Dynamic binding capacity (DBC) indicates the quantity of a specific biomolecule that can be bound during a chromatography purification step under a specific set of process conditions. Determination and optimization of this value facilitates the development of efficient and productive chromatography operations. While the same general method is used to determine the DBC for many different biomolecules, matrices, and chromatography operations, all of these variables can influence which process parameters are important. Thus, there are many different factors that must be taken into consideration and challenges that must be overcome when optimizing DBCs and ultimately chromatographic performance.

Column binding capacity can be measured in static or dynamic mode. Static measurements performed in a beaker, tube, or 96-well plate provide information on the total amount of protein that a given amount of chromatography media in a given volume of solvent can bind for a given protein concentration. Excess protein is typically used to ensure maximum binding.

DBC is determined under operating conditions (packed column or equivalent membrane/monolith device operated under flow) and is thus much more representative of actual process conditions and directly comparable to large-scale processing. Adding flow decreases the time the molecule has to diffuse into a pore at a given point as it traverses through the column, says Nathan Zeller, scientist II, process development in the Pharma Services business of Thermo Fisher Scientific. That adds a barrier to diffusion that is not present under static binding conditions. As a result, static binding capacity measurements do not characterize frontal binding characteristics that occur along the bed length of a packed column or membrane/monolith device, according to Tom Elich, manager, manufacturing, science, and technology purification process engineering, MilliporeSigma.

“DBC measurements take into account the fact that flow can greatly influence mass sorption (the capture and release of material onto solids/resin),” adds Jacob Brockerman, associate development scientist, AGC Biologics. Static binding capacity measurements also do not take into account that flow rates and other conditions are constantly changing, information that Daming Huang, development scientist, AGC Biologics, says is crucial for optimizing column performance and ensuring that the desired level of protein purity is achieved.

Furthermore, because they are performed under the conditions expected to exist during actual production and under the same specific column controls, DBC measurements provide needed information about the effectiveness of manufacturing controls over a wide range of parameters, including column performance requirements, column volume, and process flow rates or residence time, among others, notes Noah Vartanian, associate development scientist, AGC Biologics. “Designing a purification process based on data from a static binding capacity may lead to rude surprises when the scaled-up column needs manufacturing controls exerted on it, making the purification process perform outside the static binding capacity data,” he comments.

Another benefit of using DBC measurements is the fact they allow for more accurate determination of loading capacities and target protein breakthrough profiles, according to Elzbieta Pawlowska, research scientist, Astrea Bioseparations. “That means chromatography purification steps can be optimized to avoid overloading and subsequent loss of target protein,” she remarks.

Static binding capacity can, observes Tomas Björkman, senior scientist, R&D, Bioprocessing, Cytiva, be used for modeling a dynamic process, but the one measured static capacity result may not match with the capacity obtained in a flow process. “The DBC is lower than the static binding capacity,” he explains, “because of the diffusional limitations associated with chromatography resins. It takes time for the target molecules to penetrate the pores to the binding sites inside the particles.”

Alexandre Boulbrima, program technical lead, Charles River Gene Therapy Solutions, argues, however, that static binding capacity can sometimes still be used to highlight specific chromatography phenomena such as protein displacement. This behavior is characterized by an increase in binding capacity when overloading the column and is driven by the protein/compound of interest having a greater affinity to the resin than other impurities, resulting in displacement of those impurities. “Such behavior can be hard to predict using a DBC characterization approach unless previous data suggest otherwise,” he notes.

The breakthrough approach most common for determining DBC

At its simplest, DBC measurements involve loading a packed column with a known amount of sample at a set flowrate and buffer conditions (i.e., pH, conductivity) that facilitate binding of the target molecule, which, depending on the application, could be the product or an impurity. Load flow-through is collected from the column outlet, and the concentration of the target molecule is measured over the course of loading. When the concentration of the target molecule in the load-flowthrough (C) is the same as that in the feed (Cf), 100% breakthrough has occurred. The data are used to generate a breakthrough curve with load flow-through concentration (Y-axis) plotted as a function of volumetric or mass loading (X-axis). The DBC is commonly defined as the mass loading at 10% breakthrough (C/Cf = 0.1), according to Elich.

The sample loaded on the column may be pure or purified target molecule, notes Björkman. For Zeller, using representative load material is the best approach. “Harvest cell culture fluid (HCCF) is best for evaluating a Protein A affinity resin for the initial capture of a monoclonal antibody (mAb). If evaluating a resin for the subsequent bind-and-elute polishing step, such as cation exchange (CEX), then the capture elution that has been pH- and conductivity-adjusted would serve best,” he explains. Taking this approach will, Zeller says, provide the most accurate results because biomolecular impurities can affect binding of the target molecule.

In fact, Boulbrima contends that the best methods are often limited by their analytical throughput. “Ideally, a fractionation of the load flow-through should be performed, and samples should be analyzed to probe any potential breakthrough of material, indicating the maximum DBC has been reached. Nevertheless, it is important to note that the precision of this DBC value will be highly influenced by the assay precision and overall resolution of the fractionation,” he observes.

The fractionation resolution (the number of fractions to be collected per unit of volume), Boulbrima adds, should ideally not exceed the assay resolution, as it will be difficult to distinguish actual variations between samples versus the assay noise. There is often a tradeoff, however, with the greatest resolution not always being worth the cost, manpower, or time. “Ultimately, the ‘best’ methods are often a compromise of resolution (limited by the assay level of quantification, overall assay noise, etc.) and time,” he concludes.

Zeller agrees that the “best” method to evaluate or determine a DBC will depend on the resources available. “If there is a fast method for accurately analyzing concentration or titer that is readily available, then that would be the best method,” he states.

Impact of residence time for different media types

Regardless of the chromatographic media type—conventional resin beads, monoliths, nanofiber adsorbents, cast membranes, and hydrogels, the general approach for determining DBC remains the same.

The complication for non-resin-based adsorbents, according to Ian Scanlon, subject matter expert, downstream processing, Astrea Bioseparations, is specific to the device which is used to house the material, where the dead volume in the unit is generally much larger compared to a packed bed adsorbent column. “The dead volume must be accounted for in order to accurately measure the capacity of the material for scale-up purposes,” he says.

Dead volume, Scanlon continues, can be determined using a salt transition or non-interactive UV analyte, whereby the measurement of the 50% point of the transition is generally accepted as the dead volume of the housing unit and membrane bed. This value can then be used to determine the breakthrough curve.

Another factor that must be explored when using different media types is the impact of the residence time, which is related to the flowrate. “Binding capacity for resin beads is dependent on diffusive mass transfer, and it is common to characterize binding capacity at several residence times in the range of three to six minutes for bead-based technologies. For membranes and monoliths, which are more dependent on convective mass transfer rather than diffusive mass transfer, the residence time parameter is less critical,” Elich observes.

For many novel adsorbents, notes Scanlon, residence times can actually be significantly reduced. For instance, the residence times for the nanofiber adsorbent developed by Astrea range from 1 to 12 seconds compared to minutes for resin-based technologies. For this reason, Scanlon strongly recommends consulting manufacturer recommendations for flow rates and residence times to use during DBC determinations. Zeller also cautions that the flow rates for monoliths and membranes are typically near the limit of the fraction collection speed, which can create other challenges.

Potential for competitive binding of impurities

The nature of the biologic substances in the feed stream for a chromatographic purifications step will naturally have an impact on the DBC. They can also influence the DBC determination. The biggest concern that must be addressed is the potential for competitive binding effects between the product and impurities present in the feed. If competitive binding occurs, the DBC can be reduced, according to Elich.

