While monoclonal antibodies (mAbs) still dominate the biologics drug market, many novel therapeutic modalities are in the biopharma pipeline, with some already commercialized. Many of these next-generation therapeutics present new requirements with respect to analytics. Accelerated approval designations create further challenges with respect to shortened development timelines

Protein characterization is an evolving and critical application for the biopharma industry that enables the evaluation of critical quality attributes (CQAs) such as identity, potency, purity, and stability, according to Anis H. Khimani, senior strategy and product portfolio leader for life sciences at PerkinElmer. “Analytical technology platforms that can help biopharma companies deliver on CQAs and create reproducible and robust methods of development and validation are important,” he states.

Proteins are large and complex molecules that require methods with high sensitivity, accuracy, and specificity, adds Patrick Tishmack, general manager of AMRI’s West Lafayette, Ind., facility. The characteristics of such large molecules challenge the capabilities of most analytical methods, which then push the limits of available technology. “Primary, secondary, and tertiary structure, as well as purity and potency, are critical performance characteristics to be determined for protein biologics. Aggregation and immunogenicity are also critical issues to address. Protein impurities that are similar to the molecule of interest require techniques with sufficient selectivity to differentiate and separate them. Small-molecule impurities require techniques that maintain reliability over a wide molecular weight range,” he say

As a result, the biopharma industry is pushed to innovate and be at the cutting edge of analytical technology development. Drug manufacturers, contract manufacturing and research organizations, instrument suppliers, and regulators must therefore work together to advance capabilities for protein analytics to ensure the rapid development of safe and effective biotherapeutics.

Biologics continue to rise in use as therapeutics, especially in cases where small molecules have not fully addressed diseases, such as oncology. This rise has, according to Trevor Jones, head of marketing for analytical chemistry at MilliporeSigma, highlighted the need for easily integrated characterization and quality control methods that can be seamlessly integrated and transferred among sites.

“Speed and accuracy are the drivers,” Jones asserts. “Increasing the speed of assays to approach real-time release and increasing precision or repeatability of experiments provide greater data quality assurance. Structure-function relationship is always key to understanding performance and setting specifications.”

In addition to increasing the speed of workflows, Ulrik Lytt Rahbek, vice-president of assay, analysis, and characterisation with Novo Nordisk, notes that automating sample workup and workflows and increasing the resolution and sensitivity of analytical techniques are additional drivers.

For mAbs, platform characterization methods have been established. More complex biologics such as multi-specific antibodies introduce significant complexity, with the number of different charge variants—many of which can be undesirable from a safety and efficacy standpoint—increasing exponentially, according to Susan Darling, senior director for capillary electrophoresis (CE) and biopharma product marketing with SCIEX. “Many of the assumptions made about mAbs don’t apply to multi-specifics, and defined analytical tests are needed that provide as much detail for as many parameters as possible,” she says.

Other increasingly complex modalities include fusion proteins, bioconjugates, recombinants, nanobodies, and oligomeric structures, as well as newer approaches such as lipid nanoparticles, mRNA, and exosomes. Outside of the traditional biotherapeutics, there are the gene therapies that use viral vectors comprising 70 proteins and their encapsidated transgene that all require analytical characterization and development of their product quality attributes, according to Scott Berger, senior manager of biopharmaceutical markets with Waters Corporation.

“As these modalities enter the market, the challenge to assess CQAs and characterize them becomes more difficult. The challenge is to develop methods that are suited to the assessment of very high concentration drug substances, highly-glycosylated species, complex formulations/excipients, and even alternative approaches to traditional host-cell protein (HCP) coverage,” observes Todd Stone, senior manager of analytical development at Catalent.

Not only are new analytical methods required for the more diverse format of protein molecules, the chemistry, manufacturing, and controls (CMC) development cycle has decreased from approximately two years to less than six months, according to Gang Huang, senior vice-president of analytical sciences and clinical quality control for WuXi Biologics. To support CMC development within this compressed timeline, faster and higher throughput analytical methods are needed to meet such challenges. He adds that development of process analytical technologies (PAT) is essential to support continuous processing with real-time analysis solutions.

In addition, comments Berangere Tissot, director of biochem method establishment and biologics characterization with Eurofins BioPharma Product Testing, material availability is becoming an issue. “Protein characterization takes place at all steps within the drug product cycle and for many different reasons. Whether it is the first [investigational new drug] (IND)-enabling characterization or a reference-standard qualification post-commercialization, characterization data are embedded in any regulatory submission but are generally the last items to get checked off the list. The amount of material left to perform these characterization assays is therefore often limited, and the time allocated to get the results is inversely proportional to the depth of the results required,” she explains.

Novel, rapid methods are also needed in the research/discovery phase where thousands of highly complex molecules must be quickly analyzed for various properties and assessed for developability, according to Maria Wendt, head of US large-molecule research and global head of digital biologics platform at Sanofi. “More functionality is demanded from single molecules to afford multi-targeting products. Even so, protein characterization assays for these complex molecules need a certain throughput while still providing important individual molecular information. Melanie Fischer, group leader for assays and analytics at Sanofi, adds that “a comprehensive analytical toolbox composed of high-throughput compatible assets complemented with lower throughput high quality assays is thus key to success to react to the growing zoo of multi-specific drugs.”

Complexity and speed create challenges too

The complexity of new modalities also makes their production, purification, and characterization more difficult than traditional mAbs, according to Wendt. These products, according to John Rogers, director of chemistry and biochemistry, protein and cellular analysis, Thermo Fisher Scientific, require extensive characterization for structural and chemical modifications, assessment of host cell and other contaminants, and identification of reference standards and methods. “These efforts are often limited by an inability or incompatibility of existing systems to meet the characterization needs,” he observes.

Conventional purity methods such as CE and high-performance liquid chromatography (HPLC), for instance, which are used to quantify product-related variants and impurities, have been powerful tools but suffer from relatively low resolution and insufficient sensitivity for the detection of low-abundant protein product variants, particularly in the analysis of multi-specific antibodies containing new variants such as chain-mispairing and other closely related post-translational modifications (PTMs), notes Huang.

Existing methods may also not allow characterization of proteins within complex mixtures, such as in the case of gene therapy products comprising both proteins and oligonucleotides, according to Tissot. “For this new class of products, the data sets are more complicated and there are no platform methods, controls, or reference materials in place yet. Therefore, characterization of these new products requires a different perspective on the existing toolkit, with more controls built into methods and more optimization required,” she remarks.

Drug formulations, meanwhile, are a complex mix of APIs, excipients, buffers, and depending on the mode of delivery, adjuvants, lipid carriers, emulsifiers, and surfactants. “With each novel formulation component, new analytical methods are needed to detect and evaluate their effects on the stability, immunogenicity, efficacy, and safety of the drug,” comments Bettina Kehoe analytical development scientist with Catalent.

In addition, for many new analytical and characterization techniques, in fact, the maturity is often at prototype level, and additional development is required to implement new methods that have demonstrated robustness for higher throughput of sample analysis, according to Rahbek. “Technology is either moving too fast or too slow,” adds Tissot. “Some assays are still using old technology that needs to be improved, such as Edman sequencing, while complex instrumentation is being introduced that only provides meaningful information in an academic research environment,” she explains

Darling comments that the most common challenge customers are having in the development of new protein characterization methods is ensuring sufficient reproducibility and robustness. This can often mean that exponentially more effort must be invested in achieving the reliability necessary for the analysis of drugs that are injected into humans. “With the wide range of newer, highly complex and diverse modalities, that goal is becoming more critical to reach,” she says.

Furthermore, new methods may not be adequately scalable and may require unique skills, instrumentation, and software, Rogers adds. Adopting new methods requires dedication of significant time to understanding any new application theoretically as well as specific issues around successful day-to-day operation, agrees Andrew Hanneman, a scientific advisor at Charles River. The appropriate setting (quality control, process development, characterization, etc.) for new methods may also not be initially obvious. As diverse biologics and formulations move from academic laboratories toward commercialization, there is also continuous need for updated analytical methods and instrumentation, according to Kehoe.

