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

Cynthia A. Challener

The global market for highly potent active pharmaceutical ingredients (HPAPIs) has been growing rapidly in recent years due to the rising prevalence of cancer, diabetes, and cardiovascular diseases, among others. The overall market was valued at $12.6 billion in 2014 and expanding at an estimated compound annual growth rate of 7.8% from 2015 to 2023, to reach $25.1 billion by 2023, according to Transparency Market Research (1).

	The main driver of HPAPI market growth is the development of novel, targeted therapies, particularly antibody-drug conjugates (ADCs), which enable treatment with highly potent compounds that, if delivered systemically, would cause significant damage to healthy cells and tissues. ADCs consist of an antibody linked to a highly potent small-molecule “payload” that is released only once the antibody binds to the targeted cell.

	Two ADCs have been approved by FDA: Adcetris (brentuximab vedotin from Seattle Genetics) and Kadcyla (ado-trastuzumab emtansine from Genentech). Approximately 40 additional ADCs were in various phases of clinical development as of early 2015 (2).

	The production of ADCs requires expertise in both biopharmaceutical and chemical manufacturing, as well as the ability to conjugate highly potent small-molecule APIs to biologic components. This last step can be particularly challenging, given that it involves the manufacture of highly toxic compounds in a biopharmaceutical environment, according to Laurent Ducry, group leader, R&D bioconjugates with Lonza.


	A number of different linker chemistries have been explored to connect the highly potent small-molecule drug to the antibody. The linker chemistry determines the release profile of the payload, and thus is crucial to the safety and efficacy of ADCs. The complete ADC must be stable during storage and throughout the time during which the antibody circulates in the bloodstream so that the drug is only released once the antibody binds to the target cell.

	The types of conjugation chemistry that can be used are largely determined by the available functional groups that are present on the surface of the antibody and sufficiently distant from the binding site so as not to interfere with the function of the antibody. Most commonly, cysteines and lysines have been reacted with hydrazones, disulfides, peptides, and thioethers. Of the ADCs in development, more than half link to the thiol groups of cysteine residues, and at least a quarter are conjugated through surface lysines (2). The locations and quantities of cysteine and lysine residues vary from one antibody molecule to another, however, resulting in heterogeneous conjugation.

	Several new conjugation technologies for site-selective reactions that result in homogeneous conjugation for ADC production are emerging to address this issue, according to Ducry. “These techniques have the potential to provide ADCs with improved therapeutic properties,” he notes.

	Compared to traditional lysine and cysteine conjugation chemistries that produce a heterogeneous mixture of products, these novel conjugation techniques based on engineered antibodies or enzymatic reactions result in site-directed conjugation. 

	Some examples include selective N-terminal conjugation based on the differences in the reactivity of internal and terminal amino acids; site-specific functionalization of glutamines through selective enzymatic conjugation of specific residues; and protein engineering to incorporate at specific sites within the antibody unnatural amino acids with reactive moieties other than thiol or amine groups, such as aldehydes, ketones, azides, or alkynes (2). These new approaches are enabling the use of new conjugation chemistries, such as click reactions and enzymatic ligation (3).


	The design of antibody-drug conjugates, which makes them effective, targeted therapies, also makes them challenging to produce. Expertise in bioprocessing (cell-culture and/or fermentation), chemical synthesis, linker technologies, and conjugation chemistry are all necessary, as is the ability to perform much of the manufacturing process under high-containment conditions due to the high potency of the small-molecule payload.

	Different technologies and plant set-ups are required to synthesize, purify, and isolate small-molecule HPAPIs and biological targeting agents such as monoclonal antibodies. The bioconjugation step, which takes place in mostly aqueous media under aseptic conditions, requires a third set of production capabilities, according to Ducry. 

	In addition, while the conjugation of biologic compounds to non-potent chemical APIs is used for the manufacture of a number of different drug products, conjugation of highly potent APIs is a relatively new technology. “One of the key challenges for ADC manufacturing is addressing the need for high containment when working in a biopharmaceutical environment,” Ducry states. He adds that robust safety processes that ensure occupational safety but also enable effective cGMP manufacturing are required to address these challenges.

