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

Cynthia A. Challener

pills recycle | Image credit: ©Zafirah - stock.adobe.com

Improving the sustainability of small-molecule API manufacturing is essential to ensuring the continued success of the pharmaceutical industry. For most APIs, however, evaluating the global warming potential along the whole value chain or lifecycle is difficult. The greatest contributor to the carbon footprint of API manufacturing operations is the use of solvents, reagents, and precious metal catalysts, for which significant restrictions on recycling and reuse can exist. The major argument against reuse and recycling is based on product and application safety, according to Michael Nonnenmacher, director global business development differentiating technologies with Evonik.

The ability to recycle and/or reuse these materials, however, enables measurable resource savings and carbon footprint reduction. “For multi-step syntheses, it is likely that hundreds of kilos of raw materials are used to produce a single kilogram of final product. It is still common practice to incinerate most of the waste generated from these raw materials, oftentimes resulting in tons of carbon dioxide (CO2) per kilo product. The possibility to recover some of these materials or replace them with greener alternatives would enable a tremendous reduction of the process carbon footprint and improve the overall sustainability of these operations,” Nonnenmacher states.

Solvent and catalyst recycling offers several significant advantages. In contrast to incineration or other forms of disposal, which generate substantial amounts of CO

, catalyst and solvent recycling allow repurposing in the same process or in different applications with minimal energy expenditure, according to Niklaus Künzle, senior director, global head of process technology and innovation for small molecules at Lonza. In addition to the reduced direct CO

 emissions, this approach also necessitates less production of raw materials, resulting in further substantial savings in both energy and CO

Solvent recovery is the most practiced recycling solution in established large-scale processes. “For API manufacturing, the use of recycled solvent is mostly restricted to the same process and a limited number of cycles, and a thorough risk assessment to mitigate potential impurity formation and accumulation in the process is needed,” Nonnenmacher says. “Since the discovery of nitrosamine impurities, which are potent carcinogens, stemming from solvent degradation, recovery is under a lot of scrutiny, but it should still be considered in cases where it will result in no process risk,” he adds. In cases where it is not appropriate to use recycled solvent in the API production process, Nonnenmacher observes that it is possible to find other fine-chemical applications where they are suitable.

Recycling of expensive palladium, platinum, rhodium, and other precious metal catalysts helps not only to reduce costs, but the carbon footprint associated with the mining and refining operations necessary to obtain the metals, notes Nonnenmacher. While solvent recycling is often performed in-house, the transformation of used catalysts back into active species is typically performed externally by specialized companies, according to Künzle.

In addition, although filtration of heterogeneous catalysts from process wastes is typically easy, separation of dissolved homogeneous catalysts from oftentimes dilute solutions requires more specialized technologies, comments Nonnenmacher. “Evonik has established an efficient process for the concentration of precious metal-containing organic waste streams using membrane cross filtration. This approach enables the sustainable recovery of the metal at the refinery, and thus contributes to resource saving and carbon footprint reduction,” he says.

The best strategy for realizing the greatest benefits from recycling during API manufacturing is to, according to Nonnenmacher, apply the principles of green chemistry early on during process development. “In this manner, it is possible to implement eco-friendly and more sustainable raw material and API manufacturing processes,” he notes. Key considerations highlighted by Nonnenmacher include avoiding the use of large volumes of especially hazardous organic solvents that cannot be recycled, applying process intensification approaches, and controlling the atom economy for waste reduction.

“Beyond these approaches, the introduction of recycling concepts or reuse options into processes right from the beginning can facilitate acceptance by regulatory authorities,” Nonnenmacher contends. Performance of process and product life-cycle analyses to generate important sustainability metrics such as process mass intensity and E-factor values when a candidate is in clinical development can, he says, help pharmaceutical firms and API makers visualize and understand potential adverse steps in the synthesis early enough, allowing manufacturers to implement more sustainable solutions.

Of course, one of the best approaches to improving sustainability is to avoid the use of problematic processes and materials. “Ideally, the most elegant solution is to minimize or completely eliminate the use of solvents,” Künzle states. Lonza therefore strives to implement process improvements in collaboration with drug developers during the early development phase. “These process improvements aim to reduce the number of used solvents, decrease dilution, and enhance the process yield. Selection of process solvents is also optimized by considering solvents and mixtures that are feasible to recover from process waste streams,” he explains.

Recycling compounds from a complex mixture requires efficient separation techniques, such as rectification and membrane separation. Continuous liquid-liquid or liquid-solid separation may also be employed, according to Nonnenmacher. The adsorption to scavengers or filter aids can be efficient for catalysts as well. Immobilizing catalysts such as enzymes by attaching them to a solid substrate also facilitates recovery and reuse, he says.

For solvents specifically, distillation, extraction, adsorption, and membrane separation techniques, according to Künzle. “Often,” he notes, “multiple technologies must be combined to ensure the purity required for recycling these materials back into API processes.”

Nonnenmacher also emphasizes that both increasing cost constraints and sustainability ambitions are driving the development of more efficient processing techniques for separation, as well as more selective processes that result in waste mixtures that are less difficult to separate.

Recycling, whether of solvents or catalysts, is often not an easy task given the complex solutions used in API manufacturing. “Extracting valuable materials from complex process mixtures can be a significant challenge, especially when aiming to restore them to a high-purity starting quality with minimal energy consumption and at minimal cost,” says Künzle.

Another major challenge for implementing recycling solutions at both clinical and commercial scale is that recycling is usually not attractive enough for small campaigns, according to Nonnenmacher. “It is considered a given that waste is sent to incineration and, therefore, oftentimes from a cost and effort perspective, recycling strategies are not a priority,” he adds. In addition, especially for smaller API manufacturing sites that are not fully self-sustained, there are significant logistical constraints given that recycling requires significant site infrastructure. “Even if waste streams are shipped offsite for recovery, a minimum buffer capacity separate from other wastes needs to be available,” Nonnenmacher observes.

