Feliza Mirasol

Major bio/pharmaceutical companies and contract service providers leading the charge to bring COVID-19 vaccines to market are familiar with established good manufacturing practices (GMPs). To support manufacturers in the rush to develop a vaccine, FDA, in June 2020, released industry guidance (1) highlighting the development and licensure of COVID-19 vaccines.

Pharmaceutical Technology October 2020 Regulatory Sourcebook

For bio/pharmaceutical companies, quality must be more than a vague concept. Quality practices impact all aspects of drug development and manufacturing. In the 

Pharmaceutical Technology Regulatory Sourcebook

, experts share insights and best practices on a range of quality issues including the following:

Enhancing Process Validation for Sterile Liquid and Freeze-Dried Forms: Part 1

AFI representatives of the process validation working group explore and define key elements for an enhanced approach to process validation for sterile liquid and freeze-dried forms.

Enhancing Process Validation for Solid Oral Dosage Forms: Part 2

AFI representatives of the process validation working group explore and define key elements for an enhanced approach to process validation for solid oral dosage forms.

Due Diligence Assessment of CMC Activities

For planned acquisitions or licensing, a careful analysis of CMC factors is vital to ensure no problem areas are overlooked.

Do Pharmacopoeias Inadvertently Facilitate Data Integrity Violations?

Analysts should understand how a monograph, together with the associated general notices and general chapters, relate to their responsibilities under good manufacturing practices.

Tackling Cybersecurity Challenges in Legacy Systems

Ongoing and emerging cyber-risks to legacy operational technology systems in bio/pharmaceutical manufacturing must be addressed.

Ending ‘Magical Thinking’ in Compounding

Consistent product quality requires a clear understanding of the essence of CGMPs. Experts fear that the message is not always getting through to compounders.

Viral Vector API Characterization of Product-Related Impurities

FDA’s final CMC guidance sets expectations for manufacturing and quality for human gene therapy INDs. This article reviews existing analytical applications, focusing on viral vector characterization of impurities.

Viewpoint: Precompetitive Collaboration Drives Pharma Industry Innovation

The IQ Consortium marks a decade of impactful innovation of collaboration through a network of pharmaceutical companies driving change for the bio/pharmaceutical industry.

Strong Quality Culture: A How-To for Busy Managers

Building employee participation and forming good habits contribute to a company-wide quality culture that pays off.

Pharmaceutical Technology October 2020 Regulatory

www.PharmTech.com/journals/pharmaceutical-technology/pharmaceutical-technology-10-15-2020

For example, FDA noted in the guidance that no “accepted surrogate endpoints” to which clinical testing can aim currently exist. Without these target endpoints, there is likely no ability to form a reasonable prediction that a potential COVID-19 vaccine would demonstrate a clinical benefit. FDA has recommended that any vaccine development program should, therefore, follow a traditional route to approval, meaning the development programs should demonstrate the vaccine candidate’s safety and efficacy in protecting patients from infection by the SARS-CoV-2 virus and/or clinical disease that results from infection.

“The FDA Guidance highlights the pertinent CGMP [current good manufacturing practice] considerations for all vaccine manufacturing and serves as a reminder of statutory GMP expectations for COVID-19 vaccines specifically,” says Karolyn Gale, senior director, regulatory affairs, Emergent BioSolutions, a contract development and manufacturing organization (CDMO). The CDMO was granted a $628-million task order under an existing contract with the Biomedical Advanced Research and Development Authority (BARDA), part of the Office of the Assistant Secretary for Preparedness and Response at the US Department of Health & Human Services, for the
rapid, large-scale production of leading
COVID-19 vaccine candidates through 2021. Emergent will manufacture vaccine drug substance at its Baltimore Bayview facility in Maryland and will manufacture drug product and conduct fill/finish operations at its facilities in Baltimore, Camden, and Rockville, MD (2). The company currently has deals with Johnson & Johnson and AstraZeneca to manufacture their respective COVID-19 vaccine candidates (3).

“As with all FDA guidance, it gives sponsors and manufacturers clearer insight into the FDA’s current thinking on a topic and how they are leveraging and/or interpreting, in this instance, standing 21 [

 regulations for chemistry, manufacturing, and controls (CMC) in the COVID-19 vaccine development landscape,” says Gale.