Specific issues outlined by Jones include reagents for sample preparation and sample isolation/purification and derivatization, which can cause poor reproducibility and development of reference standards can be difficult due to limited availability. Biologics with newer mechanisms of action will require novel approaches to cell-based assays. Antibody-drug conjugates and protein capsids in viral vectors present their own unique sets of requirements with respect to understanding how specific properties affect functionality.

Regardless of whether protein characterization is needed for a conventional mAb or new therapeutical modality, choosing the appropriate set of orthogonal analytical techniques is crucial and challenging, because one or two methods are rarely sufficient to adequately characterize a biologic, according to Tishmack.

Consolidating assays carries risk too, though, says Tissot, because each individual technique for characterizing a protein—or a more complex biologic—has inherent bias that could lead to a mis-estimation or misleading assignment of quality attributes when combined with other methods. “Some consolidation can happen, but the only way to overcome the need for speed is to develop better interpretation software, because interpretation is often the most complex and lengthy part of a characterization study,” she asserts.

The need to conduct numerous orthogonal techniques to ensure full protein characterization is a challenge not only due to time and resource demand, but also from compliance and data management perspectives. “Addressing software challenges from a compliance perspective is critical to meet regulatory guidelines,” Khimani observes. He points to electronic records and signatures, as well as strong security and audit trails, as hallmarks for 21 

) Part 11 compliance. For characterization methods that may end up as quality controlassays in the absence of equally specific or equally performing quality control-friendly assays, Tissot notes that more may need to be done from a method-readiness standpoint than is usually performed on purified proteins, particularly with respect to 21 

The large amounts of data generated by the array of analytical technologies used for protein characterization, while welcome, is also presenting another layer of challenges. “Many organizations are struggling with both the volume of data and the capacity to leverage it fully in service of their drug-development goals, Bill McDowell, group leader and head of analytics at Abzena, observes.

“We have found that while our clients ask for their data in raw form, some of them just do not have the infrastructure in place to use, manage, and store it properly. It may be that the industry needs to lend even more scrutiny to data handling and sharing practices with labs and services providers while adopting a more common base of standards and technologies to suit the current and emerging needs of biopharma’s developers,” McDowell concludes.

Innovation and its adoption are linked to confidence in the measurement, asserts Colette Quinn, marketing manager for biopharmaceuticals at Waters Corporation. One way to build that confidence, she says, is to introduce new methods to various places within the workflow. There tends, Tissot agrees, to be less opposition to new characterization techniques for new quality control methods. “Because characterization relies on scientifically sound principles, it is up to the applicant (and its providers) to demonstrate that the method used or that the technology used is suitable for use and scientifically sound,” she notes.

Fortunately, regulators realize that protein characterization as quality control is an emerging field and, therefore, are flexible when it comes to the development of new tools and methods, according to Jones. “Drug manufacturers are often conservative in their approach to new methods because of the uncertainty it might cause in approval delays. We have found, however, that regulators are open to novel approaches so long as they are scientifically sound and meet validation and/or qualification criteria,” he comments.

The key to successful adoption of new analytical methods by regulators, according to Huang, is demonstrating that they are not only scientifically sound and robust, but also capable of generating reliable results and useful for defining control strategies. New methods should, he adds, be developed to analyze certain CQAs that are not covered by the existing panel of methods or otherwise be superior with respect to performance (accuracy, sensitivity, reliability, etc.). Ideally, says Khimani, they should also offer a broader applicability from discovery through development, including downstream process monitoring and quality assurance/quality control.

For more novel approaches such as multi-attribute monitoring (MAM), also known as quantitative peptide mapping, the emerging technologies teams within regulatory bodies often act as bridges, enabling companies to collaborate with regulators to build confidence and best practices for employing those methods, according to Berger. This approach has, he says, allowed many companies using MAM approaches to work toward deploying them beyond development and into clinical lot release.

There is also, however, Darling adds, the need to build consensus within the industry as a whole before any new method is widely adopted. First the consensus must be established between scientific experts at various biopharma companies; wider consensus can then only be built through back-and-forth with different groups within the various regulatory authorities. “Achieving critical mass is a slow process. Most companies vet a new technique or method for at least a year or two before moving it into quality control,” she says.

For regulators to accept new technologies for CMC-based characterization analysis, they need to see demonstration of equivalency or superiority of the new method for its intended purpose, Berger says. “Significant crossover studies may be required to demonstrate these outcomes, as assays in characterization not only document normal product variation, but are often employed for stability, forced degradation, and process development studies that give rise to a wider variety of product variants, often at low levels,” he comments.

Contributing factors leading to slower adoption of new analysis and characterization techniques include the need to invest in new equipment and build up necessary competencies, and the time delay for projects compared to what is possible using conventional methods, most notably due to the need to demonstrate comparability to current methods, according to Rahbek.

In addition, Darling points out that if an existing method is part of a regulatory filing for a global product, then both the old and new methods will need to be performed until all of the regulatory agencies approve the new method, which can take years. “Not surprisingly, there have to be compelling reasons and significant benefits for a drug maker to switch to a new method for a commercial product,” she says.

The resistance to adopting new analytical techniques for development candidates is often tied to the need to adhere to strict program timelines, according to Robert Vaughan, associate principal scientist in Catalent’s analytical development group. He also notes that there is additional risk associated with adopting less-established technologies given the added cost and potential for unanticipated method-specific challenges that might be encountered during development.

“Furthermore,” Vaughan adds, “adoption of any new method that requires specialized equipment would require training analysts in both process development and quality control, acquiring systems for both groups, integration into a regulatory-compliant data management system and subsequent validation. Redundancy of equipment is also important to maintaining timelines should one system go down, which can significantly increase the cost. When benchmarked against established platform methods that have a history of success within a regulatory environment (albeit lower resolution), the added time and cost can be a significant barrier.”

Rogers agrees. “Many companies already have investments in proprietary and/or established methods that would require significant commitment and investment to replace. There also may be questions regarding the validation requirements, and often there is a fundamental resistance to change that must be overcome as well,” he says.

Adding to the challenge is the fact that the benefits of new and improved methods may not initially be obvious prior to testing in one’s own laboratory, according to Hanneman. “In a fast-paced industry where experience is highly-valued, experienced developers may need to choose familiar approaches for expediency, presenting a barrier to adoption,” he observes.

Real incentives for adoption of new methods, too

It should be noted, though, that because the increasing number and diversity of new modalities is creating the need for new analytical methods, there is a growing level of incentive to adopt new techniques for these new biologics in particular. “All of these biotherapeutics would benefit from the use of orthogonal approaches that can help to reduce bias toward one method or one result,” Quinn asserts.

“Scientists tend to gravitate towards solutions with which they are most comfortable, and although data might be collected along the way, inserting a number into a cell on a spreadsheet can be a lost opportunity,” says Quinn. “Instead, the goal should be to increase confidence and look for trends that could save time when future studies are conducted. In addition to asking what has changed, it is important to consider whether that change matters. To answer both questions truly requires an orthogonal approach,” she asserts.

In addition, Quinn notes that by linking outcomes, the values from these systems can possibly become predictors of the next set of tests. As an example, she points to linking chemical outcomes like those determined via LC separation with native ion mobility or biophysical data such as differential scanning calorimetry and ultimately extending this information into interpretation of binding assays.

Berger adds that with the recent progress in International Council for Harmonisation guidelines Q12 and Q14 there is greater potential for more flexibility in analytical methods used for product analysis, which is aligning well with the growing interest in pursuing more formal analytical quality-by-design (AQbD) method development approaches. “The power of platform methods to accelerate COVID-19 molecule development and the benefits of systematically optimizing these methods is becoming very clear,” he says.

All stakeholders, such as biologics manufacturers, regulators, technology providers, and contract research organization (CROs), need to consider the properties and stability of protein drugs. A concerted collaboration and partnership between biologics developers, technology/service providers, and regulatory agencies greatly facilitates bringing a safe and efficacious protein drug to market, says Khimani.

“Collaborations between industry, regulatory agencies, instrument companies, and standards organizations during the development of new methods [are] critical,” agrees Rogers. It is key to developing technologies and methods that are robust and fit-for-purpose, adds Berger. “Organized industry meetings bring these groups together to explore technology, methodology, and its impact on regulatory science. Other more focused collaborative organizations such as the National Institute for Innovation in Manufacturing Biopharmaceuticals and the MAM Consortium dive deeper and look to establish consensus performance expectations and best practices to bring new methods into routine use,” he explains.