Another factor that impacts recycling opportunities is regulatory considerations. The recycling of solvents in API manufacturing may come with potential impurity accumulation and is therefore scrutinized by regulating authorities, especially when considering environmental- and health-critical solvents, according to Nonnenmacher.

For example, Nonnenmacher points to the decomposition of dimethyl formamide (DMF) to dimethylamine and the potential for subsequent formation and accumulation of the carcinogen N-nitrosodimethylamine, which makes DMF recycling almost prohibitive.

When developing a solvent recycling process, notes Künzle, it is therefore crucial to ensure that no by-products accumulate in the solvents, which involves extensive laboratory and pilot testing and continuous analysis. In addition, whenever recycling of less critical solvents is possible from a risk perspective, Nonnemacher once again emphasizes the importance of establishing any recycling concepts during early development and into validation and using a defined, limited number of cycles before final disposal. “As part of the process, the recycling concept needs to be filed and accepted by the authorities,” he notes.

A risk-based approach to recycling is also required for materials other than solvents. “In most cases, waste streams from API manufacturing must be incinerated due to the potential risk of pharmaceuticals or other contaminants leaking into the environment,” observes Nonnenmacher. However, he does comment that recycling of metals that in principle undergo complete reset to their original state is handled outside of the process and not questioned by regulatory agencies.

Regardless of the material or technology, the best strategy is to integrate recycling considerations into regulatory filing activities from the very beginning, Künzle concludes.

Recognition of the importance of increasing the sustainability of pharmaceutical manufacturing operations is driving investment in many new technologies and solutions, including those for recycling. “Current and future innovation in many fields will contribute to the sustainability of API manufacturing and recycling of process components. Those innovations may come from established fields like from physical separations, or from research areas less obviously related, such as more in-depth process development supported by artificial intelligence resulting in more efficient production methods and cleaner off-streams, which would be easier to recycle,” Nonnenmacher comments.

Lonza, for instance, is working to enhance its entire infrastructure to enable the realization of additional recycling solutions, according to Künzle. One example is the expansion of pervaporation capacities in Switzerland, which allow the company to process challenging mixtures with minimal energy expenditure. Lonza is also applying new oxidation and enzymatic technologies for wastewater treatment that involve significantly reduced energy consumption.

Evonik, meanwhile, is leveraging fundamentally different approaches to how pharmaceutical chemistry is performed. One example is the Chemistry in Water platform the company is developing and using for the manufacture of active ingredients. “This technology allows significant reduction of organic solvent consumption, oftentimes combined with offering superior reaction performance, such as in the field of cross-coupling reactions,” Nonnenmacher says.

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

®
Vol. 48, No. 10
October 2024
Pages: 18–20, 33

When referring to this article, please cite it as Challener, C.A. Improving the Sustainability of API Manufacturing with Recycling Technologies. 

Articles

Sustainability of small-molecule API manufacturing ensures continued success.

Improving the Sustainability of API Manufacturing with Recycling Technologies

Antibody colored by heavy and light chains against black background; glycosylated immunoglobulin in pink | Image Credit: ©Eric H Cheung-Stock.Adobe.com

Glycan modifications of recombinant proteins and antibodies can impact their function and stability and thus the safety and efficacy of drug products based on these molecules. Understanding these important critical quality attributes (CQAs) is essential, and therefore, regulatory bodies expect a comprehensive understanding of glycans for submission.

This article was published in the October 2024 print issue of 

Comprehension necessitates overcoming inherent challenges such as positional isomers and heterogeneity, as well as method-induced artifacts that influence sensitivity and specificity, according to Paula Orens, senior global market development manager, Biopharma, at SCIEX.

Multispecifics, including bi-, tri-, and tetraspecific antibodies, present additional challenges to glycan analysis beyond those of traditional monoclonal antibodies (mAbs) because the complexity of these molecules is much greater.

“Site-specific and intact mass analysis may become more challenging for increasingly complex molecules, particularly when the number of N-glycosylation sites, post translational modifications, and potential degradation products increases,” observes Oscar Potter, biological chemistries R&D manager in the Chemistries and Supplies Division at Agilent.

Structurally, multispecific antibodies are a combination of different antibody domains that allow the multispecific to bind at multiple sites. “The various domains will have their own glycosylation patterns (integral to efficacy, safety, and stability) and their own impurities, all of which need to be characterized comprehensively, effectively multiplying the glycosylation analysis complexity by the number of binding sites,” Orens explains. She adds that as the number of binding sites increases, so too does the risk of intra-protein cross talk.

Multispecifics can also include candidates in which an antibody-portion is combined with some other protein-based component. Chimeric proteins, where the domains are not only antibody in origin, are complicated to analyze due to the individual properties of each component, according to Orens. “For example,” she says, “fusion proteins are typically heavily glycosylated with N- and O-linked glycans to improve biological activity, properly stabilize the structure, or improve solubility.”

“Overall,” Orens concludes, “while the complexity of analysis is dependent on the types of domains used in a multispecific, it is certain that the analysis of these molecules will be more complicated than that of a mAb.”

Consequences of incorrect glycosylation analysis

The greater complexity of multispecifics raises the stakes for achieving correct glycan analysis. “Not only can the glycosylation patterns in multispecifics impact the stability, pharmacokinetics, off-target effects, and effector functions such as complement activation and antibody-dependent cellular cytotoxicity, conceivably the additional functions that multispecific antibodies are capable of might result in unique biological consequences from incorrect glycosylation,” Potter comments.

