suitable for oral solid-dosage forms, according to Mueller-Albers. Particle-size reduction, or particle-size design, amorphous dispersions, and lipid-based formulations are all suitable techniques for improving the bioavailability for solubility-limited HPAPI, according to McNaughton

	“Particle size reduction is likely to lead to an increase in the rate of solubility for an API and may provide some supersaturation effects, but is unlikely to result in the same level of potential increase that can be achieved by amorphous dispersions or lipid-based formulations,” he says. Lipids also offer a further advantage for low-dose HPAPIs where these can be fully solubilized because the potential for poor homogeneity is eliminated and accurate dosing can be provided, even for the lowest possible dose. “Achieving such formulations may be impossible in the solid state, so a lipid/liquid approach can be enabling for the molecule to be advanced,” McNaughton states.

	Kujath notes, though, that while lipid-based solubilizers and the formation of nanoemulsions can be an effective way to deliver an HPAPI of low solubility, these techniques should generally be combined with the use of a low-energy means of sterilization.

	If the API is not “greasy” (logP < 3), lipids are unlikely to provide the same improvements that amorphous dispersions can, according to McNaughton. In addition, processing to achieve particle-size reduction can be simpler than developing an amorphous or lipid formulation.

	“Every day the industry is improving containment solutions, making it easier to leverage HPAPI handling technologies that previously may have been less accessible,” says Kujath. He also notes that more and more focus has been placed on not just patient safety, but on worker safety. There are always new and improved excipients being developed, new approaches, and better understanding of how to use them, agrees McNaughton. He adds that improved technologies to remove the vulnerable operator from harm include more hygienic valves, in-line testing to minimize operator interaction with potent molecules, and increased automation that completely removes the operator from the vicinity of these process.

Excipients and new processing techniques can make a real difference in the development of highly potent therapies.