The R&D/analytical development departments of biologics manufacturing companies are in the unique position to bridge established, legacy-testing methods with newer technologies emerging from academic research programs and innovative instrument companies, Kehoe asserts. “Whether it is an improved reagent, a new assay, or a more efficient instrument, these departments can show equivalency to current CQA assays, demonstrate improved test performance, or provide additional orthogonal testing options,” she says.

“Instrument companies may be looking for new applications for their existing technology that have not yet migrated to the protein biologics field, or they may partner with a biologics manufacturing company or CRO who tests biologics to develop something completely new to solve an existing problem,” Tishmack comments.

Instrument vendors, meanwhile, will often introduce brand new technologies to the entire community including regulators, building a sense of urgency, according to Rahbek. In addition, Kehoe observes that as instrument companies continue to include installation qualification and operational qualification in their analysis programs, methods developed on these instruments can be transferred readily to quality control laboratories for the release of manufactured drug products.

Most CROs, biosimilar developers, and contract manufacturers often have low inducement to develop new analytical and characterization methods because they are mostly required to follow already established methods, according to Rahbek. However, Jones notes that some CROs and contract development and manufacturing organizations (CDMOs) do pursue the development of cutting-edge methods for protein characterization as a means of differentiation and to attract pharmaceutical companies seeking outsourcing partners that specialize in specific assays.

CROs with biologics expertise will know the existing challenges and continuously look for appropriate and efficient solutions, adds Tishmack. “Many times, a CRO will advance a unique application of a common technology because of the wide variety of problems they encounter and solve for their clients,” he says.

Overall, Huang observes, instrument companies manufacture analytical instruments and periodically incorporate new technologies into these analytical tools for biologics manufacturers and CDMOs/CROs. Drug developers are willing to explore new technologies and turn them into new analytical methods. Regulators ensure these new analytical techniques can serve the intended purpose while ensuring patient safety is not compromised. If a regulatory agency finds a new method for biologics characterization acceptable in a filing for one developer, this increases the likelihood of propagation of the method for use with other biologics, Tishmack observes.

While the fact that large biopharmaceutical manufacturers set the tone for biophysical characterization helps get cutting-edge techniques publicized, recognized, and exposed to regulators, there is a downside, according to Tissot: smaller companies are sometimes left in a difficult position because not all can afford highly expensive, specialty instruments nor the time to develop an optimized method internally or at an external provider. In addition, she notes that when instrument vendors collaborate with large biopharmaceutical manufacturers, they often tailor their software to the needs of those companies. Once the software is released, smaller drug developers are sometimes forced to follow the process that the software was optimized for.

Quinn argues, though, that most of the time the molecules drive innovation in the analytical instrumentation sector. “Strong collaborations between manufacturers, CROs, and analytical instrumentation companies leads to hyper-personalization of equipment, chemistry, and methods and enables faster progression of the science,” she asserts.

“What is exciting,” claims Fabio Rossi, scientific lead at Abzena, “is the increasingly collaborative role the industry is taking collectively to shape and refine protein characterization methods and study protocols. Abzena is able to improve on its clients’ methods to achieve more efficient and effective outcomes. Instrument companies are also in a cycle of continuous improvement, and over the past five years or more we have seen a tremendous improvement in study cost efficiency, speed, and the efficacy of the data analytics,” he says.

“Protein characterization techniques,” Rossi continues, “are diverse and the data each yield is too. But the information that can be derived from a well-integrated line of analytical enquiry linking the best of each technique will likely be more robust and valuable relative to the overall economics of drug development.”

The complexity of biologic products and the need for comprehensive characterization creates some unique challenges for analytical scientists. Despite all of the advances in mass spectrometry (MS) and other methods, there is still opportunity for further improvement.

For instance, there is a significant need for alternative approaches to HCP characterization and clearance during purification, according to Vaughan. “HCPs are a unique challenge given their complexity and diversity, and they pose a significant risk to any protein therapeutic given the potential of certain HCPs to impact product stability and immunogenicity of patients,” he says.

Enzyme-linked immunosorbent assay-based approaches used currently have high sensitivity against a single target, but there are thousands of HCPs present in the initial harvest, which have the potential to vary between lots. Mass spectrometry, Vaughan believes, is well-poised to fill this gap given its ability to both identify and directly quantify individual HCPs with both high sensitivity and accuracy.

Quantification of polysorbates (PS20 or PS80) in protein therapeutic formulations is another area in which advances in analytical techniques are still needed, according to Kehoe. “Regulators have recently started to increase the detail required of drug formulations, and the analytics for characterization of polysorbates is lagging behind,” she observes.

Glycosylation analysis remains a challenge as well. “Analysis of the N- and O-glycan profiles of any molecules but mAbs is an arduous task,” Tissot remarks. “Most of the methods are either highly quantitative or highly qualitative and there is no real happy medium where you can safely assign structures and report relative percentages in one go without major bias,” she explains. The ability to use LC-MS with electron capture dissociation/electron transfer dissociation (ECD/ETD) is an improvement, but most of the workflows and techniques remain in the academic realm to date.

The bi/multi-valent nature of multi-specifics bears the risk of self-association that can result in poor high-concentration solution behavior, according to Fischer. She believes high-throughput predictive assays to assess colloidal stability of biotherapeutics early on are a key advantage in the discovery of multispecific drugs. Similarly, the analytics of mispairing, in particular when compound numbers are high—in discovery but also in cell line development—is a challenge. Here again, Fischer notes that compatible MS approaches are important for effective analyses of samples with increasing compound numbers. “We have made some advances on these fronts at Sanofi,” she says.

Rogers also points to the need for tools and workflows for efficient protein variant screening and structure determination across a broad molecular weight range, amino acid misincorporation, characterization of intact proteins and protein complexes, characterization of low-abundance PTMs (natural or introduced by the manufacturing process), and the characterization and quantitation of specific proteoforms. “Workflows utilizing CryoEM, [ultra-high mass range] (UHMR)-MS, and complementary tools and software are of great interest to structural biology researchers in academia and industry because they offer great potential for improved screening, characterization, and understanding of protein structure and proteoforms,” he states.

The rapid emergence of viral and nanoparticle-based delivery has created the need for methods that address questions of composition (e.g., lipid profiling), as well as measures for proper assembly of these macrostructures (e.g., empty/partial/full analysis for the biotherapeutic molecule), according to Berger. The acceleration of gene and cell therapies reinforces and extends these needs.

In addition to developing capabilities in advanced MS techniques to address such challenges, Waters is working with emerging technology companies such as MegaDalton Solutions to bring new technologies like charge detection-MS (CD-MS) into the commercial realm. The company is also, Berger notes, investing significantly in new generations of HPLC and ultra-performance liquid chromatography (UPLC)/ultra-high performance liquid chromatography

(UHPLC) chemistries and consumables for the next generation of size- and charge-based separations for application to new modalities because these methods will also play a key role in routine analysis.

Advances in mass spec-coupled methods having biggest impact

The possibility to automate and increase robustness of current manual workflows has led to expanded use of existing technologies, which has in turn led to measurable advances in protein characterization capabilities, says Rahbek, pointing to the increased use of CE techniques and MS-based analysis. MAM, Rahbek says, is of particular note because has the potential to replace traditional methods with one LC-MS based method.

The main overall trend, says Jones, is the movement of LC-MS out of the laboratory setting and into the quality control lab because of its value in biotherapeutic characterization. “We can start to contemplate having fewer assays moved into quality control that are faster and more accurate,” he states. The ability to monitor product and process variation at the intact, subunit, or peptide levels with greater specificity and sensitivity while conducting fewer assays is an objective many labs are now investigating for both development and manufacturing/quality control applications, Berger agrees.

“It comes back to the value statement of what can you discover if you can see more,” observes Quinn. “Not only is it the high resolution of new mass spectrometers but also the incorporation of ion mobility into quadrupole, time-of-flight mass spectrometers that is enabling scientists to deconvolute complex spectra and better identify peaks that were overlapping,” she explains.