In addition, because glycosylation is a critical and highly regulated quality attribute of all antibodies, misunderstanding or incorrectly characterizing glycosylation patterns could lead to a variety of consequences, depending on the development stage of the candidate, notes Orens. She highlights a couple of examples. “At the hit-identification stage, a mis-characterized but suitable candidate could be eliminated due to poor efficacy or stability. At later development stages, meanwhile, a drug metabolism and pharmacokinetics study could exhibit faster-than-expected clearance or undesired concentration in the target tissue. An incorrect analysis could also result in promotion of a misunderstood candidate through to late-stage toxicology studies, causing unnecessary investment in time and resources for the project only to be shut down later than expected.”

There are several different techniques used for the glycan analysis of antibody-based candidates. “Fundamentally, the same glycan analysis approaches are used for multispecific antibodies as mAbs, most commonly liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry (MS). Each analytical approach offers a plethora of techniques which provide complementary or alternative details about the glycosylation profiles,” Orens observes. She does note, though, that the inherent complexity of the molecule will impart different challenges on each approach.

Typical analytical methods for glycosylation analysis of multispecifics are similar to those used for more traditional therapeutic mAbs, agrees Potter. “Released glycan analysis methods generate a glycan profile, usually based on separation by CE or LC. Intact LC/MS analysis can provide limited information about the major glycoforms, while LC/MS of protease-digested samples can give site-specific information for molecules with more than one glycosylation site,” he outlines.

Released glycan analysis relies on high-resolution separation of fluorescently labeled glycans by hydrophilic interaction liquid chromatography (HILIC) or CE. HILIC is used in conjunction with fluorescence or mass detection to separate and quantify individual glycan species over long gradients, according to Orens. Similarly, CE systems can offer comparable quantitation details with an alternative separation mode, often with faster separation. “These approaches are often used as monitoring techniques to ensure batch consistency during manufacturing,” she says.

Released glycan analysis once suffered from tedious sample prep, according to Potter, but protocols have been greatly simplified in recent years through rapid-release and instant fluorescent labeling sample prep kits. He observes that HILIC methods enable the most exact assignment of glycan structures, either using retention time reference standards or retention libraries, or by hyphenating with MS detection. “HILIC separations can often distinguish between isomeric structures, including potentially harmful alpha-galactose (α-Gal) structures masquerading as beneficial galactosylated complex glycans,” he says.

Measuring the intact mass of multispecific antibodies via LC/MS does not require any sample prep, which can be advantageous. A well-optimized method run on a high-resolution instrument such as a time-of-flight (TOF)-based system is needed, however, Potter observes.

Orens adds that TOF systems can be used as the detection method for several techniques to help identify and localize glycans, as well as quantify, resolve isomers, and structurally elucidate the different species.

Potter cautions, though, that while very convenient, measuring the intact mass may only reveal the sum of masses of the two or more glycans on each molecule, which may confound assignment of exact glycan structures. Distinguishing between isomers is also not possible.

The most information can be obtained by first digesting the multispecific with a protease such as IdeS (immunoglobulin G-degrading enzyme of 

) or trypsin into smaller peptides, according to Potter. “Digestion allows for site-specific glycosylation analysis by LC/MS, which may be particularly useful when distinguishing between glycosylation in the fragment antigen-binding (Fab) and fragment crystallizable (Fc) regions, the latter of which greatly impacts the Fc effector functions of the antibody,” Potter contends.

Orens further notes that when peptide mapping is performed using a MS system with specialized fragmentation capabilities such as electron activated dissociation (EAD), it is possible to identify unknown modifications or impurities, then localize the species to a specific domain or amino acid of the protein. “EAD,” she says, “is integral to this process, as it differentiates between isomers and maps specific amino acids, and thus provides a comprehensive understanding of the protein with its modifications.”

Ultimately, Orens emphasizes, the appropriate analytical approach is entirely dependent on the purpose of the analysis.

Given the importance of accurate glycan analysis for multispecifics, the best strategy for ensuring success according to both Orens and Potter is to use a combination of available methods.

“While using top down, sub-unit, or peptide mapping methods and state-of-the-art MS systems enables comprehensive glycan analysis of multispecifics since it provides the most information in a single step, including molecular weight, domain/site localization, species identity, and species quantity, most organizations employ multiple analytical approaches to support their characterization efforts and obtain secondary, confirmatory evidence,” says Orens.

“The best way to ensure accurate and comprehensive characterization,” agrees Potter, “is to use more than one orthogonal method. Released glycan analysis, intact mass LC/MS, and LC/MS after protease digestion needn’t be mutually exclusive—they can be complementary,” he concludes.


October 2024
Vol. 36, No. 9
Pages: 28-30

When referring to this article, please cite it as Challener, C.A. Glycosylation Analysis for Multispecifics. 

The best strategy is to use a combination of complementary methods.

Glycosylation Analysis for Multispecifics

3d rendering molecule on a displayed on a medical interface | Image Credit: ©Production Perig- stock.adobe.com

The potential benefits afforded by molecular modeling when applied to formulation development are, in fact, numerous. Molecular modeling tools, while initially applied to enhance drug-discovery efforts, are increasingly used to inform other aspects of drug development. Formulation development, which traditionally has been performed using physical experimentation in a time-consuming trial-and-error approach, is one area that can particularly benefit from 

“While the use of molecular modeling tools has historically been used extensively in the drug discovery setting, application to formulation development has been slowly evolving,” says Sanjay Konagurthu, senior director of science and innovation in the pharma services business of Thermo Fisher Scientific. Molecular modeling, he observes, can accelerate many aspects of formulation development.

“Compared with trial-and-error approaches, the application and intentional use of the scientific method is a superior approach to formulation development,” notes Nathan Bennette, director and scientific advisor, pharmaceutical development at Catalent. “In this context,” he contends, “molecular modeling tools are extremely useful to increase the efficiency of the scientific process, from problem-statement definition to experiment design and through the development of a formulation strategy.”

For instance, to tackle the challenge of poorly soluble compounds for oral drug delivery, Thermo Fisher Scientific has developed artificial intelligence/machine learning (AI/ML) models specifically to identify the most effective combinations of solubility-enhancement technologies and formulations in early drug development, according to Konagurthu.