For drug substance and drug product manufacturing, the guidance emphasizes that data be provided showing adequate control over all source material used in the manufacturing of the vaccine, including the history and qualification of cell banks and virus banks as well as the identification of all animal-derived materials that are used for cell culture and virus growth.

“It is recognized that this guidance will not cover all the considerations necessary to satisfy all statutory and regulatory requirements for licensure. It further acknowledges that in some instances, platform knowledge could be leveraged, the specifics of this potential and the science they are based upon must be considered carefully, thus highlighting the importance of early engagement with the agency further,” Gale states.

Three important challenges to overcome include being able to validate currently unproven platform technologies, demonstrating that the vaccine confers protection against SARS-CoV-2 infection/COVID-19 disease, and targeting an appropriate vaccine design (4).

Vaccines being developed with unvalidated, unproven platform technologies (e.g., DNA- and messenger RNA-platform based vaccines) raise uncertainties about long-term safety and the ability to confer long-term immunity against COVID-19. These newer platform technologies may offer unique advantages (e.g., accelerating timelines), but vaccines developed from them may face greater regulatory scrutiny than vaccines coming from traditional established technologies.

In designing a vaccine, developers must establish a sufficient “indicator of protection” (4) (e.g., antibody response levels) to first demonstrate that the vaccine protects against COVID-19 and take into account the SARS-CoV-2 virus’ mutation potential, which can impact the vaccine’s relevance and delay launch.

Ensuring that the overall regulatory strategy for a vaccine product is understood by a CDMO facility/partner chosen by a vaccine developer is one of the biggest regulatory challenges currently, says Gale. This transfer of knowledge and strategy is especially challenging considering the speed with which a COVID-19 vaccine is needed. “This is accomplished by ensuring the appropriate contacts are in place between the two organizations and that the lines of communication are open between the two entities that allow for exchange of pertinent information in real-time. Pertinent information includes FDA (or other health authority) feedback that informs or changes the overarching strategy for either the facility or the product development,” Gale says.

“Another challenge we face currently is pursuing FDA feedback for the facility and its capabilities while keeping the interests of more than one client and/or COVID-19 vaccine candidate in mind,” she continues. “The landscape is constantly shifting, and new information is coming in daily; however, decisions for the facility and/or process need to be made to move forward. Despite these challenges, FDA, BARDA, and innovator companies have been supportive throughout the process.”

“Manufacture of vaccines is a complex process that requires multiple unit operations to be executed in accordance with CGMPs,” Gale adds. “The development and scale up of a commercial manufacturing process takes up to several years. As we work with our partners to support their COVID products, we are accelerating these processes from years to months. The technical challenges we face relate to ensuring we have a robust and reproducible manufacturing process that results in high-quality product.”

Guidance for Industry: Development and Licensure of Vaccines to Prevent COVID-19

 (CBER, June 2020).
2. Emergent BioSolutions, “Emergent BioSolutions Joins US Government’s Warp Speed Program in Landmark Public-Private CDMO Partnership for COVID-19 Vaccine Development and Manufacturing,” Press Release, June 1, 2020.
3. Emergent BioSolutions, “Emergent BioSolutions Reports Financial Results for Second Quarter and Six Months Ended June 30, 2020 and Revises Upward Full Year 2020 Guidance,” Press Release, July 30, 2020.
4. G. Agrawal, et al., “On Pins and Needles: Will COVID-19 Vaccines ‘Save The World’?” www.mckinsey.com, July 9, 2020. 

Feliza Mirasol is the science editor for 


Vol. 44, No. 11
November 2020
Pages: 50–52

When referring to this article, please cite it as F. Mirasol, “

GMPs Guide COVID-19 Vaccine Manufacturing

Articles

Recent industry guidance aims to anchor rapid COVID-19 vaccine development in good manufacturing practice protocols.


Cell therapies are a relatively new type of biotherapeutic in the marketplace. As such, their commercial manufacture poses unique challenges in biomanufacturing that necessitate the need for new thinking and innovative solutions. Adding to the pressure of maintaining a sterile manufacturing environment are the restrictions imposed by the current COVID-19 pandemic, which has had wide-reaching effects across all industries. Additional manufacturing practices or operating protocols may be required to ensure aseptic manufacturing in the cell therapy arena during and post the pandemic.

Speed, safety, reliability, and cost management are among the normal challenges faced by cell therapy manufacturing. But a unique challenge that cell therapies face is the variability in donated cells that form the starting material of the therapy.