Important recent advances in protein characterization include intact protein analysis with ultra-high mass range (UHMR)-MS, and simpler and less costly instruments for functional assessment of binding activity and protein secondary structure as, says Rogers.

Sensitive detection and quantitation of sub-visible viral particles and protein aggregates are improving in several areas as well, according to Hanneman. In addition to instruments capable of measuring several aspects of protein structure within a single run and MAM solutions, specific examples include improved 

 bioassays, analytical methods runnable under good manufacturing practices (GMPs) using fully 21 

 Part 11-compliant instruments and software, native LC-MS of large intact proteins and glycoproteins by various HPLC methods including ion-exchange (IEX) and size-exclusion chromatography, and alternate fragmentation approaches for MS/MS of modified peptides and branched structures, including ECD/ETD-MS and multi-stage MS.

Ion mobility-mass spectrometry (IM-MS) and hydrogen-deuterium exchange-mass spectrometry (HDX-MS) are two other MS-based techniques that should find many applications with protein biologics, according to Tishmack. He notes that IM-MS adds a unique dimension to MS because the shape of the molecule can differentiate multiple conformations, aggregation states, or small mass differences that cause heterogeneity of proteins. HDX-MS, meanwhile, is useful for studying protein-protein and protein-ligand interactions, conformational changes related to protein activity, and protein stability among other phenomena. “The detailed understanding of protein behavior that HDX-MS affords therefore has direct application in biologics drug discovery and development,” Tishmack observes.

In Sanofi’s research lab, there is real need to access high-throughput methods that perform at a high level even for highly complex biologics. One recent advance noted by Wendt, is a method coupling size-exclusion chromatography with MS for the rapid characterization of tri-specific antibodies. “With this beautiful characterization capability for such complex molecules, Sanofi is placed in a much better position to deliver more complex drugs with a higher likelihood of success for addressing more challenging diseases,” she states.

“It has been difficult to date to couple really high-resolution techniques with mass spectrometry, but real progress has been made, which is giving scientists the ability to achieve deep characterization quickly,”agrees Darling. She highlights capillary isoelectric focusing (

)-MS as a method providing rapid separation and identification of charge variants with resolution high enough to identify isoforms on intact proteins.

The possibility of identifying both charge and size variants at high resolution down to the PTM level for peptides is a game changer, according to Stone. “Efficiency gains from the elimination of bench-scale fractionation and sample preparation are enormous, while the increased depth of data provides a much more comprehensive view of potential molecule degradation pathways and subsequent adjustments to CQA control strategies,” he says.

Biophysical analysis developments important too

In addition to such techniques with better resolution and sensitivity, Huang emphasizes the importance of the development of methods for 

 bioactivity analysis that better reflect the clinical effect of biologics products in humans. “Through the study of 70+ specific cellular models of pathologies, WuXi Biologics has developed proprietary technologies enabling the best linking of 

 responses for biologics. These technologies allow effective reduction of complicated clinical effects into simple, clear CQAs at the cellular or molecular levels, and are therefore becoming an integral quality tool for us,” he comments.

Considerable advances have also been made in biophysical instruments, according to Tissot. “Manufacturers are striving to produce new models that are more versatile, more resolute, and more efficient from a sample-volume-requirement standpoint,” he says. The latest analytical ultracentrifugation (AUC) instruments, for example, allow analysis using up to 20 discrete wavelengths. “This advancement has the potential to revolutionize the field of AUC by enabling more precise analysis of complex systems,” Tissot asserts.

Cryoelectron microscopy (CryoEM), adds Tishmack, is a powerful method for molecular structure determination that has certain advantages over crystallography, but the instrument has been very expensive and not readily available until recently. Today, however, a somewhat less powerful CryoEM instrument that is relatively affordable is available that, he says, should result in more applications of the technique for developing biologics particularly in the early stages where structure-activity relationships must be determined.

Data analysis developments noteworthy as well

Some of the biggest developments, Darling asserts, have not occurred with particular assays, but rather with data processing capabilities, which have allowed more rapid evaluations. McDowell, agrees. “Although advanced MS techniques are attracting researchers’ attention, what is advancing the refinement of analytical techniques more recently is the introduction of increasingly smart information and data analysis and sharing systems and automation solutions that speed up things like sample prep and analysis,” he asserts.

“The refinement of both the specific techniques and their supporting technologies, as well as the refined and focused application of the methodologies themselves, has evolved to a point where the industry’s confidence in these studies is extremely high because the data adds such tremendous value,” concludes McDowell.

Improvements still needed in method robustness, sample prep, data analysis

Although many advances have been made in new technologies such as CE and MS, there is still, Rahbek underscores, a need to develop these methods to a sufficient robustness level for a regulated setting such as a quality control lab.

Some of the biggest needs, according to Jones, are around the tools to support LC-MS, including reagents for derivatization. Derivatization is necessary for several tests, including peptide mapping and glycan analysis. Chromatography columns for specialty applications continue to be developed as well as mass spec standards, including isotopically labeled standards that meet quality control and regulatory standards.

As an example of a dramatically improved protocol, Wendt points to a new method Sanofi uses during cell-line development that allows for protein analysis following a simple clarification step and no need for Protein A purification. “This method truly enables high-throughput work,” she notes.

Analytical data management solutions are also needed that enable comparison across drug proteins as well as constant evaluation and update of metrics that can enable standardization across various types of protein drugs, according to Khimani.

There is also a need, according to Rahbek, for automating data analysis as the automation of instrumental workflows becomes a reality. Indeed, Tissot would like to see improvement in automation of both sample preparation and data interpretation so more characterization methods can be introduced in process-development studies. “Such a move would require characterization assays be turned into semi-quality control assays where some elements of precision or linearity would have to be demonstrated. The MAM workflow is a good example of this area of improvement but being able to extend this workflow to other complex quality attributes would be valuable,” she observes.

Wendt adds that the highly sophisticated technologies in use today provide very rich, multidimensional data that are not being optimally used. “Making use of big data, artificial intelligence, and machine learning will certainly bring us to the next level,” she states. “Data science is commonly realized as an area with high potential, and investments are needed to ensure appropriate data management and programming of 

 tools and algorithms to leverage the data that are already being generated,” Wendt adds.

For instance, these approaches could be used to provide information about proteins much earlier in the development process, and in particular in the form of predictive models that can be used during biologic drug discovery. “If we are able to quickly screen thousands of molecules and accurately assess the potential of each based on a whole host of properties, candidates with much higher likelihoods for success in development could be identified more rapidly, or we could intervene earlier, for example via engineering, saving tremendous time, money, and resources,” asserts Wendt.

Protein characterization, believes Wendt, is one of the most important parts of the drug discovery business. “If we do not have good characterization of proteins, then we can’t know the potential products we are working on. We can’t achieve the best interpretation of functional assays nor develop the best, most reliable and most consistent products,” she states.

For the most part, according to Rossi, the industry is working hard to implement advanced protein analytic inquiry techniques to ensure therapeutic goals are met and the required biologic activities of key components of biologics of all kinds are demonstrated.

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Mass spectrometry and automation are growing in importance for protein characterization, but further improvements are still needed.

Evolving Analytical Technology Unravels Protein Characteristics

Vaccines are known to be hugely important in maintaining global public health and minimizing the impact of infectious diseases worldwide. Despite their worth, vaccines are subject to intense scrutiny, and, due to the fact that they are intended for large patient populations—including healthy patients and infants—the assurance of their safety, prior to approval and administration, is of paramount importance.

According to recent market research, the vaccines market is expected to grow at a compound annual rate of 14.7% for the forecast period of 2020–2026 (1), the growth of which has been accelerated by the recent COVID-19 pandemic. “Heightened by the recent COVID-19 outbreak, vaccine innovation has become a top priority worldwide,” confirms Elham Blouet, strategic portfolio manager, Roquette—a specialist in plant-based ingredients and provider of pharmaceutical excipients.

“In recent years, a paradigm shift has been observed in the way vaccines are being developed,” says Smruti Phadnis, biopharma marketing manager at Agilent. “The focus on disease-causing pathogens has been well integrated with intensive studies on human immunity. This research has opened doors to allow advances against complex noncommunicable diseases as well as cancers. However, inherent challenges, such as antigen selection and genetic diversity, need to have a broader immune response that includes T-cell protection, and alternative routes of vaccine delivery still exist during vaccine development.”