Molecular modeling can play a crucial role in various aspects of formulation development through its ability to offer valuable insights on molecular behaviors and its predictive capabilities. “One of the primary applications is the visualization and understanding of physical and molecular processes which yields deeper intuition of the underlying mechanisms,” says Bennette. “This mechanistic understanding allows for the generation of rational hypotheses and more informed decision-making,” he adds.

Molecular modeling tools also enable formulators to explore and evaluate various formulation options, according to Bennette. “Such models allow scientists to simulate scenarios, analyze the performance of different formulations, and predict outcomes, which aids in the selection and optimization of formulations against the target product profile and critical performance attributes,” he explains.

Several different modeling tools can be applied during formulation development, from molecular mechanics to advanced quantum mechanical (QM) calculations. Examples highlighted by Konagurthu include density functional theory (DFT), molecular dynamics (MD) simulations, quantitative structure activity relationship (QSAR) models, and statistical techniques. These tools are leveraged within Thermo Fisher Scientific’s AI/ML models to aid in formulation development, including the company’s proprietary Quadrant 2 platform for addressing formulation challenges associated with solubility and bioavailability.

Bennette points to physiologically- based biopharmaceutics modeling (PBBM) for understanding absorption and the Flory-Huggins and newer, more sophisticated, perturbed chain statistical associating fluid (PC-SAFT) models for estimating the miscibility of solid solutions and phase boundaries in dynamic systems.

Once the rate limiting step for absorption of a drug substance for an oral formulation is understood using PBBM modeling, these parameters can be used to estimate the impacts of different formulation approaches, such as the use of bioavailability-enhancing technology, before any laboratory testing is needed, says Bennette. “If, for example, the rate limiting step is identified as low solubility, then an amorphous solid dispersion (ASD) might be selected as an enabling technology to improve bioavailability,” he notes.

The Flory-Huggins or PC-SAFT models can then be incorporated to identify formulations with suitable physical and chemical stability and provide insight into manufacturing conditions, according to Bennette. Understanding the miscibility of solid solutions is key to successful development of stable and metastable ASD formulations. For formulations in which the composition and thermal history change throughout the manufacturing process, the PC-SAFT model is of particular use.

Once a formulation is selected, Bennette comments that modeling tools such as FreeThink’s ASAPprime software can be used to predict the shelf-life of the product, including the impact of packaging, through its application of fundamental Arrhenius rate laws.

“Such a comprehensive modeling approach to formulation development ensures a higher probability of clinical and eventual commercial product success,” Bennette states.

Gaining a fundamental understanding of drug–drug and drug–excipient interactions is critical to developing stable formulations that can perform and do not pose manufacturing challenges. Modeling tools are beginning to be used to understand chemical stability and excipient compatibility, according to Konagurthu. “The use of these tools for both small and large molecules has been evolving and increasing with the advent of high-performance computing,” he notes.

A key current challenge to using modeling for predicting drug–drug and drug–excipient interactions is the need to ensure the models are validated using real-world, experimental data. Often, however, only limited and sparse data sets are available given the relatively new use of modeling in this application, observes Konagurthu. “Lack of access to sufficient, high-quality data can hinder the prediction capacity of such models,” he says.

Using modeling to predict molecular interactions is an evolving and growing field, though. “With advances in software, hardware, and AI/ML techniques, we anticipate this technology will continue to be an exciting and expanding area that could result in better outcomes and accessibility of life-saving therapies for the patients who need them the most,” Konagurthu states. “It is important that scientists embrace these new technologies that can help them overcome long-standing challenges in the pursuit of more effective treatments. We believe that these molecular modeling tools hold great potential for the pharmaceutical industry and patients around the world,” he continues.

Thermo Fisher Scientific, for example, has developed some of these tools for tackling formulation challenges with ASDs.

Enabling time, cost, and materials savings

The potential benefits afforded by molecular modeling when applied to formulation development are, indeed, numerous. “Mechanistic models enable visualization of molecular processes, formation of rational hypotheses, and interpretation of data against the backdrop of fundamental kinetic and thermodynamic principles,” comments Bennette. “Predictive models—both physics- and statistics-based—are useful for defining specific experimental conditions, rationally selecting formulation candidates, and informing key risk factors for formulations and processes,” he says.

“Modeling tools thus make the whole process of formulation development more efficient by enabling formulators to use a rational, mechanistic approach to defining the key problem statements, developing hypotheses, and interpreting data,” Bennette remarks. “Within the framework of these models, developers can ‘work smarter, not harder’ and avoid time- and resource-intensive trial-and-error approaches,” he emphasizes. “Indeed, by leveraging predictive molecular modeling tools that enable in silico formulation development, significant savings in time, costs, and materials can be saved, especially for expensive active pharmaceutical ingredients,” Konagurthu concludes.

®
Vol. 48, No. 9
September 2024
Pages: 15–16

When referring to this article, please cite it as Challener, C.A. Molecular Modeling in Formulation Development. 

Insights into molecular behaviors and predictive capabilities are bringing numerous benefits.

Molecular Modeling in Formulation Development

modern digital tablet pc near test tube in laboratory | Image Credit: ©donfiore - stock.adobe.com

Decreasing the cost and time of drug development while advancing differentiated therapies with improved efficacy and safety is a priority for drug makers. Automation of activities across all aspects of drug development and manufacturing can help achieve these goals. Indeed, “automation can help the API industry with improved safety, productivity, product quality, and optimized costs, making it an essential tool for the industry to remain competitive and meet regulatory requirements,” states Raghavendar Rao Morthala, chief scientific officer, Navin Molecular. These benefits can be realized at all stages of development as well as across development and manufacturing.