	In biomanufacturing, it is important to prevent contamination of the large-molecule API by biologic material originating from operators or the environment. As a result, a cleanroom environment with positive pressure is used. When working with small-molecule HPAPIs, however, protecting operators and the environment from exposure to the API is the main concern. Specialized containment, handling, and personnel protective equipment, highly complex operating procedures, and the use of negative air pressure are employed to ensure a high level of safety.

	The needs for bioconjugation of HPAPIs are therefore complex, because conflicting measures (positive and negative air pressure) are typically taken to prevent contamination and protect operators. Developing manufacturing conditions that ensure both operator safety and product purity is challenging. “Very few contract manufacturers have the unique set of capabilities necessary to achieve the balanced approach required for facilitating bioconjugation with HPAPIs,” observes Ducry.

	Other challenges when performing conjugations with antibodies and highly potent payloads include the need for rigorous equipment cleaning and waste management. Cleaning to necessary levels can be difficult because cleaning limits are calculated based on the toxicity of the material. The cleaning limits for the ADC as well as the small-molecule API component are often quite low as a result, according to Ducry.

	Waste treatment is also complicated by the presence of highly potent compounds. Again, protection of workers, the public, and the environment are the prime concern. Lonza has addressed this issue by incinerating all toxic waste on site at its Visp, Switzerland facility where toxin synthesis and conjugation are performed. “This solution provides optimal control, improved safety, and higher flexibility,” Ducry says.


	“Full-length antibodies remain the primary targeting agent used to selectively deliver cytotoxic agents to cancer cells because they have long circulation times,” Ducry says. He also notes that this approach has been validated by approved drugs such as Adcetris and Kadcyla, both of which Lonza manufactures.

	Nonetheless, smaller scaffolds such as antibody fragments, bispecific antibodies, proteins, and synthetic peptides are being explored with the goal of improving penetration rates into tumors. Additionally, conjugated polymers and nanoparticles are emerging as new drug candidates, according to Ducry. “Based on the R&D activity occurring at many pharmaceutical/biopharmaceutical companies, we expect that the number of APIs being developed with targeted drug-delivery mechanisms will continue to grow.”


	The challenges posed by cleaning permanent stainless-steel equipment and managing the toxic waste generated in these processes can be minimized through the implementation of single-use technologies. “Use of disposable systems eliminates equipment cleaning and the need to develop sensitive analytical assays. The change-over time is shorter, and compared to dedicated equipment, there are no storage or start-up costs,” Ducry explains. He does add, however, that single-use equipment lacks the robustness of stainless steel, and therefore appropriate risk-management strategies must be employed to ensure that the necessary level of containment can be maintained.

	Lonza began manufacturing ADCs in fully disposable systems in 2015 in order to simplify change over and cleaning processes, and has focused on managing the safety risk associated with highly potent cytotoxic compounds in accordance with its safety program. To date, the company has successfully produced five of its ADC products in fully disposable systems without any safety issues, according to Ducry.

Global High Potency Active Pharmaceutical Ingredients Market: Growing Demand for Targeted Therapeutics to Boost Market Growth at 7.8% CAGR

,” Jan. 6, 2016,
	2. N. Jain, S.W. Smith, S. Ghone, and B. Tomczuk, 

. 32(11), 3526-40 (2015).
	3. J.S. McCombs and S.C. Owen, 

Articles

The production of antibody-drug conjugates requires biopharmaceutical and chemical manufacturing, and conjugation capabilities.

ADCs Challenge Manufacturers’ Skill Sets

Biologic drug substances must meet numerous specifications based on critical quality attributes that impact the safety and efficacy of the active ingredient. These important properties must be consistent from batch to batch and within batches if, after final filtration, the API is collected in multiple containers rather than one large vessel. Biologic API uniformity can, however, be influenced by many aspects of the production process, including the quality and variability of the raw materials. Effective harvesting and purification processes play an essential role in ensuring that biopharmaceutical manufacturing processes provide biologic drug substances with uniform and consistent properties.


	When formulating biopharmaceutical drug products, it is essential that the biologic API have consistent product properties, regardless of the production batch or container in which it was collected following final filtration. Variation in the concentration of product- and/or process-related impurities is of paramount importance as it can affect the safety and/or efficacy of the product, according to Brian G. Turner, senior director of manufacturing sciences and technology with the Specialty Care Operations group within Sanofi. “Product-related impurities such as aggregates can be difficult to control at low levels, and certain process-related impurities can be challenging to reduce to acceptable levels if their physicochemical properties are similar to the desired product,” he notes.