A constant challenge facing pharma companies developing vaccines is the wide range of potential pathogens that could cause serious illness, explains Kai Lipinski, chief scientific officer of Vibalogics. “Great strides have been made over the years to develop and commercialize effective vaccines for a wide range of infectious diseases,” he adds. “However, there are still particular illnesses that remain difficult to prevent or treat, such as HIV, malaria, norovirus, tuberculosis, and Zika virus.”

Particularly when trying to develop vaccines for difficult to treat illnesses, development obstacles can arise due to the inherent properties of the responsible organism, Lipinski continues. “Organisms displaying high rates of antigenic shift, such as RNA viruses including influenza and even the recent coronavirus (COVID-19), pose one such challenge,” he notes. “Antigenic shift of organisms yields frequent new variants, which develop very quickly, subsequently requiring surveillance, identification, and recomposition of the modified vaccine antigen or strain, often with a seasonal cyclicality.”

As vaccines are derived from pathogens, there are myriad antigen or target types to identify and select for development, emphasizes Phadnis. “Each type has its own degree of safety and efficacy,” she says. “To add to the complexity, non-live vaccines need to be combined with adjuvants (carriers) to induce immune response and slow release. Target/antigen selection is complicated further by genetic diversity across strains, including post-infection evolution and geographic variability.”

A key challenge in vaccine development for Anissa Boumlic, head of global vaccine and plasma segments, bioprocessing, within the life science business at Merck KGaA, is the fact that there is not a singular manufacturing template available to be widely used. “Manufacturers need to adapt constantly to tackle new pathogens and, sometimes, under tight deadlines,” she says. “A number of processes need to be optimized to increase scalability, robustness, efficiency, and cost. Additionally, formulations of certain vaccines need to be improved to increase efficacy and stability while ensuring patient safety, and facilitation of distribution and access.”

Lipinski concurs that, in addition to the challenges associated with vaccine candidate discovery, companies need to develop vaccines that are stable, can be stored, and administered in a variety of climates to ensure widespread distribution and use. “For example, emerging countries often lack appropriate facilities for vaccine storage at sustained sub-zero temperatures at or near the point of treatment, which can pose issues in launching vaccination programs that require cold-chain storage,” he says. “With this in mind, developers are challenged to devise vaccine formulations capable of being stored and transported at temperatures that don’t restrict their widespread use. Formulations must also allow for in-use stability, in the brief window between removal from storage and administration. They must be able to remain stable in tropical environments, where temperatures might exceed 30 °C.”

“Lyophilization or spray drying of vaccines can provide stability during transport and introduce alternate vaccine administration routes,” continues Phadnis. “Packaging is also heavily impacted by liquid vaccines. Durability of glass vials at very low temperatures and permeability of plastic vials has complicated the packaging decisions as well.”

Since the beginning of the pandemic, the bio/pharma industry has been under pressure to produce stable formulations for effective vaccines in accelerated timescales, Blouet asserts. Moreover, the drive for a COVID-19 vaccine has occurred during a period of increased basic scientific understanding, such as in genomics and structural biology, supporting a new wave of vaccine development and production, she says.

“As well as bringing about much needed innovation in a historically conservative field, [COVID-19] has also raised questions about whether conventional vaccines—developed by attenuating or inactivating the respective pathogen—are enough in today’s landscape. Although mostly successful in the past, these established methods may not always be suitable or even feasible in outbreak situations because they simply don’t allow for the fast response required,” Blouet reveals.

There are numerous tools that have been developed to help accelerate vaccine development, and novel technologies and platforms are emerging. “Manufacturing platforms, such as viral vectors, virus like particles, and messenger RNA, allow a reduction in process development time once established,” Boumlic remarks. “Systematic methodologies such as design of experiments (DoEs) and quality-by-design (QbD) have demonstrated the ability to increase process robustness and scalability.”

Processing has been simplified through the implementation and availability of single-use and filtration technologies, Boumlic continues. These tools have enabled an increase in speed of development and are easy to use. Boumlic also notes, however, that flexibility in scale, technologies to manufacture different modalities, more process simplification, and modernization are areas that require improvement still.

According to Phadnis, in addition to single-use technologies, automation for high throughput and robust analytical assays are necessary for rapid turnover during development and manufacturing of vaccines. “Automation technologies can speed up vaccine research as well as the manufacturing process by efficiently reducing manual errors. Automation also gives the flexibility of scaling up or down depending on the external demand. Simultaneous high-throughput workflows supported by robotic automation can swiftly generate consistent data across samples and users,” she says.

Using robust analytical assays for the characterization and validation of vaccine components, and to measure potency, are critical for inducing immunogenicity, Phadnis emphasizes. “Right from antigen selection and functionality testing at laboratory level to its final manufacturing, an integrated workflow of several analytical methods is required to ensure efficacy and safety of the vaccine,” she notes. “Innovations in mass spectrometry (MS) and capillary electrophoresis (CE) combined with in-line detection methods, such as high-performance liquid chromatography (HPLC), provide high sensitivity, selectivity, and accuracy for multidimensional analysis of samples. This innovation aids robust monitoring and control of key attributes of vaccines during development and enables a quick and reliable decision-making process during product development and manufacturing.”

For Blouet, the critical role of functional excipients, specifically cyclodextrins, is gaining interest for those in vaccine development seeking to overcome formulation challenges and produce a safe and efficacious vaccine quickly. “Cyclodextrins are a family of oligosaccharides produced from starch using enzymatic conversion,” she states. “2-hydroxypropyl-beta-cyclodextrin (HPβCD), in particular, is reaching into therapeutic biologic processes, final formulations, and delivery of vaccines, and is being used in some promising new areas where work is still at a very early stage.”

“To shorten formulation development timelines, accelerated temperature predictive modeling is key,” adds Lipinski. “However, this requires robust and well-designed analytical assays to assess a formulation’s performance.”

Both physical and chemical stability of vaccines are ongoing formulation challenges, notes Blouet, particularly given the novel technologies being employed and demand for increased shelf-life for convenience and cost-efficiency. “Physical and chemical instabilities may lead not only to the loss of biological activity, but also potentially to the formation of products that detrimentally affect toxicity and immunogenicity,” she says.

Using adenoviruses as an example, Blouet highlights that a “robust formulation, with precise optimization of buffer type, pH, and specialized excipients, are critical to ensure stability over a wide range of conditions. But options for addressing stability challenges in adenoviral vector formulations are limited to those few excipients approved for parenteral use.” Here, she emphasizes the utility of HPβCD, which is already approved for existing commercial formulations.

“At the same time, a key challenge in recombinant subunit vaccines is their stabilization for delivery,” Blouet continues. “These are typically protein-based and very few approved excipients are available to formulators working in this area.” Again, she emphasizes HPβCD as a potentially useful excipient to stabilize subunit vaccine proteins.

“Certain vaccines are less stable than others depending on their inherent properties,” specifies Boumlic. “For example, viruses or mRNA can be degraded through the process and a number of approaches need to be considered to minimize product loss—gentle purification, specific reagents, degradation inhibitors, stabilizers, delivery vehicles, and so on.”

It is imperative that the stability of process intermediates, or the product matrix as it moves through the manufacturing process, be understood and addressed, emphasizes Lipinski. “As downstream purification processes can span multiple days, a vaccine that is sensitive to the process itself, or cannot be subjected to freezing due to freeze-thaw processes, will present complications or limitations,” he says. “Developers must link drug substance and drug product manufacture, often within one facility, or within the limitations of the stability of the fresh (unfrozen) product, which can pose issues with often desired manufacturing flexibility.”

Combinatorial platform excipient banks can be used to speed up formulation development while overcoming stability issues, Lipinski states, which involves developing each vaccine in the same design space to optimize stability while reducing time-to-market. “Vaccine technology platforms (materials, processes, formulations) can also be used to minimize the development time for new products, as this approach offers considerable advantages where one universal formulation creates efficiencies that can be utilized across new products minimizing changes and allowing for a more predictive development paradigm,” he adds.