Many applications for automation in API development and manufacturing

Automation in many forms has, in fact, been used for API development and manufacturing for many decades, according to Morthala. He points to examples such as electronic lab notebooks and electronic batch manufacturing records, statistical software for design-of-experiment studies employed in process optimization efforts, control systems for API manufacture in both batch and continuous mode, and inventory management and material movement solutions.

“Automation is used extensively in API synthesis,” emphasizes David Ford, senior director of chemistry at Snapdragon Chemistry, a Cambrex company. “While batch manufacturing processes are primarily executed under direction of a human following a batch record, automation is used for temperature and flow control (reagent dosing), as well as for safety interlocks. Continuous flow manufacturing processes use automation more extensively, as automation can be used for reliable start-up and shut-down of the reactor, clean-in-place operations to recover from a clog, and to divert reactor effluent to waste if the quality is expected to be poor,” he explains.

The above forms of automation, Ford says, make work more convenient, but an operator is still required to be present to initiate operations and complete the batch record. “In contrast, automation in the R&D lab can be used to run processes completely without operator intervention,” he observes. In this context, Ford explains, whether batch or flow processes, all steps including reagent charges, sampling, on-line or off-line liquid chromatography, and storage of product for further analysis can be automated.

Such automation tools can also be coupled with statistically powered experimental design or auto-optimization algorithms. “This type of automation can accelerate tasks that can be labor-intensive, such as process optimization and process characterization. Automation can, in addition, free a process development chemist or engineer to research more broadly in the literature or execute more high-level analysis of the data,” Ford comments.

In addition to accelerating physical experiments, literature search engines are available that continuously update comprehensive databases of identified and potential chemical substances, chemical transformations, and newer methodologies. “Using these automated systems, research scientists can quickly identify desired organic transformations and optimum reaction conditions, as well as gain insights into reactant and product properties and raw material sourcing options,” says Morthala.

It is important, cautions Jens Schmidt, process expert and high-throughput experimentation (HTE) manager at Lonza, to apply phase-appropriate automation techniques to ensure highly effective use of the technology across the entire lifecycle of a project. In the initial phase of a project, according to Schmidt, automation is particularly potent and extensively utilized in process development, commonly in the form of HTE.

“This technique facilitates the miniaturization and parallelization of chemical reactions, allowing for the rapid testing of a broad spectrum of reagents, solvents, catalysts, and conditions. It enables the assessment of various routes, thereby increasing the likelihood of identifying optimal conditions early in the project’s timeline,” he explains.

For example, Ford highlights the use of automation for screening of reagents, catalysts, or solvents to find beneficial combinations and automated workflows for measuring solubility and partitioning in liquid-liquid extraction to investigate isolation strategies for intermediates and APIs. “Such a screening approach can enable identification of useful combinations that would escape attention if experiments are performed manually because a much larger number of combinations can be screened in an automated context,” he notes.

As a project matures, HTE continues to play a crucial role, albeit in a slightly different capacity. The main goal is to aid in process optimization and characterization, thereby enhancing process robustness, notes Schmidt. He adds that automation during this phase also encompasses the standardization and automated execution of individual experiments, with the objective of delivering reliable and high-quality data.

“Upon reaching the commercial stage, automated experiment execution or HTE supports a diverse range of activities, including the optimization of established processes and the enhancement of process understanding,” Schmidt comments. Automation also contributes to the attainment of sustainability goals, the securing of supply chains, and the resolution of unforeseen issues. Of course, automation of large-scale manufacturing is a significant aspect that warrants consideration due to the numerous advantages it affords.

“Automation is transforming the landscape of API development and manufacturing. By automating tasks like route exploration, reaction optimization, and data analysis, researchers can significantly accelerate the identification of optimal synthetic pathways and fine-tune processes for maximum efficiency. These benefits translate to faster development times, reduced errors, and more consistent API production. For drug developers, that means reduced risk and uncertainty in API development, enabling a greater focus on clinical trials and regulatory approvals,” Schmidt concludes.

Using automation in synthetic route development

The concept of automated multicriteria evaluation of new process routes, combined with route development, is based on data mining, according to Morthala. “There is an increasing prevalence of chemical processes being developed based on automated experiments that make use of data science, which can provide multiple numeric criteria for scientists to reach a balanced decision on the suitability of a novel synthetic route,” he says.

Information generated via data mining can be used for chemical route development and evaluation, including planning of hypothetical synthetic routes, Morthala contends. Such efforts can take into consideration not only process conditions, but environmental indicators, such as energy consumption, E-factor values, solvent scores, reaction reliabilities, and route efficiencies allowing for multi-criteria environmental sustainability evaluations, he observes. “This type of automated workflow is based on deep data mining and algorithmic decision-support tools to enable multi-criteria decision making,” he says.

Small-molecule APIs are becoming more and more complex, and the resulting longer synthetic pathways pose challenges for process chemists to develop efficient API manufacturing processes. Overcoming these challenges is crucial to avoid delays in investigational new drug applications and expenses in transitioning new drug candidates from early development to commercial production, according to Simon Wagschal, associate director of advanced chemistry technologies, Lonza Small Molecules. “As the industry faces new drug candidates requiring over 20 synthetic steps on average, AI [artificial intelligence]-enabled predictive route design technologies have become essential for creating efficient retrosynthetic routes,” he says.

Indeed, AI-enabled API development tools are already being used by several R&D companies for the identification of optimized synthetic routes, according to Raghavendar Rao Morthala, chief scientific officer, Navin Molecular. “An AI platform has the potential to design and synthesize APIs in a matter of weeks or months, compared to the years it can take using conventional methods,” he says.

Currently, however, the application of AI to API synthetic route design is in its early stages, contends David Ford, senior director of chemistry at Snapdragon Chemistry, a Cambrex company. Synthetic route design by AI is used primarily at the brainstorming stage of route development, he says, with identified options narrowed down by humans. “Given the importance of the choice of a route or routes and the quantity of resources used to pursue them, the role of the synthetic chemist remains central in route design,” he states.