	API consistency begins upstream with cell line selection, according to Tim Hill, director of upstream process development for Fujifilm Diosynth Biotechnologies USA. “If a mammalian cell line is not entirely clonal or is sensitive to small changes in processing controls such as pH, temperature, gassing, and feeds, we typically see wide swings in cell growth, metabolites, and production profiles and impacts on product quality attributes such as aggregates, charged variants, and glycans,” he explains.

	The use of undefined media components such as soy or yeast hydrolysates are particularly problematic for API product quality consistency. Lot-to-lot variability of the undefined components impacts cell cultures more than fermentation processes and typically requires raw-material use testing prior to acceptance into manufacturing, according to Hill. Fortunately, he notes that several media vendors have developed very good chemically defined (CD) alternatives to traditional growth media and feeds for common cell culture applications.

	Isolating a stable cell line with robust growth and protein expression capability is still the major challenge in cell culture, Hill adds. “Because of the increased interest in the manufacture of biosimilar monoclonal antibodies, cell clone selection with a close eye on product quality is even more important to ensure that undesirable qualities such as aggregates, half-antibodies, and high mannose-5 glycan derivatives are screened out of the process,” he says.


	While some products may benefit from maintaining tight control of harvesting pH and temperature, Turner finds that the impact of harvesting on product purity is often minimal for secreted proteins, but can be significant for proteins expressed intracellularly, such as insulin expressed as insoluble inclusion bodies in 

	The harvesting process must, however, provide a relatively uniform in-process material to the start of downstream operations with each production batch. To achieve this goal, according to Hill, requires consistency in processing times to avoid over exposure to proteases from lysed cells and unfavorable physiochemical conditions for the protein. Tighter control of the harvest tank environment to minimize degradation of certain products may also have a positive impact on API purity, according to Turner. “When raw materials or process controls vary too much, the consistency of the harvested material can change, which can impact downstream process performance,” he says. “Clearance of potential contaminants including viruses and chemical process impurities during downstream processing can often be impacted by upstream variability, leading to failure in release specifications related to process safety measures,” Hill continues.

	Some advances in technology are helping biologic API manufacturers improve the harvesting process with respect to achieving more consistent harvest cell-culture fluids (HCCFs). For instance, Turner notes that the use of flocculants to improve flows (throughput) in centrifugation can improve the efficiency and increase the capacity of post-centrifuge depth filters. In addition, charged depth filters can reduce process-related impurities such as DNA and basic proteins. Other technology advances are leading to improved efficiencies while also ensuring product consistency. “High-capacity depth filters that can process high-density cultures without centrifugation provide increased efficiencies, while process intensification (e.g., concentrated fed-batch culture) using alternating tangential flow (ATF) filtration allows for the continuous harvest of clarified supernatant,” Turner notes.

	For Hill, single-use tubular bowl centrifuges represent a key technology development for harvesting. These centrifuges collect cells in a gentle manner to minimize cell breakage. “Because this type of centrifuge enables harvesting at high-cell viability, it allows for lower protease, host-cell protein (HCP), and DNA content in the clarified harvest, and thus better capture column performance downstream,” he observes.

	With respect to future developments in harvesting technology, Turner would welcome advances in depth filter construction that would enable the effective processing of high-density cultures. The key remaining challenge, according to Hill, relates to the need to reduce impurity loads associated with in-process streams; lower impurity concentrations would enable the reduction in size of purification columns. Fujifilm Diosynth Biotechnologies is, in fact, working with 3M to evaluate the capability of 3M’s new Emphase AEX Hybrid Purifier to decrease DNA and HCP concentrations in cell-culture harvests. “This additional filtration technology appears to significantly reduce DNA loads onto Protein A capture columns, thus enabling the capture columns to more effectively separate host-cell proteins from biologic APIs,” comments Hill.