“The complexity of feedstocks should be accounted for with all biologicals, but particularly where competitive binding is possible,” adds Scanlon. He notes that the issue is less of a concern with affinity interactions (e.g., when using proteins A, G, and L) because they are highly specific, but for many of the newer targets these types of affinity ligands are not yet commercially available.

Competitive binding occurs when two or more biomolecules have similar physicochemical properties. Elich uses plasmid DNA and RNA as one example. In this case, the molecules have similar sizes and isoelectric points, which creates the risk for reduced plasmid DNA binding capacity when using anion exchange resin. “If the chromatography conditions are not properly optimized, RNA impurity will compete with plasmid DNA for binding sites, limiting available capacity for product binding,” he says.

Ion-exchange chromatography for viral-vector platforms suffer particularly from competitive binding issues, according to Elich. “Many process- and product-related impurities feature acidic isoelectric points that are comparable to the viral vector product, posing risk to competitive binding effects. This makes optimization more challenging as compared to traditional mAbs that feature a basic isoelectric point.

“In these cases, it is important to determine loading volumes with the appropriate feed rather than using a purified target, as this may lead to overestimation of capacity,” Scanlon states. He adds that it is useful to ascertain if the loading conditions can be optimized to avoid binding as many of the present impurities as possible. “A salt concentration and pH screen at the DBC stage can lead to better capacity for the target and cleaner elution pools,” Scanlon contends.

Another important point to remember, Boulbrima emphasizes, is that the product of interest might not always be the determinant of the DBC. “In the case of a polishing step using a flow-through method,” he explains, “the product of interest will flow through by design, whereas impurities will remain bound to the medium. Sizing can then be achieved through targeting the maximum DBC of the impurity(ies) screened for removal.”

Additional considerations needed for new modalities

Finally, Scanlon notes that DBC determination for novel therapeutic modalities for which quantification of the target is not possible using UV alone (e.g., viral vectors, vesicles, etc.) can be problematic. “In these cases, incremental collection of the flowthrough fractions during load and offline measurement of concentrations of the target can be used to determine the breakthrough and calculate the DBC per unit volume,” he says.

Similarly, obtaining DBC values for new mAb-derivatives such as bi/tri-specifics is not as straightforward as it is for conventional mAbs and recombinant proteins, according to Zeller. “The mismatched mAb impurities (homodimers, half mAbs, mismatched light chains, etc.) tend to exhibit similar binding characteristics to the target molecule. It is therefore harder to get a DBC value that maximizes the binding of the molecule while reducing these specific impurities.

As importantly, Scanlon notes that stability of novel therapeutics is often an issue, and thus collected flowthrough fractions should be treated appropriately to avoid inaccurate quantitation.

Selection of monitoring methods important

The methods used to monitor breakthrough must be appropriate for the type of chromatography being performed. “The principle for determination of DBC is independent of the target molecule, but the analytical methods required for detection of breakthrough may differ,” notes Björkman.

For instance, for viral-vector purification, there is a debate about which assay to use for DBC determination and optimization. “In the case of lentiviral particles, both total particle titer (often achieved via an ELISA [enzyme-linked immunosorbent assay] against the p24 capsid protein) and infectious particle titer (cell-based assay) could be used, but both have displayed great levels of inconsistency so far,” Boulbrima comments.

Methods typically differ for capture vs. polishing steps as well. “HCCFs are full of impurities, so a titer method is typically required to get the concentration values for breakthrough analysis for capture steps. For cleaner material loaded onto polishing resins, however, a microplate spectrophotometer can often be used to get the concentration values of the effluent fractions,” Zeller comments.

Elich agrees.“Because of the low feed purity for capture steps, the UV signal at the column outlet can become saturated with unbound impurities (i.e., host-cell proteins [HCPs], DNA, and cell debris), making UV absorption an imprecise measurement tool for this application. A better method might be to collect fractions for concentration measurement by offline high-performance liquid chromatography (HPLC) analysis.” For post-capture polishing steps, however, online UV absorption might be a viable analytical tool for breakthrough curve assessment.

Here again, it is important to remember that for polishing steps in flow-through mode, the goal is to understand impurity binding capacity as opposed to product binding capacity. “Molecule-specific analytical tools should be used to construct breakthrough curves of the target impurities that bind the chromatographic matrix as the product flows through (i.e., HCPs, DNA, virus, product-related impurities, etc.),” Elich says. Typically, ELISA or HPLC methods are used for offline analysis.

Competitive binding not the only challenge

While competitive binding is generally considered the biggest challenge to optimizing DBCs, there are other issues that must be addressed. For instance, Björkman notes that the binding capacities of affinity steps are usually more robust within a certain pH and salt range and are therefore not necessary to optimize in the same way as non-affinity steps, which require careful selection of pH, salt concentration, and salt type. Boulbrima adds that capture steps are also usually robust with respect to the compositions of the feed streams employed, while the performance of ion-exchange and hydrophobic interaction chromatography steps can often be far more influenced by the feed stream quality and composition.

Elich also points out that it is important to consider the impact of media chemistry on DBC determinations. Binding capacity experiments may be designed differently for affinity, ion exchange, and hydrophobic interaction technologies, for instance. As an example, he notes that the DBC for affinity chromatography is less dependent on conductivity, whereas that for ion exchange and hydrophobic chemistries is heavily dependent on this feed property.

Using dilute crude samples is also more difficult than using purified material, according to Björkman. “Often when using a crude sample, you need to collect fractions and perform off-line analysis and that adds to the work needed and also to the uncertainty,” he explains. Björkman also observes that limited access to sample material may require use of miniaturized techniques, small columns, or even batch experiments to estimate binding capacities.

In addition to the flowrate, Gráinne Dunlevy, R&D director delivery, Astrea Bioseparations, points to the importance of considering the delta column pressure, which increases with flow rate for traditional adsorbents. This issue is not a concern for next-generation matrices such as nanofibers because there is little back pressure, which is one of the reasons higher flow rates are possible.

Another challenge noted by Zeller relates, once again, to whether the operation will be run in bind-and-elute or flowthrough mode. Bind-and-elute is generally easier, because monitoring involved detection of the single target molecule. Flowthrough mode can be trickier, he observes, because of the heterogenous pool of impurities, which typically requires a cell line-specific protein detection assay, such as an ELISA for host cell proteins. If the flowthrough operation will be claimed as a viral clearance step, then it is also necessary to determine how much material can be loaded without virus breakthrough.

Optimizing DBCs can be achieved using a variety of approaches. In some cases, static binding experiments performed in 96-well plates are useful as a first step because they enable high throughput screening of relative binding capacities for a wide range of traditional resins and/or buffer conditions, according to Elich. He also notes that more recently predictive modeling software has emerged for optimizing the performance of a chromatography step using mechanistic and thermodynamic models.

Screening is helpful because optimization of the DBC requires identification of parameters that affect it and then optimizing those parameters, according to Brockerman. “The specific set of important parameters varies from process to process (resin vs membrane, affinity vs ion exchange, etc.), but typically include load buffer composition (pH, salt, etc.), load concentration, flow rate, and residence time. Given the number of parameters, Brockerman notes that multivariate analysis (design of experiment [DoE]) approaches have proven exceptional for efficiently identifying relevant parameters and their optimization.

Ben Galarza, research assistant, Astrea Bioseparation, adds that the DoE approach is applicable across all media and purification steps and is a powerful tool because it allows selection of both numeric and categoric factors. “For instance,” he observes, “different types of media can be input as categoric factors alongside any numeric factors if there is uncertainty as to which media to select.” He does note that DoE strategies may be more relevant for the development of chromatography processes for new modalities.