For instance, autologous chimeric antigen T-cell (CAR-T) cell therapy requires a sophisticated manufacturing process and uses cells collected from an individual cancer patient, says Ryan Larson, senior director, cell therapy product and analytical development, Bristol Myers Squibb. “During the autologous process, a patient’s cells are collected through a process called leukapheresis, in which blood is drawn and white blood cells are separated out to use for manufacturing. The cells are then shipped to the manufacturing facility where they are reprogrammed to become CAR-T cells to recognize and fight cancer cells and then sent back to the patient’s certified treatment center where they receive their CAR-T cells via a one-time treatment,” Larson explains.

“One key challenge in manufacturing autologous CAR-T product is between-patient variability in starting material,” Larson continues. “In some cases, a CAR-T manufacturing run may fail due to the detrimental impact of multiple rounds of chemotherapy for prior cancer treatments on T cells that serve as the starting material for the manufacturing process. Manufacturing process robustness to handle heterogeneous autologous starting material between patients and across disease indications is an important part of process design and development.”

Further, autologous cell therapy manufacturing is much more challenging than the manufacturing process for allogeneic cell therapies. The manufacturing process for an autologous cell therapy must accommodate for the variability in the source patient’s cells in order to consistently produce final product with desired quality attributes, notes Dr. David Chang, CEO of WuXi Advanced Therapies, the cell and gene therapy contract development and manufacturing organization business unit of WuXi AppTec. Manufacturers need to have shown “proven and qualified” end-to-end manufacturing capacity to regulators for commercial production. In addition, practices such as right-first-time are essential to ensure patients can receive drug production for treatment reliably in the shortest possible turnaround time, according to Chang.

In addition, many general cell therapy manufacturing operations are conducted in “open” settings, which require aseptic processing in a biosafety cabinet (BSC) in a Grade B cleanroom, Chang says. The extensive use of sterile disposable components and complicated raw materials requires intensive supplier management effort. “There are also complicated analytical methods (e.g., flow cytometry, potency assay) for in-process control and release testing that are difficult to develop and qualify/validate,” Chang adds.

Specifically, cell-therapy manufacturing processes are most vulnerable where there is open processing under a BSC. Fortunately, significant progress has been made to date in “closing” these operations through the use of tubing welding/connections that allow the manufacturing processes to take place out from under the BSC, thus reducing vulnerabilities, notes Chang. There remain a few steps, however, such as viral transduction and cell harvesting, that are harder to “close”. 

Safeguards for sterile operation include going through formal aseptic processing validation (APV) to further assure sterility throughout processing. “It is important to conduct extensive in-process and final release sterility testing to ensure final drug product sterility,” says Chang.

Allogeneic CAR-T cell products, in comparison, are produced in large batches from normal healthy donor starting material (e.g., stem cell bank or primary T cells). With allogeneic cell therapies, a single batch can be used to treat many patients, Larson explains.

“These products may be easier to produce than autologous products, because the starting materials are derived from healthy donors rather than heavily pre-treated patients. However, challenges remain with regards to gene-editing strategies that mitigate risk of graft versus host disease and engraftment,” Larson continues. “In addition, stem-cell based allogeneic T-cell therapies are being developed in which a CAR-T cell product is manufactured from a master stem cell bank, which has the benefit of lot-to-lot consistency in starting material. Allogeneic cell therapy products will reduce manufacturing costs while providing patients with quicker access to ‘off-the-shelf’ treatment and more consistent therapeutic doses.”

In both allogeneic and autologous cell therapy, Chang points out that manufacturers have to contend with requirements for maintaining chain-of-identity throughout the manufacturing and transportation processes to prevent mix-ups or cross-contamination among patients’ lots. “Highly manual operations demand a high proportion of direct labor, which results in a high cost of manufacturing and is susceptible to human error,” Chang states.

Working with what are fundamentally “living therapies” in the cell (and gene) therapy field also creates one of the most complex supply chains in medicine, says Tamie Joeckel, global business lead, cell and gene therapy, ICON. “Orchestrating the cell therapy logistics for both source materials and final doses requires detailed planning and hands-on management. The end-to-end processes of tracking and tracing the movement and handoffs of these shipments among stakeholders are rife with risk and potential points of failure,” she says.