	“A well-designed purification process should deliver a consistent biologic API,” states Michael Murray, director of downstream process development with Fujifilm Diosynth Biotechnologies. Each purification step in the downstream processing of biologic APIs serves a particular purpose, according to Turner. For monoclonal antibodies, he notes that Protein A chromatography effectively separates the product from most process-related impurities. Anion-exchange chromatography, meanwhile, is an excellent method for reducing DNA levels. For some products, hydrophobic-interaction chromatography can reduce aggregates to acceptable levels. Nanofiltration is typically included in modern purification processes as a dedicated viral clearance step.

	“If a process is challenged to obtain multiple logs of clearance of an impurity, then multiple removal steps are often required not only to meet the needed quality attributes but also to ensure process robustness, which leads to product consistency,” Murray observes. If the demand for clearance is lower, he notes that a single step can often be designed to ensure that quality attributes are met, but process robustness must be built into the step, either through thoughtful design or characterization of the design space.

	Advances in chromatography technology have been crucial to the improved performance of downstream purification operations. “Chromatography manufacturers continue to increase their offerings of different functional groups and bead structures from which a process development scientist can choose. For instance, resins are now available with increased capacity, improved selectivity, and tolerance to a wider range of conditions (i.e., salt tolerance),” Murray notes. A wider range of membrane chromatography options are also available that are enabling more optimized purification processes.

	There remains, however, growing pressure to increase the throughput of downstream processes to match upstream improvements in productivity and cell-culture titers. “The need is particularly true for monoclonal antibodies that are approaching commercial manufacturing. The challenge for development, engineering, and manufacturing will be to create unit operations that consistently achieve the desired quality attributes for a given step, but at a higher output,” says Murray.

	Further advances in downstream purification technologies could have positive operational and financial impacts and also influence biologic drug substance quality and consistency, according to Turner. These advances include low-cost alternatives to Protein A chromatography (e.g., synthetic affinity ligands), high-capacity membrane adsorbers to replace column resins, spray drying or bulk lyophilization of APIs, and room-temperature storage of APIs for increased shelf life. Murray adds that the combination of multiple unit operations into a continuous mode shows promise for improving throughput while still maintaining quality.

: 
	When referring to this article, please cite as C. Challener, Upstream and Downstream Operations Can Impact Biologic API Uniformity," 

Bioprocess operations-from cell line selection to final filtration-can influence the consistency and purity of biologic drug substances.

Upstream and Downstream Operations Can Impact Biologic API Uniformity

Solid-dosage drugs remain the preferred dosage form due to ease of administration and typically lower manufacturing costs compared to parenteral and other dosage forms. Many of the properties of a chemical relate directly to its physical form, and solid compounds can adopt numerous different crystalline forms (e.g., polymorphs, solvates, and hydrates) and non-crystalline (e.g., mesophases or amorphous) forms. Extensive screening must be completed to identify the different potential stable and meta-stable forms that an API may adopt during manufacturing of the drug substance and drug product, packaging, storage, and within the body. Potentially stable salts and cocrystals of the API may also be evaluated. In addition to the skills and knowledge required to conduct and evaluate the large quantities of structure data generated during polymorph screening projects, the ability to design appropriate screening studies has a direct impact on their success.

Toxicology, efficacy, and stability are important criteria when selecting an appropriate candidate for the development of a solid-dosage drug. “These properties can vary depending on the physical structure of the API. It is necessary to understand the physical properties of each potential solid form and the relationship between these different forms in order to identify the candidates with the greatest likelihood of success,” says Brett Cowans, director of materials science for SSCI (Solid State Chemical Information), a division of AMRI.

	The chances of selecting the best candidate improve considerably the earlier these factors are known. “The challenge for companies is to balance the cost of development and risk of selecting the wrong solid form. Ideally, this balance will include understanding the true physical properties of the selected candidate compound, including polymorphism, and how these properties will affect the API manufacturing and drug product efficacy and stability for each form, as well as the intellectual property position for the formulated drug,” Cowans observes.

From a technical point of view, the main challenge of polymorph screening is to identify all crystalline forms relevant and potentially crucial to development. Solid forms may, for example, be observed in the presence or absence of certain impurities in the final isolation step of the API, upon storage of its intended dosage form, or 

 when administered orally, according to Patricia Andres, director of particle engineering for SSCI. “The screening of such forms typically requires a rational design of experiments often not amenable to high-throughput screening, and the unambiguous identification of new phases necessitates the preparation of pure phases in sufficient quantities for characterization,” she notes.