In addition to optimization of typical process conditions, other strategies highlighted by Huang include column packing and particle size optimization, use of the appropriate column conditioning steps, such as equilibration, and column scaling to the appropriate size. “These strategies are applicable across different media, purification steps, and drug substance types, although the optimal approach may vary depending on the specific needs of each purification process,” he says.

More general approaches to optimizing the dynamic binding capacity of chromatographic purification steps in biopharmaceutical manufacturing noted by Huang include the use of multimodal chromatography, which can increase the DBC while reducing the number of chromatography steps; membrane chromatography, which can significantly increase the DBC while reducing the amount of resin required for purification; continuous chromatography (vide infra); and automated purification systems, which can increase the DBC while reducing risk of operator error and increasing process consistency and reproducibility.

Zeller agrees that using a liquid handling robot capable of performing chromatography is the most efficient method for optimizing the loading conditions that then effect the DBC, especially during complete development of a polishing step. “This approach can screen multiple resin types, buffer conditions, and molecules in one programmed method and can usually be complete in less than 24 hours compared to
days or weeks using a conventional fast-protein liquid chromatography instrument,” he says. Zeller does note that these systems are not yet compatible with membrane chromatography.

The scaling of chromatographic purification steps can impact the DBC in many ways. “As processes are scaled up, certain physical parameters (such as decreased capillary action at large scale) can start having a much greater influence on the chemical interactions in the exchange between static and mobile phases,” Boulbrima explains. This behavior, he says, can potentially be predicted for a specific biomolecule and a specific chromatographic support, using either a robust scale-up characterization strategy or a robust small-scale model qualification strategy and/or historical data at all scales. “The latter will especially be key for continuous improvement of the platform, knowledge gathering, and outlier characterization,” he contends.

Maintaining an optimum DBC when scaling chromatography steps is generally possible if the bed height and residence time are kept constant by increasing the column diameter, according to Björkman. “While there are some cases where the residence time is of no importance within a reasonable range, maintaining the same residence time used for determination of DBC as processes are scaled is always a good rule of thumb,” he says.

A properly designed scale-down tool should enable consistent binding capacity between small- and large-scale operations, agrees Elich. In practice, he notes that it is common to employ a safety factor to account for any process variations between scales, resin lots, cleaning cycles, or product batches. As an example, he notes that during operation, a chromatography matrix may be loaded only to 80% of the DBC measured at 10% breakthrough (80% of DBC10%).

Dunlevy agrees that because the DBC for most chromatographic processes depends on the residence time, keeping it constant during scale up is important. She suggests, however, that it is possible to keep the bed height and linear flow rate constant or adjust for any increase in the bed height by increasing the flow rate. The downside of these approaches, Dunlevy notes, is that increasing the flowrate and bed height lead to higher delta column pressures, and thus changes are limited by the properties of the resin being used. Newer, alternative chromatography matrices such as fiber have an advantage here, because they can be kept at very short bed heights with low delta column pressures and thus can be scaled more easily.

Running chromatography steps in continuous mode does not have an impact on DBC determination and optimization. “Continuous chromatography with columns operated in overload mode (i.e., a second column collecting the target flowing through the first column) will not complicate the optimization,” remarks Björkman. “Conditions are optimized using the first column and then depending on system, the operation can be quite simple, especially if on-line sensors such as UV-cells are placed before and after the column and software controls the flow path,” he adds.

Loading multiple columns in series allows for not only continuous loading, but a DBC that is closer to the media’s static capacity and without sacrificing yield, according to Elich. In fact, it enables use of the full DBC of the column, remarks Dunlevy. “In standard batch chromatography, loading is typically to 80% of the DBC to minimize any breakthrough during the loading phase. That decreases the yield and prevents leveraging of the full DBC of the column,” she says. Elich notes that typically only 50–60% of the static binding capacity is used in batch chromatography. In continuous chromatography, because the breakthrough is loaded onto the next column in series, reaching nearly 100% of the DBC of the column is possible, Dunlevy adds. In the end, continuous chromatography actually lessens the importance of the DBC due to the efficiency of the processing, observes Zeller.

In fact, Zeller emphasizes, one of the major drivers for increasing binding capacity is to reduce the number of cycles needed to purify a product. “Since the simulated moving bed process is quick and efficient, multiple cycles can be completed in the time it takes for one conventional batch chromatography unit. Therefore, DBC is less important in that aspect,” he comments.

For large-volume processes and/or when cycle count is important, however, Zeller stresses that high DBC values help to concentrate the product and reduce the number of cycles that would otherwise be needed, which results in a lower buffer footprint.

There are some caveats. Design of continuous chromatography operations requires accurate characterization of the complete breakthrough curve profile during DBC assessment, which according to Elich is best accomplished using a column bed height representative of process-scale operations (typically near 10 cm). The life cycle of the chromatographic support (resin, monolith, others) must also be characterized and the right cleaning procedure put in place, Boulbrima states. These steps can be achieved via a resin-reuse study, which would not differ much from what is required for conventional processes using chromatography multi-cycling.

Developers of chromatography purification processes for biologic drugs should think about a few other aspects of development activities to ensure greater likelihood of success. For instance, the DBC for any specific biologic should be determined at the start of a process and characterized over as many of the variables present in the process as possible, according to Scanlon. In addition, if any part of the process is changed at any time, reevaluation of the DBC should be performed to identify any impact of the change. Furthermore, the DBC should be checked during lifetime studies that reflect the use-case of the adsorbent.

Specifically for polishing (IEX, HIC) steps, Zeller recommends that the load material be adjusted to the pH and conductivity conditions of the first wash buffer to mitigate product loss with buffer matrix transitions.

Finally, Elich notes that DBC is only one factor considered when optimizing bioprocess chromatography steps. Purity and yield are two other important factors, and it is possible that conditions that provide an optimal DBC do not optimize purity and/or yield. “A balance of these parameters must be achieved,” he states.

Björkman agrees that the DBC is an indication of the maximum quantity of the target molecule (or impurity/impurities) that can be bound under certain conditions (buffer composition and column residence time), while the optimal load value also takes the desired purification objective into account. “If the chromatography step is used to remove impurities that also bind to the column, it may be necessary to reduce the loading significantly below the DBC of the pure target molecule. Consequently, the column load is often one of the optimized factors for a purification step,” he concludes.

Cynthia A. Challener, PhD is a contributing editor to 


Vol. 36, Number 4
April 2023
Pages: 10-14, 34

When referring to this article, please cite it as Challener, C.A. Determining and Optimizing Dynamic Binding Capacity. 

Articles

Cover Story

The breakthrough approach is commonly used, but the devil is in the details.

Determining and Optimizing Dynamic Binding Capacity

Hydrophobic interaction chromatography (HIC) is a separation technique widely used for the purification of therapeutic proteins. The main leverages identified to operate HIC processes are solution pH and ionic strength. In this article, the authors show that simplistic mathematical models derived from statistical analysis cannot describe the impact of pH and ionic strength observed in HIC processes. A new mechanistic model accounting for the protein charge in solution (varying with pH) and the solution non-ideality (activity coefficient varying with the ionic strength) is proposed here. This simple model is shown to accurately describe experimental data and paves the way for numerical optimization of HIC processes.

Submitted Sept. 15, 2022
Accepted: Dec. 8, 2022.

Lucrèce Nicoud*, lucrece.nicoud@ypso-facto.com, is head of product; Yohann Le Guennec is project manager; and Edouard Nicoud is head of customer success; all at Ypso-Facto, France. Antonio Cardillo, PhD, is expert scientist & GSK fellow; Elisa Innocenti is scientist; and Alessandro Pieri, PhD, is scientific leader & GSK associate fellow; all at GSK, Italy. Elena Lietta, PhD, is from Politecnico di Torino, Duca degli Abruzzi, Italy.