An added dimension to this supply-chain challenge lies in the fact that, in some programs, the patient and site schedules are co-dependent and require close coordination. “Many patients must travel to specialty centers, requiring the additional coordination of concierge services for both the patients and their caretakers. Any change or disruption in schedule ripples through the entire workflow and can affect the product chain-of-custody, chain-of-condition, and chain-of-identity that are required to ensure patient and product safety,” Joeckel says. “New disruptions caused by the COVID-19 pandemic exacerbate the risks of these critical shipments.” 

The challenges posed by cell therapy manufacturing have pushed forward biomanufacturing innovations already under development or in use for monoclonal antibody-based and protein-therapeutic-based production.

“In my opinion, innovations in cell therapy are actively taking place in several fields, such as disposable technologies, automation, process integration, and digital solutions,” notes Chang. The effort of automation and process integration is addressing the challenges faced with the highly manual cell therapy processing steps, such as sampling, medium exchange, in-process monitoring/testing, and cell manipulations (e.g., activation, transduction, harvest, etc.), Chang explains.

“I believe that there has been some progress made in automation, but cell therapy needs significantly more innovations in this area in order to transform the manufacturing paradigm. There are innovations in process integration that are aimed at fully integrating end-to-end manufacturing steps into a turn-key solution,” Chang says.

“Digital solutions are also evolving, such as cell-therapy-manufacturing-friendly MES [manufacturing execution system], LIMS [laboratory information management system], and ERP [enterprise resource planning] systems that are being actively developed and/or optimized,” Chang continues. “These digital solutions need to be fully integrated to be able to communicate in real time during manufacturing and release testing. It is also critical to connect manufacturers to clinics electronically to ensure the chain-of-identity is fully maintained.”

Ultimately, there is an urgent need for innovation, especially in automation, to transform cell therapy manufacturing, Chang concludes. The primary goals of innovation are to lower the cost of goods, reduce human error, and accelerate timelines with greater predictability.

“At Bristol Myers Squibb (BMS), we embrace digitization and have invested in the use of next-generation technology,” says Chris Ramsborg, vice-president, cell therapy process science and technology, Bristol Myers Squibb. BMS has invested in the development of its proprietary NEX-T manufacturing platform, for example, with which the company may be able to create a manufacturing environment that is optimized to potentially reduce turnaround time and increase product control.

“BMS currently has two active clinical trials evaluating our NEX-T technology for a CD-19-directed CAR-T cell therapy in relapsed or refractory B-cell non-Hodgkin lymphoma and an anti-BCMA CAR-T cell therapy in relapsed and/or refractory multiple myeloma. We recently dosed our first patient using this technology and look forward to seeing more progress,” Ramsborg says.

“In addition to NEX-T, we are also continuing to invest in, evaluate, and develop new manufacturing platforms to advance scientific innovation in cellular therapies, researching technologies for overcoming resistance to CAR-T cell therapies, investigating use in solid tumors, and potentially creating allogeneic CAR-T cell therapies for patients,” Ramsborg adds.

The manufacture of cell therapy product carries vulnerability and risk, which have been further pronounced during the COVID-19 epidemic.

For CAR-T cell therapies, every stage is vulnerable to scientific and technical challenges stemming from the inherent variability of the autologous starting material, states Larson. To mitigate these manufacturing vulnerabilities, BMS is creating a simple digital platform that streamlines handling, chain of identity, ordering, and scheduling to expedite delivery of CAR-T cell therapy, he says.

“We are also optimizing manufacturing through automation to make processing units more efficient while supporting product consistency,” Larson says, explaining that current CAR-T manufacturing processes require delivery of a transgene encoding the CAR, which induces T cell activation and co-stimulation in response to target antigens expressed by the tumor cells.

“Sourcing and control of the gene delivery raw materials, whether it be viral or non-viral modalities, introduce challenges to the CAR-T manufacturing process. Prioritizing this area to increase knowledge through identifying biologically relevant critical quality attributes and building a supply chain for GMP [good manufacturing practice] gene delivery, raw materials will continue to increase the technical maturity of this element of CAR-T manufacturing,” Larson continues.