QbD and DoE-based principles, supported by orthogonal analytical methods, are significant when optimizing and streamlining the assessment of formulation stability, Lipinski remarks. “As an example, using physicochemical analytical methods to assess formulation performance as an indicator for infectious titer in the case of live-attenuated vaccine development can potentially reduce development timelines,” he says. “If sufficient knowledge is not available in-house to perform formulation stability studies to a high standard and with minimal delay, collaboration with experienced [outsourced service providers] is vital.”

The majority of commercially available vaccines are formulated as liquids, which, if kept in cold temperatures, can maintain their potency for about three years, asserts Phadnis. “However, freeze-thaw cycles can lead to immediate loss of potency,” she stresses. “Alternate formulations such as lyophilization and spray drying are being considered for alternative administrative routes as well as for ease of distribution and stability of the vaccines. However, dehydration needs antigen-protective excipients such as a combination of amino acids and glucose derivatives to replace the water molecules.”

“The route of administration has a significant impact on the development pathway of a vaccine,” Lipinski asserts. “Oral vaccines, such as those utilized for poliomyelitis or rotavirus, offer a number of advantages when it comes to development compared with parenteral products.” However, oral administration is not suitable for all vaccine candidates.

Administering vaccines nasally can also provide advantages over the more traditional parenteral approach, such as the avoidance of needles, ease of administration, and convenience for respiratory diseases, Lipinski continues. “One further delivery device being utilized is the micro-needle-based vaccine,” he says. “This [route] overcomes patients’ syringe needle anxieties while providing further benefits in its ease of use, and lack of cold chain distribution and storage need, due to its solid-state nature. This technology can also be self-administered by patients. However, sufficient production capabilities and capacities for such products are not available yet.”

The majority of vaccines are delivered intramuscularly with other routes including subcutaneous, intradermal, oral, or nasal, confirms Boumlic. The administration route of choice is determined by the type of vaccine being developed (i.e., whether the vaccine is adjuvanted or not) and whether the efficacy versus side effects can be balanced, she adds.

“The route of administration will influence the process to meet the associated safety requirements or to trigger sufficient immune response. For instance, injected vaccines will need to be sterile, and this can impact process design and cost efficiency,” Boumlic says. “Certain vaccines will need to be injected in high quantities to be efficacious and, therefore, this will impact the dimensions of the process (more volumes to handle, more scalability challenges). Messenger RNA vaccines need to be delivered in lipid nanoparticles to remain protected and be translated to antigens.”

The route of vaccine administration is dictated by the formulation and potency requirements, notes Phadnis. “Technologies that enable lyophilization, spray drying, and use of inert stabilizers in the form of lipids and nanoparticles are being developed for ease of administration and distribution of the vaccines,” she reveals. “A careful optimization of stabilizers as well as different formulation parameters during the first stage of vaccine product development should be done along with antigen screening and validation.”

“A glaring constraint with injectable delivery systems for large-scale vaccine programs is a complete reliance on cold storage, which directly escalates operational costs and diminishes market reach,” emphasizes Blouet. “For example, to ensure that each patient benefits from the injectable vaccine, meticulous logistical planning is required to successfully maintain the cold chain at every step, often involving collaborations with supply chain partners.”

The COVID-19 pandemic has presented industry with significant and unprecedented challenges in terms of vaccine development; however, opportunities have also arisen. “Historically, vaccines took about 10 years of development to reach the market and were mostly restricted to affected areas,” states Phadnis. “With the COVID pandemic, we have seen a paradigm shift in shortening the vaccine development process to less than a year. mRNA vaccine technology to make highly potent and safe vaccines was a breakthrough during this pandemic. This technology was implemented for the very first time in commercial vaccines.”

Additionally, the fact that the pandemic has raised questions about whether or not conventional vaccines are sufficient in today’s landscape has challenged developers to push the boundaries of vaccine development, Blouet notes. “Rapid development and accelerated large-scale vaccine manufacture are both critical factors in outbreak situations, key to combatting pandemics and protecting individuals worldwide by getting solutions to market faster,” she says. “To get vaccines to market quicker and overcome complex formulation challenges, more developers than ever are moving away from traditional vaccine development models.”

For Boumlic, the pandemic has demonstrated the requirement of standardized vaccine manufacturing and has highlighted the need for expanded capacity to ensure biopreparedness. “A clear trend is the development of platforms that can be easily adjusted for new targets. Using recent technologies, such as viral vectors and mRNA, single-use technologies and tapping into contract manufacturing organization capacity has also been beneficial to increase productivity and will shift vaccine development and manufacturing in the future,” she says.

“One key development that has proven useful during the pandemic and will change the way we operate in the future is the creation of vaccine platform technologies,” concurs Lipinski. “The advantage of such a platform approach, in the example of adenoviral vector platforms, is that the different vaccine products are, with the exception of the disease-specific transgene, the same. This shortens the production timeline for a number of reasons, as the producer cell bank and expression technology are already in place and the vaccine design tools and manufacturing infrastructure are already established. Moreover, formulation and stability data are available from products previously developed with the same platform technology.”

Furthermore, collaboration by all stakeholders has been highlighted as crucial during the COVID-19 pandemic, Lipinski emphasizes. “Greater collaboration should continue into the future. Not only will it support national governments in preparing for future outbreaks, but it will also allow knowledge-sharing between businesses and academia to devise the technology and infrastructure needed to develop treatments more quickly,” he says.

“As mentioned before, the inherent challenges would remain as different pathogens emerge,” Phadnis concludes. “However, the rapid development of the COVID vaccines and their usefulness to control the pandemic has given us hope for a better future in vaccine development.”

For research use only. Not for use in diagnostic procedures. Agilent products are NOT authorized for COVID-19 testing, diagnosis, treatment, or mitigation. Agilent has not validated a product to detect the novel coronavirus.

Vaccines Market Size by Age Group (Pediatric, Adult), by Disease (Cancer, Hepatitis, Pneumococcal Disease, DTP, Dengue, Influenza, Human Papilloma Virus, Meningococcal Disease, Polio, Rotavirus), by Technology (Conjugate, Live, Inactivated, Recombinant, Toxoid), Industry Analysis Report, Regional Outlook, Application Potential, Price Trends, Competitive Market Share and Forecast, 2020–2026

Felicity Thomas is the European editor for BioPharm International.

When referring to this article, please cite it as F. Thomas, “Overcoming Vaccine Development Challenges,” 

Development

Vaccine development is inherently challenging; however, in light of the COVID-19 pandemic, innovations have been prioritized, leading to accelerated development processes.

Overcoming Vaccine Development Challenges

The global market for recombinant cell culture supplements is forecast to grow more than 6% from 2021–2031 as demand rises, and specific demand for microorganism-based recombinant cell culture supplements is expected to grow at an even higher pace, driven by rising demand for animal-free cell culture supplements (1). Microorganism-based recombinant cell culture supplements offer the benefit of eliminating the risk of contamination from cell culture media and are animal-free derived material.

Challenges in traditional media and supplements

Among the ongoing challenges in commercial-scale cell culture using traditional media and supplements has been a reliance on further supplementing traditional media with material such as human AB serum, human serum albumin, or platelet lysate, says Alexis Bossie, director of Media R&D, Lonza. Often, these undefined supplements are bound to various cytokines, hormones, and other growth factors necessary for primary cell growth and function, but, unfortunately, lot-to-lot variation of these components often leads to differential responses from cells and can produce unwanted side effects, she states.

“Additionally,” Bossie continues, “as the number of cell and gene therapy clinical trials and commercial applications increases, the supply of these critical raw materials is becoming increasingly limited and can cause delays in patient treatments.”

Trissa Borgschulte, head of Bioprocessing Upstream R&D at MilliporeSigma, also points out that having enough capacity to supply critical, high-quality raw materials that are needed for traditional cell culture media and supplements (e.g., animal sera) continues to be a major ongoing challenge. Specifically, COVID-19 has negatively impacted many critical supply chains. “There just aren’t enough materials to supply manufacturers with what they need for their bioprocesses,” she says.

Meanwhile, one of the longer-lasting challenges surrounding traditional cell culture media and supplements is related to patient safety and stems from the potential risk of adventitious agents potentially introduced into bioprocesses from animal-derived raw materials. “To reduce this risk, there is a strong push to remove animal-derived components from modern bioprocesses whenever possible. As a result, today’s standard production processes for recombinant therapeutic proteins are chemically defined and non-animal origin (NAO),” Borgschulte says.