*To whom all correspondence should be addressed.


Vol. 36, No. 4
April 2023
Pages: 22–29

When referring to this article, please cite it as Nicoud, L.; Le Guennec, Y.; Nicoud, E.; et al. Purification of Protein by HIC: Mechanistic Modeling for Improved Understanding and Process Optimization. 

Peer-Reviewed Research

In this article, a simple chromatographic model is proposed that is capable of predicting the impact of pH and ionic strength on HIC chromatograms.

Purification of Protein by HIC: Mechanistic Modeling for Improved Understanding and Process Optimization

With the explosion of RNA-based therapeutics on the scene, development projects are ramping up to bring these and other nucleic acid-based therapeutics to market. But what exactly are nucleic acid-based drugs and what advantages do they hold over conventional therapeutics (biologics and small-molecule)?

According to Carl Schoellhammer, PhD, principal at DeciBio Consulting, a US-based life science strategy consulting firm, nucleic acid-based therapeutics are drugs that are made up of a string of nucleotides; these include DNA and RNA-based medicines. These types of therapeutics can act at the genetic level to potentially correct or restore healthy function at the level of a patient’s genes.

Vasant Jadhav, PhD, senior vice-president of Research and head of the RNAi Platform group at Alnylam Pharmaceuticals adds that a common feature of nucleic acid-based therapies is that they are “programmable” drugs; a nucleic acid sequence provides the “code” for the intended target. Examples of approved nucleic acid-based therapies include RNA interference (RNAi), antisense oligonucleotides (ASOs), and the now-familiar messenger RNA (mRNA) therapeutics.

RNAi therapeutics utilize a post-transcriptional gene silencing mechanism in which the active ingredient is double stranded short interfering RNA (siRNA). The siRNA targets and degrades mRNA encoded by a disease-causing or disease-associated gene, explains Jadhav. “RNAi therapeutics leverage a naturally occurring protein complex, called RISC [RNA-induced silencing complex], to accomplish gene silencing,” he states.

Meanwhile, ASOs, also a gene silencing medicine, are composed of single-stranded oligonucleotides that hybridize to target RNA and degrade it by “either recruiting RNAseH [ribonuclease H] (so called gapmer design) or modulate splicing to produce desired mRNA (so called splice switching olignucleotides),” according to Jadhav.

Finally, as has been seen with the COVID-19 vaccines, mRNA therapeutics are experimental genetic medicines in which the mRNA itself is the therapeutic entity; it is introduced with the aim of expressing a functional/therapeutic protein or antigen. However, there are currently no approved mRNA therapeutics outside of the COVID-19 vaccines, Jadhav observes.

The COVID-19 vaccines are currently the most well-known type of medicine that utilizes a nucleic acid-based active ingredient (i.e., mRNA). However, as described previously, nucleic acid molecules can be used for other types of medicines (e.g., as gene-silencing medicines).

Schoellhammer emphasizes the fact that nucleic acid-based therapeutics “are a powerful set of medicines with the capacity to turn signals on, or off, at a genetic level.” He points out that there are well over 10 nucleic acid-based therapeutics that are approved by regulators. “These medicines are, broadly speaking, divided by the action they take in the body to either turn signals on or off,” he states.

ASOs, as well as siRNAs, for example, target genes to turn them off or limit their expression. And while most of these approved nucleic acid-based therapeutics are for rare diseases (i.e., small patient population), some have been indicated for vast patient populations: Schoellhammer points to a Novartis product, Leqvio (inclisiran), which received approval by FDA for treating a type of high cholesterol in late 2021 (1).

Inclisiran, Schoellhammer explains, is a siRNA medicine that shuts off a signal in the liver, leading to a lowering of lipids. While it is currently approved for a subsegment of individuals with high cholesterol, he expects that the therapeutic could in the future be applied to anyone with high cholesterol, therefore making these medicines mainstream.

“On the other end of these therapies sits mRNA, which tells the body to produce, or turn on, specific proteins that the mRNA encodes for. [Messenger] RNA is like a blueprint, telling our cells how to make certain proteins. If a certain protein is desired, the corresponding mRNA can be made and delivered so the body will produce it,” Schoellhammer adds.

This is how the COVID mRNA vaccine operates; it is a sequence that encodes for a small fragment of the spike protein in the SARS-CoV-2 virus, for example. “When our body’s cells produce this small protein fragment, they recognize it as foreign, and produce an immune response, just as a traditional vaccine does,” Schoellhammer says.

“The pandemic demonstrated the power of this therapeutic and interest has flourished, with 100s of companies working to create new mRNA-based medicines and well over 200 public programs in development,” Schoellhammer continues. “These programs are tackling a diverse set of applications, ranging from oncology through the development of immune-stimulating treatments to help fight the cancer, to other indications leveraging CRISPR [clustered regularly interspaced short palindromic repeats]-based editing whereby the mRNA encodes for the CRISPR machinery.”

Nucleic acid-based therapeutics hold a definite advantage over conventional biologics or traditional small-molecule APIs. According to Schoellhammer, one of the greatest benefits of nucleic acid-based medicines is their specificity.

“In the case of those that turn off signals, they must find and bind with the exact complementary sequence in our body to turn it off. This means that other genetic signals can be unaffected. Similarly, for mRNA, the sequence, and therefore the protein it encodes for, is controlled. These features greatly limit any off-target effects that are often seen with small molecules or biologics,” Schoellhammer states.

Compared to small molecules and monoclonal antibodies, nucleic acid medicines, such as RNAi therapeutics work “upstream,” asserts Jadhav, treating diseases at their genetic source—that is, at the RNA/transcript level—rather than by addressing the symptoms/manifestations of the disease. Furthermore, Jadhav adds, “small-molecule drugs and monoclonal antibodies have inherent limitations when it comes to the speed with which they can be developed and the type and breadth of diseases they can be used to treat.”

Jadhav points out that the development of small molecules is an arduous process that relies on understanding the structure of the target disease-causing or disease-associated protein (vs. the genetic sequence that encodes that protein). “Oftentimes the structure of the protein is not known and needs to be solved. Identification of the right small molecule requires screening large libraries of chemical compounds to identify a candidate that can interact with the target protein in a ‘lock and key’ fashion. This is a painstaking process that requires extensive trial and error to achieve efficacy, tolerability, and safety,” he emphasizes.

As far as biologics such as antibodies are concerned, Jadhav explains that, while antibodies as a class of medicines have enjoyed much therapeutic success in the past decade, one of the drawbacks here is the inability to access and act on intracellular protein targets. “In contrast to monoclonal antibodies, nucleic acid medicines, such as RNAi therapeutics, can act on disease-causing or disease-associated proteins that are expressed both on the inside and outside of the cell,” he explains.

Meanwhile, Schoellhammer points out a limitation in nucleic acid-based therapeutic development: nucleic acid-based medicines are difficult to deliver, which is a drawback. To work, he specifies, they must be taken up by the right cell in the right place in the body. At the same time, nucleicacid-based medicines are extremely delicate and susceptible to being destroyed in the bloodstream.

“While this can somewhat limit the indications that scientists are using these medicines to treat, it further helps improve these medicines’ specificity and lack of off-target effects. For example, if one uses an siRNA therapeutic to ‘silence’ a target, dosing someone with more of that siRNA should have little effect. This is not the case for small molecules, such as aspirin, where taking more of it could eventually lead to significant, negative side effects,” Schoellhammer says.

Jadhav, meanwhile, explains that nucleic acid-based therapeutics, such as RNAi-based medicines, leverage a platform technology that gives rise to consistent and predictable drug properties. “This is not the case for small molecules, where each individual small molecule compound is completely unique, both in terms of its design elements and its profile as a drug candidate,” he states.