	Andres cautions, however, that while thorough and targeted screening will considerably reduce the chances that a new critical form is discovered later in development, it is not possible to know without a doubt that all relevant forms have been found. She cites ritonavir as a classic example of a crystalline form not discovered during development. The product was developed using a form designated as Form I, which was later found to be metastable with respect to a new form exhibiting considerably lower solubility and rendering the initially developed drug product bio-unavailable.

“The ability to gauge the level and type of screening that is adequate based on considerations of development phase, dosage form, physicochemical and biological properties of the compound, and regulatory requirements is paramount,” Andres asserts. Risks associated with the discovery of a new crystalline form at a later phase of development must be weighed against the possibility of the compound failing in development. This risk assessment requires both experience of solid-state issues that can affect the performance of the drug substance and drug product and intimate knowledge of the drug substance and drug-product processes.

	Another practical challenge in polymorph screening, according to Marco Gil, a general manager with Hovione, is to identify processes for isolation of different forms that can be scaled in a robust manner to yield the selected polymorphs (or cocrystals).

Polymorph screening is a complicated process that involves many individual experiments and significant data analysis. Significant resources have been invested in efforts to accelerate the process. The dream of displacing rational experimental design in pharmaceutical form screening (e.g., salts, cocrystals, and polymorphs) with high-throughput screening was pursued for about a decade by mid-sized and large pharmaceutical companies, as well as material science contract research organizations (CRO), according to Jon Selbo, director of preformulation with SSCI. “Significant internal resources were dedicated to developing home-built screening instruments and/or purchasing equipment and software to implement this new strategy,” he says.

	The justification for high-throughput screening was that significant improvement in identification of otherwise unknown forms could be more easily obtained by conducting large arrays of experiments in parallel. In most cases, however, this strategy failed for both identification of new forms and desired forms for development. “In general, crystallization quality was poor, there was a lack of experimental diversity (regardless of significant increases in solvents used in crystallization attempts), the analytical results were inadequate, and analyzing the large quantities of poor quality data generated in the experiments often proved difficult,” Selbo observes. In addition, polymorphs identified using high-throughput screening must be considered as general lead compounds and must be thoroughly assessed, including evaluation of their pharmacokinetics, solubility, and other properties, according to Gil.

	The driver for large-scale screening in early development has, for the most part, also disappeared with the new molecular entities failing more often today due to efficacy and toxicity issues rather than biopharmaceutics properties. “Only a few larger pharmaceutical companies and small CROs continue to employ high-throughput screening as a first pass strategy,” says Selbo.

	What is important, according to Cowans, is how the results of high-throughput screening can contribute to better understanding of the properties of potential solid-form candidates. “As a screening tool, high-throughput technology provides a platform to evaluate the potential for crystallizing new solid forms under certain conditions, albeit with some limitations. The information obtained solely from high-throughput technology is not sufficient to select a solid-form candidate for development, however,” he says. Consequently, rational experimental design with fit-for-purpose screening strategies based on rational screening or mixed screening approaches have become the norm for the industry, according to Selbo.

	Large-scale screening is still employed as a part of intellectual property (IP) strategies for later-stage programs where there is more certainty of compound survival. “High-throughput screening is today an essential technology for mapping polymorphic forms, generating IP, and broadening the protected space in terms of polymorphs, and helping to avoid surprises as much as possible in advanced development phases,” Gil comments. For these programs, mixed screening services with rational, mid-, and high-throughput screens may be employed in an attempt to cover all bases, according to Cowans.

With respect to advances in technology for polymorph screening, Cowans points to improvements in x-ray powder diffraction (XRPD), which he refers to as the “quintessential tool for distinguishing different crystalline forms.” Most importantly, current-generation instruments are capable of providing the sharp, high-quality patterns that are necessary for identifying whether a material exists as a single crystalline phase and are amenable to indexing algorithms.

	The combination of indexing software and advanced XRPD technology improves the ability to identify crystalline phases or mixtures, increases screening efficiency, and provides critical information about the various crystalline forms obtained, according to Cowans. SSCI has developed and patented its own indexing software specifically targeting organic molecules and integrated this tool into its routine screening activities.