Sonjoy Mukerjee, PhD, principal application scientist, Cell Culture and Cell Therapy, Thermo Fisher Scientific, adds that scale-up, manufacturability, reproducibility, and achieving consistent performance are also among the challenges encountered with using traditional cell culture media and supplements. He explains that this variability in performance can result in sudden spikes in titer, variability in product quality, and difficulties with developing a consistent manufacturing process.

“Additionally,” Mukerjee continues, “there is an increased need to prevent mycoplasma contaminations through the use of 0.2-micron or even 0.1-micron filtration steps at the end of the final formulation. This poses a challenge because it adds additional steps to the process that could be avoided with the use of raw materials that are validated to more stringent bioburden specs.” Finally, variations in glycosylation profiles can limit the ability of quality control (QC) teams to release final batches because it is difficult to set these kinds of limits.

“These challenges highlight the importance of manufacturing supplements and media specifically for biomanufacturing to provide better and more consistent performance,” Mukerjee says.

Benefits of microorganism-based recombinant material

Several recombinant proteins and peptides are already used in many cell culture media, including, for example, transferrin and insulin, says Bossie, who explains that using defined components in place of the alternative form that is isolated from serum leads to less lot-to-lot variability and can produce more robust cellular responses. “Elimination of human or animal sources for media components reduces the risk of virus or other contaminants and eases the regulatory burden on the final therapeutic product,” she says.

“As mentioned above,” adds Borgschulte, “today’s standard bioprocesses for the production of recombinant therapeutic proteins are chemically defined, NAO, and, in some cases, protein-free. Optimized media formulations and robust bioprocesses have resulted in manufacturing templates that only require low-molecular weight, recombinant peptide growth factors for the cell to thrive.” By contrast, however, the rapidly growing field of novel gene and cell therapies still requires more sophisticated media formulations owing to the diverse nature of cells (i.e., primary cells, embryonic and tissue-specific, regenerative stem cells, induced pluripotent stem cells) used therein, she points out.

Thus, for these close-to-the-patient cell expansions and/or differentiation media, sophisticated supplementation will need to closely mimic the bodily environment from which they are taken, Borgschulte further explains. “Not surprisingly, high-quality bovine and clinical-grade human sera, or fractionated components thereof, are still at the core of these protocols. Replacing sera with supplement cocktails containing only NAO recombinant growth factors and transport proteins is an achievable goal,” she states

Recombinant supplements are already used in commercial cell culture media. Recombinant insulin and transferrin are two examples of widely used recombinant supplements in many cell culture media, Bossie says. She also points out that cytokines and interleukins (e.g., IL-2, IL-7, IL-15) as well as other growth factors (e.g., Rho kinase inhibitor, TGF-) are common recombinant proteins used in cell culture media.

While microorganism-based substitutes for undefined serum-derived components could alleviate some of the challenges associated with commercial-scale cell culture, care must be taken, however, to also supply the additional growth factors that are non-specifically associated with the native forms of these components, Bossie cautions. For instance, trace elements, lipids, interleukins, and as-yet-unidentified other proteins might also need to be added to the cell culture medium when recombinant supplements are used in place of the native form isolated from human serum.

“Determining the optimal concentrations of all of these ingredients can be a time-consuming discovery effort,” Bossie says. Moreover, the costs associated with additional components can result in an extremely expensive medium, resulting in an additional cost burden to the therapeutic product.

Demand for recombinant animal-free components in cell culture media is growing especially on the back of concerns regarding contamination in raw materials. Borgschulte points to the threat to the supply of porcine trypsin (African Swine Fever) and general animal-origin concerns, as well as the current need to accelerate COVID-19 vaccine programs, for example, as driving factors for increased recombinant animal-free cell culture components.

Meanwhile, peptone hydrolysates, specifically manufactured for biomanufacturing, are being widely used in both pre-clinical and all phases of clinical trials as well as many marketed molecules, emphasizes Mukerjee. “Successful applications include monoclonal antibodies, microbial fermentation applications, and various vaccines for both human and animal health,” he says.

In the area of cell therapies, there is need for greater consistency as well as higher regulatory standards, which is also driving the increased use of recombinant growth factors wherever possible, says Bossie. “This trend should be expected to continue,” she continues, adding that “hurdles to greater utilization of recombinant proteins revolve around the need to more completely understand the metabolic demands of the cells to be able to provide all of the necessary ancillary factors bound to or associated with the serum-derived components.”

Once there is greater utilization of the recombinant proteins in place of serum-derived ingredients, however, the economies of scale in their manufacture and increased competition among manufacturers should serve to drive down costs of these materials, Bossie adds. Until then, the high cost of some recombinant proteins might limit their adoption, she says

Furthermore, NAO alternatives reduce the risk of introducing adventitious agents to the biomanufacturing process, Borgschulte confirms. Additionally, recombinant cell culture supplements can offer more consistent purity and activity levels, she explains. “In the growing field of cell and gene therapy, high-quality animal derived supplements are still commonly used. However, replacing these supplements with NAO recombinant growth factors and transport proteins is the direction of the future,” she states.

“Many companies in this space are looking for lot-to-lot consistency for the media and supplements that they use in their processes,” adds Mukerjee. These companies also want full traceability of the components that make up the media and supplements. “These are a couple of the drivers towards microorganism-based recombinant cell culture supplements and media. It’s possible that these type of supplements and media could replace traditional supplements and media, but it would come at a higher cost. The incremental added benefits of these newer supplements might not outweigh this increased cost and given that there are many high-quality options on the market right now, it’s not likely that companies will be making the switch anytime soon,” he concludes.

1. Persistent Market Research, “Recombinant Cell Culture Supplements Market,” 

When referring to this article, please cite it as F. Mirasol, “Recombinant Supplements Offer Lower Risk of Cell Culture Contamination,” 

Upstream Processing

Demand for recombinant microorganism-based cell culture supplements is rising on the back of lower contamination risk.

Recombinant Supplements Offer Lower Risk of Cell Culture Contamination

The past few years have brought significant cost and competitive pressures to biopharmaceutical manufacturers. Process intensification and integration have allowed manufacturers to address these challenges, improving yields and reducing timelines.

Conditions are now set for change. “For monoclonal antibodies, outputs continue to increase, kilograms per batch are getting lower, and we’re getting higher titers out of cell cultures. In addition, there is a need for more flexible, adaptable manufacturing plants, particularly multiproduct plants, that can be run more flexibly,” says Darren Verlenden, head of bioprocessing at MilliporeSigma.

More companies are moving beyond intensification and evaluating continuous processing, which would usher in a future of lower manufacturing costs, real-time product release, and predictive control. “Continuous bioprocessing promises to transform biomanufacturing by reducing inter-campaign downtime, removing time- and labor-consuming steps, and reducing risk by chaining or combining serial steps in current processes,” says Phil Vanek, chief technology officer at Gamma Biosciences, which specializes in tools and technologies for advanced biotherapeutics development and manufacturing.

However, Vanek says, “in order for continuous biomanufacturing to work, we need to think about each manufacturing process in its entirety and seek areas where product can be harvested in real time without disrupting the ongoing process.” He visualizes highly productive expression systems under tight process control, chained to efficient cell harvesting and subsequent downstream technologies, which can be developed into modular continuous biomanufacturing platforms, all within a highly efficient and compact footprint. An example of this, he says, is Univercells Technologies’ NevoLine biomanufacturing platform for intensified and automated viral production.

Currently, most companies are applying continuous processes upstream, particularly to perfusion. Within 10 years, such efforts are expected to drive reactor productivities from 0.05–1 g/L per day to 0.5–10 g/L per day (1). Technology providers, including Cytiva, Sartorius, and MilliporeSigma have all released new automated perfusion systems and bioreactors. However, the continuous concept is also being developed downstream, enabled by new and improved alternatives to traditional chromatography. WuXi Biologics is using continuous perfusion and downstream processing in its WuxiUp platform.