Development of nucleic acid-based therapeutics supports the bio/pharma industry’s shift to precision medicine. For instance, there are examples of nucleic acid-based medicines (e.g., RNAi therapeutics) that are approved to treat rare and ultra-rare genetic diseases for which there were no previously available therapeutic options, points out Jadhav. He has observed the therapeutic landscape of rare diseases dramatically evolve in large part due to the development and availability of genetic medicines, which now have the potential to help patients with more common diseases for which treatment options are limited or inadequate.

Schoellhammer says that, because these medicines are based on genetic sequences, they are highly specific. They can be used to treat rare diseases that are caused by a mutation in our genes. He points out that Biogen’s Spinraza (nusinersen), an antisense therapy for treating spinal muscular atrophy, was a significant accomplishment that has drastically improved the quality of life in patients for whom the disease often presents in newborns and is fatal by age two.

“Beyond presently approved nucleic acid medicines, companies are working on using these therapeutics in combination with CRISPR machinery to enable highly precise edits to genetic signals in patients. In theory, this could one day be used to target a drug for a unique individual,” says Schoellhammer.

Adding to the complex tapestry of nucleic acid-based therapeutics are the different moieties, or species of molecule, within the nucleic acid space. According to Schoellhammer, there are several different types based on their action. Within ASO therapies, for example, there are a variety of strategies to control the “degree” of silencing. There are certain therapeutics, such as Spinraza, for example, that are termed “exon-skipping” because, rather than telling the body to turn off a gene completely, these therapeutics instruct the body to skip only a certain part of a gene when producing the corresponding signal, Schoellhammer explains.

He also emphasizes the existence of microRNA (miRNA), which can have a similar effect to that of silencing therapies, guide RNAs (gRNA), which are used to target a desired gene sequence in the body for editing using CRISPR machinery, and, finally, certain types of RNA medicines that utilize naturally occurring machinery in the body to elicit an editing effect on defective genes. The latter is referred to as transfer RNA (tRNA).

“Beyond this, there is an almost endless further segmentation based on chemical modifications made to the nucleic acid therapy to make it more resilient to degradation by our body or make it more efficient in eliciting its desired effect,” Schoellhammer concludes.

1. Novartis. FDA Approves Novartis Leqvio (inclisiran), First-in-Class siRNA to Lower Cholesterol and Keep it Low with Two Doses a Year. Press Release, Dec. 22, 2021.

Feliza Mirasol is the science editor for 


Vol. 36, No. 4
April 2023
Pages: 15–17

When referring to this article, please cite it as Mirasol, F. Ramping Up Development of Nucleic Acid Therapies. 

Development

The COVID-19 vaccines opened the door to powerful market potential for other nucleic acid-based therapies.

Ramping Up Development of Nucleic Acid Therapies 

Innovations in bioreactor design are prompted by the need to optimize cell culture reactions. Cell culture processes must be robust and scalable, and yet economical. A look at the fixed-bed bioreactor (FBB) can help with understanding their impact on process development and scale up of biologics.

Looking at the history of bioreactors for bioprocessing, the first bioreactors were developed from the chemical industry, broadly speaking, explains Alex Chatel, senior marketing and product manager, Univercells Technologies. These early bioreactors were large, stainless-steel stirred-tank (STR) bioreactors designed for industrial production of biologics. 

“While they offer size and scale, they lack flexibility and require extensive cleaning regimes (and associated quality assurance and control activities). To accommodate more flexibility, reduce operational and capital expenditures, and de-risk commercialization, novel single-use STRs were developed,” Chatel says.

The single-use STRs are disposed of after each batch, removing the need for cleaning regimes, which in turn reduces the need for hard piping, cleaning utilities, and other associated personnel. Chatel notes that, while an improvement, the STR design itself is by nature not linearly scalable and not well suited to handling delicate cells or processes, especially at scale. This limitation leads to complex and time-consuming scale-up studies, he states.

In STRs, Chatel explains, cells are suspended in a liquid media and are constantly mixed by an impeller. In single-use FBBs, however, cells are “entrapped” in a solid but porous matrix, and cells are at a much higher density. As opposed to STRs, the cells don’t move around the bioreactor in the latter, but, rather, an impeller circulates media containing nutrients throughout the fixed-bed matrix.

“Biomass immobilization has several advantages, such as providing a low-shear environment for cell growth and sensitive products and removing the need for cell retention devices for perfusion processes,” Chatel states. He goes on to explain that first-generation FBBs are made up of randomly packed bed matrix. This construct can be problematic because it provides an inhomogeneous environment for cell anchorage and growth, which leads to variable productivity and product quality, according to Chatel. Newer FBBs, however, provide a structured fixed bed with a homogenous three-dimensional environment, ensuring homogeneous cell distribution and growth. These newer FBBs are equally suitable for adherent and suspension cells both in serum and serum-free conditions.

Finally, structured FBBs are scalable by design and offer higher throughputs than conventional STRs, an advantage that enables cost-efficient production. “In one study conducted by Univercells Technologies (to be published), producing an AAV-2 [adeno-associated virus-2] in [an FBB] (scale-X nitro 600, Univercells Technologies, which has a 600-m growth surface and 60 L working volume) equates in productivity to a STR capacity of 3600 L and results in a cost of goods (CoG) reduction of 24% compared to the STR process,” Chatel says.

Andrew Laskowski, global product manager, iCELLis Bioreactors, Pall, reinforces the notion that FBBs are compact upstream manufacturing technologies that have been optimized for the cultivation of adherent cells. The primary feature of these bioreactors is an immobilized surface where cells adhere and proliferate. FBBs are primarily used to manufacture viral vectors or viral vaccines, remarks Laskowski.

“These products can also be manufactured in suspension cells using a conventional stainless-steel or single-use stirred bioreactor, but suspension processes require additional development time to adapt suspension cells lines, and product titer is generally lower than adherent cell processes. FBB therefore enable companies to bring products to clinic quicker and with a more efficient processes,” Laskowski observes.

As FBBs are mostly used in the production of viral vectors, viral vaccines, and other cell-based products expressed by adherent cells, these bioreactors streamline large-scale workflows by facilitating easy sterile media addition, cell addition, and infection/transfection, notes Laskowski. “Cells are immobilized on the surface of the fixed bed, enabling FBB to act as natural cell retention devices, facilitating the easy implementation of perfusion,” he states.

Laskowski also points out that FBBs help simplify downstream purification processes, as the fixed bed acts as a natural size exclusion filter for turbid biomolecules, reducing the need for further downstream clarification.

Chatel adds that some structured FBBs (e.g., the scale-X family of bioreactors, Univercells Technologies) also enable cell harvest, meaning they can be used to generate the inoculum of larger-scale systems. “They thus play a central role in biomanufacturing processes, as the choice of upstream technology significantly impacts the rest of the manufacturing process,” he states.

FBBs convey specific advantages to the overall biomanufacturing process. For example, they offer a fully closed, automated system of cell culture and contain a large surface area for cell attachment in a small footprint, Laskowski explains. Furthermore, FBBs are integrated with analytical technologies, such as pH sensors, dissolved oxygen sensors, temperature sensors, and (in the case of the iCELLis bioreactor, Pall) a biomass sensor to measure cell density.

“Compared to conventional 2D flatware manufacturing methods, these properties of fixed-bed bioreactors result in a decrease in labor and operational costs, process footprint and facility utilization reduction, decreased risk of contamination due to open handling, and an increase in process assurance, consistency, and robustness,” Laskowski asserts.