Wagschal adds that AI tools have traditionally been used for early-phase discovery, often generating routes not suitable for commercial-scale production.

Other exciting applications for AI in process development, Ford says, lie in its ability to manage knowledge. “Recent advances in large language models have made it possible to index and explore diverse sets of documents like weekly updates, PowerPoint presentations, technical reports, batch records, and campaign summaries,” he explains. These AI models allow for the content to be searched semantically, based on the meaning of the search, instead of by keywords.

“Given the volume of information trapped in these documents, it is very likely that work is forgotten, and hard lessons then need to be re-learned,” Ford observes. “We see great opportunity in helping researchers make connections between their work and the work that has been done in the past,” he notes. Generative AI models may also prove useful for preparing first drafts of templated documents like standard operating procedures, qualification protocols, and batch records, according to Ford.

Lonza, meanwhile, is one company not deterred by the limitations of early AI programs developed for scouting of synthetic routes for small-molecule APIs. “The introduction of computer-aided synthesis planning (CASP) technologies with route prediction capabilities has significantly influenced the route selection process,” says Wagschal.

Nearly all CASP tools employ a form of machine learning called Monte Carlo Tree Search to rapidly prioritize computationally predicted synthetic routes, Wagschal explains. This heuristic search method evaluates the likelihood of reaction success at each step of the proposed pathway, from starting materials through intermediates to the final API. The prioritized routes are then reviewed and refined by process chemists. “This approach, guided by our experts, has increased the number of potential strategies and led to more efficient API synthesis routes,” he states.

To overcome the limitations of early AI-based models for route development with respect to commercial practicality, Lonza has combined leading AI-powered predictive route design models with proprietary supply-chain data, including raw material costs, availability, and detailed supply chain intelligence, according to Wagschal. “This integrated technology allows process chemists to quickly identify not just the shortest pathways, but also the most commercially viable ones by considering real-world supply chain constraints. These advanced AI solutions provide a holistic view that streamlines route scouting and accelerates the transition from research and development to robust manufacturing processes, resulting in significant time and cost savings in developing complex APIs,” Wagschal concludes.

There are challenges to using automation tools for API development, though. Automation systems require substantial investment in hardware, software, and infrastructure, and thus can be expensive to commission, Morthala observes.

Access to relevant, high-quality data is also important, Morthala adds. For automated route development in particular, wide implementation of automated development and evaluation methodologies requires recognition of the value of reaction data currently being held in the cheminformatics community by the wider chemistry and chemical engineering community and publication of that data in a manner that ensures it is accurate and available electronically. “If this state can be achieved, it will be possible to leverage automation for process development on a wider scale,” contends Morthala.

One of the biggest challenges to benefitting from automation in API development in Ford’s opinion is that any automation tools need to at least perform as well as the highly trained and experienced process development scientists and engineers currently executing much of this work manually. “These experts have developed very efficient ways of doing their work and can easily adjust to accommodate a very wide range of process conditions, something that is difficult to achieve in an automated platform. Given that challenge, the key to succeeding in using automation is to identify areas where automation will have a fighting chance of adding value,” he comments.

Beyond having access to the right data and tailoring automation strategies to the specific stage of the project lifecycle, best practice for using automation in API development activities is to take a holistic approach. “First and foremost,” underscores Schmidt, “is to define clear objectives that allow for prioritization of tasks and processes that will most benefit from automation.”

It is also important, according to Schmidt, to ensure that automated workflows are reliable and thoroughly understood. Applying appropriate analytical techniques and where possible integrating advanced analytical tools into automation solutions will enable monitoring and optimization of processes. “Finally,” Schmidt observes, “promoting interdisciplinary collaboration to leverage diverse expertise and ensure cohesive integration of automation solutions, including those for data handling and treatment, across different phases of API synthesis will allow for full realization of the benefits of automation.” 

®
Vol. 48, No. 8
August 2024
Pages: 14–17

When referring to this article, please cite it as Challener, C.A. Automating Development of Small-Molecule APIs. 

A holistic approach to automation can provide benefits at all stages of development and manufacturing.

Automating Development of Small-Molecule APIs

Antibodies floating. 3D illustration | Image Credit: © Steven - stock.adobe.com

Successful biomanufacturing processes not only provide the desired product in high yield and purity, they provide consistent results whether implemented in the lab or the production plant. For many monoclonal antibody (mAb) products, processes must consistently perform at scales ranging from hundreds of milliliters to thousands of liters. Lack of scalability can result in significant project delays and added cost due to the need for further optimization. Developing scalable processes from the outset is therefore the most effective approach to ensuring the smooth advancement of mAb projects through development to commercialization.

The main technical challenges faced during the scaling of upstream mAb processes include variations in cell densities, cell viabilities, and product concentration, as well as potential offsets in product quality attributes, according to a spokesperson from Boehringer Ingelheim. These attributes can be impacted by the bioreactor design, process parameters such as temperature and oxygen transfer, media composition, supplement and feed strategies, and numerous other factors. Obtaining process understanding is therefore essential to establishing robust, scalable processes.

“Addressing these scale-up considerations in upstream mAb process development ultimately comes down to the timeline, budget, and priority of the developer,” says Leslie Wolfe, site head and vice-president (VP) of process development at KBI Biopharma. She notes that it is imperative to fully understand what knowledge exists and what is needed as part of the scope to help support effective process design. Because time and cost are often the most important drivers during early phase development, there may be a necessary trade-off in the form of reduced process knowledge and understanding of the design space. “One potential consequence,” observes Lorenz Hasler, VP of global manufacturing, science, and technology at KBI Biopharma, “may be reduced knowledge of key inputs to process design regarding quality attributes and process performance and underestimation of considerations as they relate to commercial manufacturing equipment.”