Eliminating clarification and product hold time

Transcenta Holdings is working to improving continuous processing both upstream and downstream functions. Based in China and United States, the company was formed in early 2019 when two drug development companies merged. MabSpace brought a focus on novel technology in discovery and research, while Hangzhou Just Biotherapeutics contributed strengths in chemistry, manufacturing, and controls development and bioprocessing platform. “The idea was to combine two companies with complementary cutting-edge technologies in R&D and biomanufacturing. The goal is to use innovative approaches to develop and deliver affordable innovative drugs to patients with unmet medical needs across the globe,” says Chris Hwang, chief technology officer of Transcenta, and a 25-year veteran of Genzyme and Sanofi. Although there is still much work ahead, continuous bioprocessing offers a number of advantages, he says.

Transcenta’s manufacturing platform, Integrated Continuous Bioprocessing (ICB), uses continuous perfusion that can achieve very high productivities by maintaining cells at high densities in a productive state for the duration of each run to reduce downtime, he says. The company plans to rely on process integration with intensified and connected/continuous downstream to remove process bottlenecks where production takes place.

By implementing ICB, Transcenta will also be able to eliminate harvest clarification and product hold steps, says Hwang. Those changes, combined with robust process control and automation, promise to result in significant reductions in footprint, labor, materials, consumables, and operator errors and overall cycle time.

Transcenta envisions a future based on small, closed, and highly automated single-use flexible facilities that use continuous processes and can be set up quickly and inexpensively, much like its facility T-BLOC in Hangzhou China. T-BLOC was built in January 2018, using G-CON’s prefabricated modular clean room technology, the first of its kind for protein production. Also, the facility will fully leverage single-use technologies, allowing it to support multi-product and scale-out as necessary to support clinical and commercial production needs.

“Since we opened our T-BLOC facility, we have invested significant efforts in developing our own cell lines, chemically defined and low-cost cell culture media (< 1/10 of commercial media), and as well as our own continuous perfusion platform. Since then, we have demonstrated a more than 10-fold increase in process output when compared to conventional fed-batch processes for multiple cell lines and molecules,” says Hwang. He estimates that it would take 22 2000-L single-use or four 12,500-L stainless-steel bioreactors in fed-batch mode to match the output of four 1000-L single-use bioreactors using the same cell line in their plug-and-play perfusion platform.

To handle such a high cell-culture output efficiently without large-scale equipment, Transcenta is also developing continuous downstream bioprocesses in parallel. To accelerate implementation of ICB for good manufacturing practice production in its T-BLOC facility, Transcenta established a strategic technology collaboration with MilliporeSigma in mid-2020, leveraging its BioContinuum platform, as well as its expertise in continuous processing (2). However, rather than go all in to end-to end continuous, the team decided to implement in a stepwise manner, to minimize any risk to manufacturing operations.

The first phase of this multi-year partnership, the team will co-develop a first-of-its-kind single-use flow-through polishing system, says Hwang, and the second phase will focus on expanding the boundary of continuous to the rest of the process, as well as digital technologies to optimize ICB. “To further minimize manufacturing risks, the technologies we are developing and implementing need to strike a right balance of risks and benefits with strong preference for simplicity as oppose to complex systems,” he added., “Significant progress has already been made and I expect our end-to-end continuous and automated ICB technology will be ready for operation in two years in our T-BLOC facility and ready to support clinical and commercial production,” Hwang says.

Transcenta’s work reflects greater industry interest in continuous processing across the board. Pilot-scale continuous operations are showing progress, says Verlenden. A recent report projected demand for continuous bioprocessing to increase by nearly 23% per year through 2027, reaching $198 million by 2027 (3). At this point, the only dampening factor appears to be concern about regulatory acceptance of continuous, Verlenden says, noting a recent company survey on BioPharma 4.0, in which respondents found regulatory concerns to be the largest hurdle for companies to overcome. “This may prevent some companies from moving beyond pilot level,” he says.

At this point, savings are attracting more companies to continuous manufacturing approaches. “Continuous processing has been shown to demonstrate cost-of-goods reductions for monoclonal antibody purification, especially for orphan drugs,” says Dmitrii Sorokin, drug product development engineer at Exothera, a contract development and manufacturing organization affiliated with Univercells that specializes in viral vector manufacturing. “In these cases, the cost of Protein A resins is pushing developers and manufacturers to explore alternatives to big expensive chromatography columns, and continuous multi-column chromatography (MCC) instruments offer an opportunity to decrease investment during development, clinical trial material manufacturing, and commercialization of such products,” says Sorokin.

For products that are difficult to produce using traditional technology—often due to stability issues or cytotoxic/cytostatic effects on cell culture—a combination of upstream perfusion, continuous downstream processes using MCC, and in-line conditioning and tangential flow filtration (TFF), are being used in development, says Vasily Medvedev, head of development at Exothera. “In viral processes, more efforts are using continuous purification for sensitive viruses, to allow purification to take place more quickly, since target recovery remains one of the industry’s top challenges,” he adds.

At the same time, advances continue to be made in downstream bioprocessing technology itself. In January 2021, Sartorius Biotech acquired Novasep’s chromatography business, while, in April, the company opened a new center of excellence for downstream bioprocessing in the United Kingdom.

Improvements are also being seen in traditional products such as Protein A resins. “Next generation medicines are complicated systems ranging from in-vitro transcribed RNA, purified extracellular vesicles, viral-based vectors, and vaccines, to genetically modified cells,” says Vanek.

Although these therapies promise transformational health outcomes for patients underserved by conventional medicines, Vanek explains, their complexity translates into manufacturing challenges that range from inefficient production steps to difficulties in packaging and distribution, he says.

The introduction of affinity technologies, most notably Protein A in the monoclonal antibody workflow, has boosted productivity by simplifying the number of steps necessary to achieve functional purity. Affinity methods will continue to be applied to all forms of advanced therapies with the goal of improving recovery and quality of final product, as well as reducing cost through process simplification, Vanek says. In addition, he notes, identifying highly tunable ligands and conjugating them to appropriate chromatography substrates such as resins, magnetic ferrofluids, or electrospun non-woven membranes promises to open up a whole new dimension of bioseparation possibilities.

Nanopareil electrospun nanofibers, one of Gamma Biosciences’ newer bioseparation product offerings, combine extremely efficient chromatographical properties such as short residence time and high dynamic binding capacity with large pore sizes that are better suited to advanced therapy modalities such as viral vector and exosomes. “This platform is being developed with traditional chemistries as well as Astrea Bioseparation’s affinity ligands to address new applications,” Vanek says.

Nanofiber chromatography technology promises to improve cell and gene therapy capture and polishing applications, offering high binding capacities, fast cycle times, and scalable formats, says Fletcher Malcom, head of product at Nanopareil. As a result, this technology can help reduce processing time, chromatography facility footprint, buffer requirements, and waste significantly, shortening time to market while cutting costs and ensuring environmental sustainability, he says.

In viral-vector manufacturing, downstream processing is adapting chromatography and TFF steps to aseptic processing for viral particles that are too small for sterilizing-grade filters, says Sorokin. Currently, more chromatography materials are available as membrane capsules and monoliths, which can be gamma-irradiated and ready to use in closed processes. In addition, presterilized TFF hollow-fiber cartridges are available with aseptic connectors, he says.

“This trend is unlocking aseptic downstream processing at a large scale, which is critical for several categories of viral vector products (e.g., oncolytic viruses),” says Medvedev. “Another trend we are following closely is the availability of new modalities for separation of viruses (e.g., steric exclusion chromatography), which offers value in cases where conventional affinity or ion exchange resins would result in low recovery,” he adds.

“Many anion exchange diffusion-based media (e.g., resins based on Q-Sepharose) that are suitable for viral vector purification are being replaced by single-use convective media (e.g., anion exchange membranes or Q-adsorber capsules),” says Sorokin, “but a lot of polishing steps are required for monoliths to remove aggregates and residual DNA-VP complexes.”

More biopharma developers are also looking into the idea of combining unit operations, such as the midstream approach used in Univercells Technologies’ NevoLine or coming up with innovative approaches for sensitive targets such as Repligen’s tangential flow/depth filtration concept for lentivirus, says Medvedev.

As downstream bioprocessing improvements continue, efforts are underway to integrate downstream processes into continuous operations. 

BioPharm International-05-01-2021

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