Structured FBBs are also highly intensified, meaning that, for an equivalent production throughput, the working volume is much smaller than in STRs, says Chatel. This result is achieved through drastically increased volumetric cell concentrations thanks to the immobilization of the cell mass within the fixed bed, he explains.

Furthermore, the cell growth support matrix can act as a filter, helping pre-clarify the harvest material from cell debris and other process impurities. Most importantly, however, FBBs provide a low-shear, homogenous microenvironment for cell growth, which typically leads to higher productivities (two- to four-fold increases have been observed) (1). “The resulting harvest is therefore not only more concentrated in product and of higher quality, but of greatly reduced volume compared to an equivalent STR process, resulting in a simplified downstream process,” says Chatel.

Scale-up of the biomanufacturing process typically takes places in steps, with key process parameters and product quality attributes confirmed at increasingly large scale, Chatel explains. “For processes based on STR, the change in volume between the laboratory and the final scale can be 100 to 1000-fold. The scale-up activity is often empirical, time-consuming and can result in trade-offs between reaching the final scale and meeting target productivity, for instance,” he states.

When scaling up using FBBs, however, the change in volume does not exceed tens of liters. “The inherently scalable bioreactor design also significantly simplifies the process, thus accelerating and de-risking scale-up,” Chatel adds.

Meanwhile, Laskowski points out that FBBs are the most scalable technology currently available to cultivate adherent cells. Unlike 2D flatware, which scales out rather than scales up, he explains, FBBs are available in many different sizes (e.g., iCELLis bioreactor, Pall, available in sizes from 0.5 m

).“The performance at these different scales is maintained by increasing the diameter of the vessels but conserving the geometry of the fixed bed and the linear speed of cell culture media through the fixed bed. Contrary to 2D flatware, as batch sizes in FBB increase, the number of operators and process footprint remains relatively unchanged, providing FBB manufacturers with the advantage of economies of scale,” Laskowski states.

On the process development front, there is some influence from FBBs. As Laskowski notes, FBBs require a bit of additional upfront capital investment to purchase the process development-scale hardware and automation systems. He estimates the investment cost to be in the ballpark of approximately $100,000. After the initial capital investment, the cost in raw materials per batch is comparable to flatware at a similar scale, he says.

Yet, there are significant advantages to using FBBs for process development. Laskowski points out that it is possible to use the provided analytical tools to better optimize the cultivation conditions for cell growth and production. “The bioreactors are fully automated,” he states, “freeing up scientists to work on other projects.”

Finally, the process optimized in an FBB can be more easily scaled up to manufacture clinical or commercial batches, allowing FBB users to achieve important milestones more quickly, Laskowski adds. “For example, as they disclosed in a presentation at ASGCT in 2019, Novartis Gene Therapies used the iCELLis bioreactor to commercialize their gene therapy product, Zolgensma, in just under 38 months,” Laskowski says.

Meanwhile, Chatel explains that cells adapt well to the protective environment in a structured FBB, which means that initial process transfers from smaller-scale laboratory systems or other bioreactors is simple and effective with equivalent performance and without the extra step of conducting process optimization. Thus, the transfer of a benchtop process to clinical and commercial production is simplified, he emphasizes. He points to one study (to be published) in which a process using a suspension human embryonic kidney 293 (HEK293) cell line producing an adenovirus product in a 2-L STR was transferred to a bench-top system (scale-X hydro, 2.4 m, Univercells Technologies). This process was scaled-up to commercial production (scale-X nitro, 200 m, Univercells Technologies) in less than nine months while increasing product titers.

Thus, FBBs, in effect, do double duty in helping to optimize overall bioproduction. They convey volumetric and process development advantages in the upstream, while also producing targeted product that undergoes an initial filtering process prior to cell harvest. The impact on downstream processing can thus be seen early in the manufacturing life cycle.

1. Univercells Technologies, scale-X Bioreactor Demonstrates Superior Productivity for Vaccines and Gene Therapy. Press Release, Jan. 27, 2022.


Vol. 36, No. 4
April 2023
Pages: 18–21

When referring to this article, please cite it as Mirasol, F. Exploring the Impact of Fixed-Bed Bioreactors in Upstream Processing. 

Upstream Processing

The innovation of going from stainless-steel bioreactors to fixed-bed bioreactors shows an evolution in upstream optimization.

Exploring the Impact of Fixed-Bed Bioreactors in Upstream Processing

Just as fixed-bed bioreactors were an innovation that advances upstream bioprocessing, simulated-moving bed (SMB) technology is a technology adopted from other industries that can help advance continuous chromatography processes in downstream bioprocessing. First used in the petrochemical industry in the 1940s (1), SMB also became useful in the chemical industry, where it allows for the scale-up of lab chromatography processes in the case of high production outputs (1). In the bio/pharmaceutical industry, SMB technology offers advantages over batch chromatography and can support a shift to continuous chromatography.  

The process of adsorption has been shown to be successful for the purification of commercial products in other industries. Generally speaking, higher yield and better purity can be achieved with SMB (1). This technology can enhance process intensification; for example, fermentation products made in diluted streams have been efficiently extracted using SMB (1). 

In the purification of protein therapeutics, SMB technology shows promise. In one study, an evaluation was done in which an SMB chromatograph technique was combined with a protein capture device to see how much productivity could be increased for the purification of immunoglobulin G (IgG) (2). The results of the study showed that the yield of purified IgG product using the chromatography columns with the protein capture device combined with SMB was much higher compared to conventional chromatography columns, 79% vs. 25%, respectively (2).

Simulated Moving Bed Technology: Overview and Use in Biorefineries. Biorefineries—Selected Processes

; Biernat, K., Ed.; IntechOpen, London, UK, 

. DOI: 10.5772/intechopen.99991
2. Broedel, S.; McManaway, M.; Barrett, W. Highly Efficient Process for the Purification of IgG. 

; W.L Gore & Associates, Newark, Del., March 2020.

When referring to this article, please cite it as Mirasol, F. SMB Technology Supports Shift to Continuous Biomanufacturing. 

SMB technology in the downstream can facilitate the shift to continuous bioprocessing.

SMB Technology Supports Shift to Continuous Biomanufacturing

Drug manufacturers involved in the production and dissemination of time- and temperature-sensitive specialty biologic medications, cell and gene therapies (CGT), and other personalized medicines have a lot on their minds, as they ponder factors that could disrupt their various supply chains at any given moment. Considerable effort is required to understand, avoid, and quickly ameliorate diverse factors that threaten both the upstream supply chain connecting them with their own vendors and supply partners, and the downstream supply chain that feeding their distributors, wholesalers, clinical trial settings, physician offices, and ultimately, patients.

Consider the strong growth in CGTs, just one sector of the overall biologics market. As of early 2023, 27 CGT products have been approved for use in the United States, according to FDA, and a handful more have been approved already in early 2023 (1). CVS Health (2) projects 15 new CGT products will be approved in 2023 (including the first expected gene therapy for sickle cell disease), and 17 more in 2024 (including therapies for Duchenne muscular dystrophy, DMD).

Robust, resilient supply chains are important not only to support commercial-scale production, but far earlier during the clinical-development process, as well. Today there are more than 1500 clinical trials for CGTs registered within ClinicalTrials.gov—across many disease categories including oncology, rare diseases, regenerative medicine, and more. This signals great potential for patients—and growing complexity of the sophisticated supply chain infrastructure. “Strong, swift growth in this sector is also creating increased, parallel demand on upstream suppliers, which may lead to longer lead times for delivery,” says Alexander Aust, principal/owner of pharmaceutical consultancy, Aust Business Solutions. “This must be factored in by drug sponsors.”