	Another important advance for polymorph screening is the reduction in the quantity of API required for analytical techniques (e.g., XRPD), according to Gil. Automation has also increased the efficiency of these analyses and accelerated sample pre-characterization.

Determining the appropriate amount and type of screening challenges drug developers when identifying relevant crystalline forms.

Identifying Relevant Crystalline Forms


	When referring to this article, please cite it as C. Challener, “Conjugation Chemistry with Highly Potent Compounds," 

Manufacturing highly toxic compounds in a biopharmaceutical environment tests equipment and systems.

Conjugation Chemistry with Highly Potent Compounds


	When referring to this article, please cite it as C. Challener, " Polymorph Screening for Identification of Relevant Crystalline Forms," 

Gauging the adequate level and type of screening is the challenge when identifying relevant crystalline forms.

Polymorph Screening for Identification of Relevant Crystalline Forms

Biopharmaceutical manufacturing involves a series of complex unit operations linked together to provide high-purity, biologic actives with specified physicochemical and pharmacokinetic properties. The development of commercial-scale processes that consistently provide high-quality product that meets those specifications requires extensive examination of numerous process variables and potential process variations. Conducting the required number of studies on the commercial scale is not practical; scale-down models are therefore employed to determine optimum conditions for downstream separation processes, including chromatography and viral clearance steps. Qualification that such models provide results truly representative of commercial-scale processes becomes necessary as molecules move closer to commercialization.

A scale-down model is a bench-scale process designed to predict the results that will be obtained when the process is run at commercial scale. Unlike initial processes that are used during the discovery and early development phases to produce material for characterization, efficacy, safety, and other studies, scale-down models are intended to aid in optimization and characterization of the process that will be implemented for manufacture of large quantities of a biologic once the product has been approved.

	While process simulations are an ideal surrogate, given the complexity and inaccuracy of many theoretical models, there often remains no substitute for a physical scaled-down model, according to Adam J. Meizinger, a process engineer in Genzyme’s Purification, Manufacturing Science, and Technology Laboratory.

	Scale-down models are used in the development of nearly every unit operation in the overall biopharmaceutical manufacturing process, including both upstream and downstream steps, and separation and filtrations steps, such as chromatography and viral filtration processes. A scaled-down chromatography process, for instance, is designed considering the residence time, flow rate, and column bed height of the large-scale process. Additional parameters that are considered include buffer preparation and feed stream. “The ideal model behaves identically to the scaled-up process such that the results obtained in the lab exactly predict the results that will be obtained in the manufacturing plant,” says Paul Jorjorian, director of global technology transfer for Patheon. Because they are models, however, such a situation does not occur. The goal, therefore, is to mimic the commercial-scale process as closely as possible.

The quality-by-design (QbD) approach to process development requires characterization of the impacts of different process parameters on product critical quality attributes (CQAs), which in turn necessitates considerable experimentation, according to Meizinger. “While the consideration of up-front risk assessments, access to general scientific understanding, and the use of design-of-experiment (DoE) methodology minimizes the required number of runs and maximizes the informational output, it is impractical to conduct such characterization experiments at full scale. Qualified scaled-down models are instrumental for conducting such characterization work, as well as for viral clearance studies, continuous process improvement, and for conducting impromptu investigations in support of large-scale operations, such as when deviations occur,” he adds.


	While ideal scaled-down models correlate exactly with their large-scale counterpart processes, generally a perfect match is not achieved between the bench and commercial scales, and therefore, model processes are not perfectly scalable. The causes, according to Jorjorian, may not relate directly to the process, but can be attributed to small, nuanced differences that still have an impact on the results at different scales. “The issues are often unavoidable and thus must be addressed by developing correction factors and/or transfer functions that enable the accurate prediction of the behavior of manufacturing processes,” explains Jorjorian. “It is important, however, to minimize the need for correction factors and transfer functions, and for those that are required, provide thorough justification and clearly demonstrate their suitability,” he adds.

	Another aspect of scale-down models that can create issues when predicting large-scale behavior is the fact that models are qualified at the center point of the process design space. “This approach can lead to discrepancies in predicted and actual results when the model is applied to the entire design space,” Jorjorian observes. He adds that timing and planning of modeling runs is also important and can negatively influence results if not done effectively. For example, it is important to use the same feed used for production runs in scale-down modeling runs; therefore, modeling runs must be scheduled when the feed is available. In many cases, that isn’t possible, and the feed may need to be frozen, which introduces a new variable that can impact the reliability of the model.