Benefits of established platform processes

An important consideration for mammalian upstream processes for mAb production, notes Hasler, is that the potentially critical respective key scale-up factors are comparable between processes. “Process knowledge and expertise can therefore be leveraged between processes. Because potential key parameters and equipment limitations can be taken into consideration upfront, potential scale-up issues can be reduced,” he explains. Risk analysis tools, Hasler adds, can then be used to screen and understand other potential process variables specific to the mAb candidate and thereby mitigate potential process risk at scale.

The best approach to ensuring scalability of mAb production is to use a platform process rather than focusing on individual project variations, observes the spokesperson from Boehringer Ingelheim. Taking this approach, it is possible to manage challenges to scalability using standard equipment (bioreactors).

Wolfe agrees that working with an established and scalable platform is key to mitigating the risks associated with scaling up. She notes that such a platform is typically developed through iterative application, which helps in establishing critical process knowledge.

“This knowledge and understanding form the foundation for establishing a process control and monitoring strategy tailored to each unit operation, leading to a potentially robust and reliable scale-up approach,” says Hasler. Platform processes may also speed up development activities because process development, and facility-fit assessments can leverage process knowledge and understanding, he adds. These platforms also help balance the timeline-versus-process-knowledge conundrum for programs with aggressive development timelines.

Relying on platform processes to ensure scalability must be pursued with some caution, however. “Conditions and predictions to perform a process at scale or commercial process for any new drug substance or drug product still need to be demonstrated at laboratory or pilot scale,” Hasler states. Additional information may also be needed to support the requirements of specific mAb-based candidates. When necessary, for instance, Boehringer Ingelheim uses computational fluid dynamics and process modeling, including detailed equipment characterization, to ensure smooth scale-up.

To ensure scalability for upstream mAb processes, designing an efficient process complemented with an effective process control approach is critical and should be tackled during early process development, according to Hasler.

For Boehringer Ingelheim, a critical aspect of its scalability approach for mAb processes is the selection of a robust clone during early development. The chosen clone, says the company’s spokesperson, should not only fit the platform, but also exhibit robustness in relation to classical scaling parameters.

Even when a platform process is employed, Hasler recommends generating process knowledge for a specific mAb candidate using design-of-experiment studies, which help in defining the relationships and multivariate interactions between various process parameters and resulting outputs. He notes that it is also crucial to develop compelling analytical methods to support these small-scale studies. Demonstrating scalability of upstream mAb processes at an intermediate scale is beneficial as well, according to Wolfe, as doing so helps to de-risk scale-up to manufacturing.

Finally, Hasler comments that for mAbs with expedited approval designations, the shortened development timelines may lead to a reduced approach to process design and, therefore, reduced process knowledge and understanding. To balance the potentially reduced process development activities, he suggests that a detailed gap identification and subsequent risk evaluation may support potential scale-up considerations and success.

Consistent scale-up through platform approach

Platform processes not only streamline the development of scalable processes, they also make it possible to use a consistent approach to scale-up across all mAb-related projects. That is the case at Boehringer Ingelheim. “We do not test different scale-up strategies for our projects, as our scale-up strategy is not influenced by the specific product being expressed. Whether the product is a standard IgG1 [immunoglobulin G1] antibody or a trispecific molecule, the scale-up strategy generally remains the same,” says the spokesperson from Boehringer Ingelheim.

While general scale-up strategies also apply to more complex or advanced antibody-based therapeutics, Wolfe observes that appropriate risk assessments should be conducted and considered with more complex therapeutic targets to ensure sufficient knowledge is available. “Using this approach ensures the target product quality profile is achieved from the defined process,” she concludes.

®
Vol. 48, No. 8
August 2024
Pages: 18–20

When referring to this article, please cite it as Challener, C.A. Considering Process Scaling for Monoclonal Antibodies During Early Phase Development. 

Platform processes and effective risk assessments help overcome time and cost challenges.

Considering Process Scaling for Monoclonal Antibodies During Early Phase Development

The biologic drug segment has both expanded rapidly and evolved technologically over the past three decades. Many biopharmaceutical drugs have also reached blockbuster status. Some have already lost patent protection, and many more will during the next decade. All will face competition from biosimilars, which are increasingly accepted by doctors and patients. Biobetters—improved versions of the originator biologic offering advantages such as enhanced efficacy and safety, reduced side effects, and more attractive administration options and dosing frequencies—provide an alternative to biosimilars with greater potential for differentiation in the market. Increased understanding of disease pathologies and more advanced capabilities in the design, engineering, and manufacturing of biologics are leading to growth of the biobetters market, which is predicted to expand at a compound annual growth rate of approximately 8% over the next five years (1,2)

Manufacturing and product performance improvements

Drug developers pursue biobetters to improve the treatment of diseases, overcome the limitations of existing drugs, and better meet the needs of patients, contends Ulrike Herbrand, scientific director, global in vitro bioassays with Charles River Laboratories. “Biobetters are novel drugs that represent an improvement on the original drug and have advantages over the original drug,” she says.

Better treatment options are achieved through improved properties. Examples, according to Herbrand, include a longer half-life, higher stability and selectivity, increased specificity, reduced toxicity and immunogenicity, and/or a different route of administration. “All these factors are related to better efficacy, safety, or patient-friendliness,” she remarks.

Those improved properties can be realized by leveraging innovations in production technology, Herbrand says. “Greater understanding of human biology has enabled identification of opportunities to improve the molecular structure of originator molecules. Advances in cell-line engineering, cell culture processes, and process analytical technologies, meanwhile, enable better control of process parameters and thus more precise control of post-translational modifications and other important quality attributes,” she explains. Similarly, advances in formulation science make it possible to improve upon the formulation of the original therapeutic drug.

There is also an important market driver for the development of biobetters. “Unlike biosimilars,” Herbrand states, “biobetters are considered new drugs and therefore benefit from market exclusivity rights, even if they have similarities to existing therapeutics.”