“Manufacturers are always concerned about large orders of hundreds or thousands of kilos of critical ingredients at a time, but shortages or delays in lower-volume materials that are key to the manufacture of lifesaving biologics can still shut down an entire plant and create disruption,” notes Jim Luchsinger, vice president, business development and international distribution for Spectrum Chemical Manufacturing Corp.

“The most pressing source of risk in the timely production of biologics is manufacturing readiness including material readiness,” says Sigma Mostafa, PhD, senior site head, KBI Biopharma, a global contract development and manufacturing organization (CDMO) and manufacturer of APIs. “To address the risks our clients face and allow them to be able to take drugs to the clinic as quickly as possible, it is of the utmost importance that we maintain our own manufacturing schedule.”

During the pandemic, lead times for many standard materials went from less than a month to over 12 months, says Mostafa. Such uncertainty “created considerable upheaval to our own manufacturing schedule and forced us to slow down the cadence of runs for a short period and pivot to finding and cultivating as many alternate vendors as possible for a given product.”

To hedge risk, she says that the company ended up placing orders with multiple suppliers at a time. “In some cases, we identified smaller manufacturers further upstream (that had been suppliers to our original suppliers), and we even started to switch to raw materials and processes in favor of those that were more widely available during the process-development phase,” Mostafa continues.

“Setting up a robust material requirement planning (MRP) process is one important way for suppliers to meet their pharma customers’ drug-development and manufacturing needs and help to reduce risk,” says Mostafa. For a CDMO with a large early-phase pipeline and a considerable number of complex molecules, determining the full bill of material (BOM) a year ahead can be challenging. “Nonetheless, during the pandemic, we took action to strengthen our MRP process and our tech-transfer workflow to be able to complete 95% of the BOM well ahead of time.”

Similarly, by considerably improving material demand planning and safety stock determination process—while consolidating its own global materials demand database to allow for quicker identification of available inventory within our own network—Mostafa continues, “We have been able to transition to a leaner inventory while still retaining our agility to respond to client needs.”

The reliable supply of APIs, excipients, and critical components (e.g., vials, stoppers, tubing, filters, aseptic bags, specialized temperature-controlled packaging) is subject to disruption at any moment because of workforce shortages, weather events, geopolitical tensions, mechanical failures, fire or flooding, cyberattacks, quality or regulatory issues, transportation issues, and other risk factors that may impact suppliers. To manage risk, producers of biologics (and their third-party CDMO partners) must be imaginative during strategic planning exercises and contracting efforts with suppliers—running many ‘what if’ scenarios to develop an accurate assessment of both obvious and non-obvious sources of risk.

A regular review of best practices that are emerging among competitors, along with increased use of state-of-the-art modeling that can support improved demand forecasting, should be part of any winning risk-reduction strategy. “Biopharmaceutical companies rarely have the luxury of extra time,” says Aust. “Supply chain interruptions create costly and painful consequences for drug manufacturers that are not prepared. Long lead times could delay or interrupt clinical trials, threaten the regulatory filing, and lead to shortages in commercial therapies.”

“One of the challenges many manufacturers have is that they don’t always get transparency from upstream suppliers in terms of where their chemicals are coming from,” says Luchsinger. “A lack of transparency can compound risk because many materials come through multiple layers of the supply chain.”

How many suppliers is enough? While intuitively there is often benefit to be gained from simplifying and streamlining any complex supply chain to reduce the number of players, many experts agree that reliance on a single vendor for mission-critical supplies or components is never wise.

While supplier redundancy certainly makes sense, “many biologics manufacturers are still making the mistake of having one company designated as primary and all others as secondary,” says Luchsinger. “A better practice is to designate two primary suppliers. That way, if there is an issue arises with one supplier, the other can hopefully jump in to make up the temporary shortfall in supply with little to no lag time in the event of two scenarios—either a surge in demand on the part of the drug sponsor, or a supply interruption from one of the suppliers.” Increased use of domestic suppliers can also help to streamline the rigorous documentation that is required by various regulatory bodies worldwide when suppliers have many different countries of origin.

However, adding a secondary source brings its own challenges. “With materials that have unique traits, there may be only one type of supplier approved,” Luchsinger continues. “Approving a second source of supply within the realm of biologics is certainly more challenging compared to basic chemicals, which tend to have a relatively straightforward set of specifications.” Given the complexity, the objective for drug makers should be to identify and qualify new sources of supply and straighten out all of the non-disclosure and other agreements as early as possible, he adds.

Meanwhile, vertical integration is another way that some CDMOs are working to increase overall visibility and reduce the potential for supply chain surprises. For instance, in recent years, science and technology innovator Danaher has acquired both Pall Corp. (a maker of specialized filtration, separation, and purification systems), and Cepheid (a molecular testing and diagnostics company). Danaher also acquired Integrated DNA Technologies (which develops, manufactures, and markets DNA products to support the life sciences industry), and Cytiva (which offers products and solutions that allow for the development and scaling of biomanufacturing operations), and many more, with a goal of keeping under one roof critical suppliers that support steriledrug development.

“When planning for safety stock inventories, drug sponsors must take into account total lead time required, so that items can be replenished in an efficient manner without creating potential disruptions in production,” says Aust. “Since supply planning is a function of demand planning, the need for visibility up and down the supply chain is essential.” He notes that electronic quality management systems (eQMS) from competing vendors can help drug sponsors keep track the status of all raw materials and integrate product demand signals, but also be alerted to audits, deviations, and status updates and more from suppliers and other vendors.

When it comes to kicking the tires with critical suppliers and third-party CDMOs, biologics manufacturers should carry out regular audits and inspections. “The goal is to verify their quality systems, to be sure they have their own contingency planning in place, and to ensure that all quality systems are robust,” says Aust. Importantly, when partnering with key suppliers or a CDMO, Aust recommends drug sponsors establish three critical documents—a Business Continuity Plan (which specifies the supplier’s own business-interruption plans), a master service agreement (which specifies all agreed-upon terms and conditions), and a quality agreement (which is a legal document that outlines when the
supplier or contractor must report a certain deviation, how long that company have to deliver a batch, and how often the drug company can go onsite to carry out its own GMP qualification and quality testing).

When it comes to CGTs, the downstream supply chain that connects these highly personalized therapies with patients is fraught with peril and calls for the right mix of high-tech and high-touch support considerations. For instance, there is zero tolerance for failure in any one of the discrete, yet tightly intertwined steps required to produce autologous (“vein-to-vein”) CGT products that are customized for a single patient using blood or tissue extracted from them as the first step of the process.

Each step requires tight coordination and handoffs among parties, all the while working to ensure proper temperature-controlled packaging and swift transportation to reduce delays and temperature excursions that could spoil the high-value product.

When it comes to allogeneic (“off-the-shelf”) CGT products, even greater logistical planning is required. This involves scheduling the collection of the starting materials from the designated donor, delivery to the manufacturing site and then interim storage and distribution to multiple, geographically disparate patients—each of whom must also be scheduled to receive their therapy infusions in different clinical settings. Each step involves specialized packaging and shipping requirements, with robust chain-of-custody and chain-of-identity documentation. With allogenic CGTs, there is also a need to plan for multiple interim temperature-controlled storage, time- and temperature-sensitive transit legs to reach each of the multiple, geographically diverse recipients.

Advanced monitoring and tracking systems that track individual shipments to the individual vial and individual patient and provide user-friendly dashboards to all stakeholders en route help to increase real-time visibility into location, temperature, time in transit, and more. Today’s state-of-the-art data-analytics capabilities help stakeholders to coax actionable insights from the real-time data and identify weak links and potential vulnerabilities with enough time to act upon them.