	Specifically for chromatography operations, some key considerations in developing scaled-down models are minimizing the differences in the extra-column volume, which varies considerably with scale, contending with increasing wall effects inherent in small-diameter chromatography columns, and accounting for differences in detector-specific path lengths and the associated non-linearity of detector output, according to Meizinger. He also notes that some steps (e.g., gas overlays for intermediate holds) may be challenging to implement as part of scaled-down model development, and mixing dynamics and heat transfer often remain different between scales. “The impact of these differences must be carefully assessed for their resulting impact on scaled-down model performance,” he says.

	Scale-down modeling of membrane chromatography processes can also be difficult, according to Jorjorian. The reason often relates to the designs used for small- and large-scale membranes; they are often different, with large-scale membranes having added flutes or pleats or other features that affect their performance such that it is difficult to develop bench-scale processes that can reliably predict production-scale outcomes.

There is nothing new about the use of bench-scale process models during the development of biopharmaceutical manufacturing processes. What has become an important trend in process development is the need to demonstrate that scaled-down models truly represent their large-scale counterparts. “It has only recently become standard practice to demonstrate that scaled-down models provide results that are statistically equivalent to those obtained from commercial-scale versions of the same processes,” states Meizinger.

	Often during earlier stages of development, even through Phase I clinical trials, scale-down models of downstream biopharmaceutical processes are generally not formal, qualified models. “For these models, the basis for use of each model and the rationale for why it is expected to be representative of the process at large scale are documented,” notes Jorjorian. As a molecule progresses toward commercialization, however, formal, qualified models are required. The ability of the model to predict results at large-scale must be demonstrated by performing at least triplicate runs. The use of any correction factors and/or transfer functions must also be justified and their application demonstrated to provide robust results.

	Qualification of most models for chromatographic separations is fairly straightforward, according to Jorjorian. “There is a lot of documentation and analyses to complete, so in essence, qualification of scale-down models is often an exercise in coordination, rather than presenting a substantial technical challenge,” he observes. Meizinger agrees that the use of statistical equivalence for testing quality attributes and process performance indicators between scales is generally not an issue. “Demonstration of both individual unit and linked unit output (recovery, product quality attributes, product purity, chromatographic profiles/transition analysis, etc.) comparability between small and large scales remains both practical and accepted,” he states.

Qualification of scale-down models can be challenging, however, when very small column sizes are involved. There is significant interest in using high-throughput approaches for scale-down modeling to speed up development times. There are, however, technical issues that arise due to the different behaviors observed in very large and very small columns, such as the wall effects mentioned previously. 

	“It is definitely more challenging to establish robust scale-down models using high-throughput methodologies because typically transfer functions and correction factors are required. As a result, it is more difficult to adequately demonstrate the scalability of the models to the level required for qualification,” Jorjorian says. He adds that there is a lot of effort being devoted to overcoming these issues to enable high-throughput modeling of downstream bioprocesses.

	A few groups are also investigating the application of first principles calculations to the scale-down modeling of both upstream and downstream unit operations, and particularly for chromatography, for which there is significant understanding of separation behaviors and existing mathematical models. “This approach to scale-down modeling is in the earliest phases and largely limited to large pharmaceutical companies and university research groups with the resources and computing power required,” Jorjorian says.


	“Despite the limitations of existing scale-down modeling methods, the use of scaled-down models is recommended wherever possible, including for process characterization, viral clearance studies, continuous improvement, manufacturing-related investigations, evaluation of changes in raw materials, and so on, barring only validation efforts and clinical trial/commercial manufacturing requirements for performance of large-scale runs,” asserts Meizinger. “Today, scaled-down models serve as efficient and cost-effective representations of full-scale processes. While the inability to demonstrate equivalence between scaled-down and large-scale operations is often inevitable for some metrics, further understanding of these scale-related differences, particularly through the development/refinement of associated engineering models, promises to continuously improve the accuracy and utility of scaled-down models,” he states.

Scale-down modeling accelerates the development of downstream biopharma manufacturing processes.