Common chemical modifications include revising the glycosylation profile with the intention of improving the stability, pharmacokinetics, and immunogenicity of the originator biomolecule, according to Herbrand. PEGylation (attachment of polyethylene glycol to proteins) and sialylation (covalent addition of sialic acid to the terminal end of glycoproteins) are also well-known to improve the half-life, reduce immunogenicity, and enhance solubility, she adds.

Another important strategy is to modify the amino acid sequence of the protein. “Such changes can achieve enhanced efficacy and/or reduced side effects as well allow for engineering of the protein structure so it exhibits improved folding properties, which lead to both better stability and function,” Herbrand comments.

If the originator biomolecule is derived from proteins not of human origin, another chemical modification that is often employed is humanization to make the drug substance more like human proteins, according to Herbrand. This change reduces the risk of immune reactions.

Beyond changing the amino acid sequence, more significant structural modifications are often employed in biobetter development to enhance in-vivo performance. One of the most common structural changes, says Herbrand, is optimization of the binding site to improve its interactions with the target receptor and thus overall function.

As an example, Herbrand points to engineering of the complementarity-determining regions (CDRs) of antibodies. “These are the variable regions of an antibody that directly interact with the target antigen. Rational engineering of CDRs can improve binding affinity and specificity, which can lead to better therapeutic outcomes,” she observes. It is also possible to introduce charged amino acids (so-called charged mutations) near the edges of CDRs to enhance solubility and reduce aggregation. The latter can also be reduced/prevented by introducing other mutations to generate aggregation-resistant domain antibodies, Herbrand adds.

Increasingly, computation approaches are used for structure-based optimization during biobetter development. For instance, predictive computational strategies support the assessment of developability risks early in the lead candidate discovery phase, according to Herbrand. Computational modeling also helps identify key residues involved in binding to optimize binding affinity and predict solubility and stability properties and the potential risk of aggregation.

Biobetters can offer advantages other than those related to their quality, stability, safety, and therapeutic efficacy. A key goal of drug developers today is to provide medicines that take into consideration the specific needs of the target patient population so they are easy and convenient to use. As a result, a key aspect of biobetter development includes modification of the formulations of originator drugs to allow for greater patient-centricity.

“Changes in a formulation to support another route of administration, such as allowing the switch from intravenous to subcutaneous delivery, can make the patient experience more convenient and lead to healthcare savings,” notes Herbrand. “Higher concentration formulations, for instance, lead to a reduced application volume and thereby allow for administration via auto-injection devices, which is crucial for patients with chronic diseases (e.g., asthma, diabetes, colitis, or arthritis) who are dependent on regular drug administration,” she adds.

Formulation changes also involve using excipients that enhance drug stability, solubility, and bioavailability, Herbrand comments.

Protein engineering, including site-directed mutagenesis, molecular dynamics, PEGylation, sialylation, and bioconjugation, remains a foundational technology for biobetter development, with advances occurring in many of these areas, according to Herbrand. “Application of nanotechnology to drug design and development is a newer addition to the portfolio of engineering capabilities, with examples including formation of drug products as encapsulated or immobilized nanoparticles to improve pharmacokinetics,” she says.

One development area of particular interest to Herbrand is antibody-drug conjugate (ADC) biobetters. ADCs combine the tumor-targeting properties of antibodies and the cytotoxic potency of chemotherapeutics with the goal of delivering destructive compounds specifically to cancer cells while minimizing damage to healthy tissue. On the innovator side, Herbrand notes that manufacturers are currently working to identify new targets and/or payload mechanisms of action (MoAs).

ADC biobetter development, meanwhile, is focused on ADCs that leverage established target/payload MoA combinations but incorporate novel delivery components to achieve a best-in-class profile. “Non-specific and insufficient payload delivery remains a challenge with approved ADCs. Issues include premature payload release, poor tumor penetration, variable drug-to-antibody ratios, and aggregation,” Herbrand explains. Next-generation ADC technology aims to improve these aspects (3).

Always a balance between costs and benefits

Biobetters are designed to be an upgrade from existing therapeutics with structural or chemical changes that lead to improved efficacy and safety, lower side effects, and other advantages. “These improvements must lead to measurable benefits for patient and healthcare systems to justify the necessary developmental efforts and costs and to support regulatory approval,” states Herbrand.

As a result, the choice to pursue biobetter development depends on both safety and cost-benefit considerations, according to Herbrand. “Intensive research is needed to optimize therapeutic products, but it is not always the case that attempts to change and optimize an originator biomolecule will lead to better results. The efficacy or safety may not be sufficiently improved, and without superior performance there is no point in advancing such projects. It is also possible the cost-benefit ratio will not be clearly in favor of the biobetter, leading healthcare systems and patients to prefer the originator product or a biosimilar,” she explains.

The benefits, therefore, must be carefully weighed against the resources invested in biobetter development, Herbrand concludes.

Grand View Research. Biobetters Market Size, Share & Trends Analysis Report By Drug Class, by Application, by Route of Administration, by Distribution Channel, by Region, and Segment Forecasts, 2023–2030. 

https://www.grandviewresearch.com/industry-analysis/biobetters-market-report

Mordor Intelligence. Biobetters Market Size & Share Analysis–Growth Trends & Forecasts (2024–2029). 

https://www.mordorintelligence.com/industry-reports/biobetters-market

Sasso, J. M.; Tenchov, R.; Bird, R.; Iyer, K.A.;, Ralhan, K.;, Rodriguez, Y.;, and Zhou, Q.A. The Evolving Landscape of Antibody-Drug Conjugates: In Depth Analysis of Recent Research Progress. 

 34 (11), 1951–2000. DOI: 10.1021/acs.bioconjchem.3c00374

®
Vol. 48, No. 7
July 2024
Pages: 14–15, 19

When referring to this article, please cite it as Challener, C. Biobetter Development. 

Weighing development costs/resources and performance benefits is essential.