Just as important is the rise in the use of outsourcing of critical functions. The right third-party partners bring to bear their own specialized expertise and infrastructure to provide end-to-end coordination of services for every dose and every patient. This option is especially useful for smaller companies that lack the financial resources and managerial and tactical bandwidth to manage all of the complexity on their own.

“Working with a specialized service provider to deliver dedicated case-management services for complex supply chains—to take advantage of their scale and scope, as well as established relationships with multiple premium couriers so sponsors don’t have to navigate this—can reduce strain on the pharma company’s limited internal resources, and enables greater program scalability, as the number of indications, patients, and clinical settings continues to add complexity to the overall picture,” says Trish Demko, director of case management services for Catalent Clinical Development and Supply.

When the service provider takes responsibility for the downstream supply chain for CGT products, “we will have already identified multiple backup plans ahead of time and have the infrastructure in place that enables a quick pivot when any situation arises.” She continues, “We’re doing the heavy lifting—managing all steps from scheduling of starting material collection to delivery of the finished therapy for infusion—so pharma and biotech companies don’t have to navigate this on their own and can instead continue to focus on the science and ongoing innovation while still knowing that the product is in good hands at all times.”

By leveraging this specialized service, customers retain complete visibility at all times, knowing that a dedicated case manager is always on call 24/7 for emergency response and issue resolution. “This service is critical because many pharma and biotech companies may not have sufficient resources, experience or long-term relationships with courier services to confidently manage these complex logistics on their own—and may not be comfortable managing these shipments given the potential impact on the patient if the therapy is not received in time,” says Demko.

“Outsourcing supply chain management to an experienced service provider can provide peace of mind,” Demko adds. The goal is to expect the unexpected and then plan accordingly.

FDA. Approved Cellular and Gene Therapy Products. 

CVS Health, White Paper, Gene Therapy Pipeline 3Q 2022–1Q 2027: Projected Treatments and Approval Timelines.

Suzanne Shelley is a contributing editor to 


Vol. 36, No. 4
April 2023
Pages: 30–34

When referring to this article, please cite it as Shelley, S. Reducing Vulnerabilities in Upstream and Downstream Supply Chains. 

Supply Chain

Makers of specialty biologics, gene therapies, and other personalized medicines are working to identify and reduce potential supply chain risks both upstream and downstream.

Reducing Vulnerabilities in Upstream and Downstream Supply Chains

As patients and the public health community mark the 40th anniversary of the Orphan Drug Act of 1983, bio/pharma companies and FDA officials are acknowledging continued challenges in testing, approving, and producing new treatments for critical conditions affecting small patient populations. The program has led to market approval of more than 600 therapies to treat serious diseases affecting fewer than 200,000 patients in the United States. It has also spurred investment by providing grants and tax credits to help fund very small clinical trials, exemptions from hefty user fees, plus seven years added marketing exclusivity for approved orphans, alongside more incentives for developing rare disease treatments for children (1).

FDA notes that orphan therapies accounted for more than half of novel new drugs approved in 2022, and the agency already has approved two notable rare disease treatments this year. At the same time, clinical development and approval of orphan drugs is often difficult, as trial results may be unclear, and the high cost of research and manufacturing can limit industry investment. Tension continues between funding the development of these much-needed therapies and ensuring their affordability for government health programs as well as individuals, particularly with the development of more gene therapies to treat “ultra-rare” diseases and “bespoke” conditions.

Although FDA marked Rare Disease Day in February by approving an important new rare disease drug, the lengthy path to market for Reata Pharmaceuticals’ Skyclarys (omaveloxolone) to treat the neurological disorder Friedreich’s ataxia illustrates both the promises and the challenges in this field. The drug gained approval only after years of development because its initial pivotal clinical trial did not convince FDA of its effectiveness in slowing disease progression. Approval finally came when the agency determined that Reata’s three-year open-label extension, or natural history, study of patients demonstrated notable gains for individuals taking the drug vs. placebo (2). Reata has set the list price for Skyclarys at $370,000 a year, which will be watched closely for its impact on coverage, costs, and patient pushback.

Meanwhile, FDA is moving on several fronts to provide additional assistance to sponsors in designing studies and obtaining needed data to bring rare disease therapies to patients. An initiative from the Center for Biologics Evaluation and Research (CBER) aims to provide increased interactions with agency experts for firms developing promising rare disease gene therapies, an approach that proved highly effective in accelerating the development of critical vaccines during the pandemic. This “Communications Pilot” will aim to answer questions quickly to speed the development and approval of orphan breakthrough therapies or regenerative medicine advanced therapies.

Similarly, the Center for Drug Evaluation and Research (CDER) reports advances in its Accelerating Rare disease Cures (ARC) program—sort of an “operation warp speed” for orphan drugs that coordinates CDER offices in collaborating with industry and patient groups to facilitate product development (3). CDER notes that ARC has formed several consortia to advance treatments for specific rare disorders, and the initiative may support development of new biomarkers and outcomes measures for clinical trials, as well as natural history studies to serve as external controls for developing treatments for less well-known conditions where historical controls may be inadequate.

Both CDER and CBER are involved in the Rare Disease Endpoint Advancement (RDEA) Pilot Program, another initiative offering sponsors more assistance in developing efficacy endpoints for orphan therapies (4).

Supported by recently reauthorized user fees for drugs and biologics, the RDEA pilot will support a handful of sponsors that have initiated rare disease development programs involving novel efficacy endpoints or natural history studies utilizing a proposed endpoint.

And recent legislation has established an initiative to accelerate the development of more effective treatments for Amyotrophic Lateral Sclerosis (ALS) through an FDA Rare Neurodegenerative Disease grants program (5). The awards aim to cover R&D costs of new therapies for ALS and a range of rare neurogenerative diseases in adults and children, as spelled out in an FDA Action Plan issued in June 2022.

Efforts to advance rare disease treatments, however, may be stymied by the ongoing legal battle that has raised challenges to FDA policies governing the scope of orphan drug exclusivity policy. FDA has opposed a recent federal court decision, Catalyst v. Becerra, that granted an orphan drug producer broader exclusivity for treating all aspects of its target condition, and not just the specific uses or indications cited in its application. FDA approved a Catalyst drug for use in adults, but the company sued the agency later for approving another drug for pediatric treatment for the same condition. A federal Court of Appeals supported Catalyst and rejected FDA’s claim that it has authority to make its more limited approval decision.

Despite that court ruling, FDA has insisted that it will continue to tie orphan drug exclusivity only to approved uses or indications (6). The agency has pressed Congress to enact legislation to clarify this stance, but so far has failed to gain action on Capitol Hill. One result is that some manufacturers are delaying investment in orphan drug development, waiting for further clarification on expected exclusivity terms from FDA or the courts.

FDA. Office of Orphan Drug Products Development. 

Reata Pharmaceuticals. Reata Pharmaceuticals Announces FDA Approval of SKYCLARYS (Omavaloxolone), the First and Only Drug Indicated for Patients with Friedreich’s Ataxia. Press Release. Feb. 28, 2023.

FDA. Accelerating Rare disease Cures (ARC) Program. 

FDA. Rare Disease Endpoint Advancement Pilot. 

FDA has spurred investment to create and develop 600 therapies.

BioPharm International, April 2023

Determining and Optimizing Dynamic Binding Capacity

Purification of Protein by HIC: Mechanistic Modeling for Improved Understanding and Process Optimization

Ramping Up Development of Nucleic Acid Therapies

Exploring the Impact of Fixed-Bed Bioreactors in Upstream Processing

SMB Technology Supports Shift to Continuous Biomanufacturing

Reducing Vulnerabilities in Upstream and Downstream Supply Chains

Kudos and Hurdles in Tackling Rare Diseases

Zauberkugel: Pfizer’s Seagen Acquisition

Who’s Got Game?