The company’s investigational autologous CAR-T cell therapy, lisocabtagene maraleucel (liso-cel) for treating multiple hematologic B-cell malignancies, for example, uses a manufacturing process designed to support robustness and consistency for a diverse patient population across different B-cell malignancies. “The process employs technologies for purification, activation of the starting material, and gene delivery that aims to reduce the variability between patient populations, supporting consistent and predictable manufacturing duration and product quality,” states Larson.

Another manufacturing vulnerability lies in maintaining chain of identity, especially in autologous cell therapy manufacturing. “To address this challenge, manufacturers should establish procedures and trainings to ensure operations maintain end-to-end chain of identity throughout manufacturing process. In addition, it is strongly desired to use fully integrated digital solutions (e.g., MES, LIMS, ERP, etc.) to achieve this goal,” Chang states.

Furthermore, cell therapies are both time and temperature sensitive, leaving them at the mercy of transport logistics. “[Cell therapy] logistics is an already complex, finely tuned, orchestrated process involving numerous stakeholders—and disruptions caused by COVID-19 in site procedures, patient re-scheduling, and shipment delays can ripple through the workflow, placing both the product and the patient at risk,” indicates Joeckel.

For example, the end-to-end supply chain involves a large number of handoffs between internal departments at the sites and external service providers. “More importantly, for many patients, these therapies may be their final hope—and patients in the final stages of their disease may not be able to physically withstand an additional procedure should their cell shipments be damaged or lost. It is truly a ‘zero defect’ environment. Mapping out the detailed workflows and validating the entire process is critical,” Joeckel asserts. 

Moreover, because autologous therapies involve cells from the patient—which are used to manufacture the final dose that goes back to the same patient—another element that needs to be safeguarded is that of “track and trace.” Safeguarding “track and trace” activity is important for maintaining the chain of identity of the material—from the initial collection of the patient’s cells to transportation from the apheresis center to the manufacturer—all throughout the manufacturing process of the final dose and transport back to the site of administration, Joeckel continues.

“A common delimiting factor in autologous programs is managing limited manufacturing capacity. Manufacturing schedules must be coordinated between sites, labs, patients, apheresis centers, specialty couriers, and the manufacturing center to collect the cells and ship them overnight to the manufacturing facility. These refrigerated shipments are highly time sensitive, and the manufacturer must be ready to start the process upon receipt,” says Joeckel.

In the face of the current COVID-19 pandemic, manufacturers are learning to navigate not only the complexity of cell therapy manufacturing and development, but also restrictions and necessary shifts in daily operating protocols.

“During the initial COVID-19 outbreak, the majority of clinical trials, especially in cell therapy, were put on hold,” says Chang, who goes on to say that “since some restrictions now have been eased or lifted, hospitals and clinics are once again able to receive and treat patients for clinical or commercial cell therapy applications.” WuXi Advanced Therapies (WuXi ATU) proactively increased critical inventory and is evaluating alternative suppliers for future inventory.

Autologous cell therapy clinical trials are complex in nature, and COVID-19 adds to this complexity, stresses Ramsborg. Because the cell therapy requires transportation of cells from patients to manufacturing sites and back, travel restrictions both in-country and across countries have a significant impact, he emphasizes.

“Lastly, in contrast to clinical trials of many investigational products, initial treatment with a cellular therapy product requires a lead time of several weeks. Because of the uncertainty created globally by COVID-19, healthcare professionals may be conservative about starting patients on an investigational therapy in a clinical trial, given that the trial, and thus the treatment course, may be interrupted by the uncertainties of the evolving pandemic,” notes Ramsborg.

In response to COVID-19, BMS had early on made the necessary but difficult decision to temporarily suspend screening, enrollment, and apheresis in its cellular therapy clinical trials. “That pause has since been lifted, but we are continuing to monitor the COVID-19 situation to ensure that we are supporting the safety of study subjects, staff at clinical trial sites, and our employees while also ensuring regulatory compliance and the scientific integrity of trial data,” Ramsborg states.

Disruptions varied by region, says Joeckel, but can start with the cell collection step. For example, some apheresis centers had reduced schedules or stopped operating altogether to limit the exposure of their staff and donors to COVID. Sites concerned about the transmission of the virus, especially to vulnerable patients, also had to cancel or delay appointments, and many sites reassigned personnel, which affected patient enrollment, access, and capacity. As a result of these measures, restricted access to healthcare facilities and new COVID safety and screening procedures should be included in protocols and training manuals going forward, says Joeckel.

A critical issue being faced currently is receiving shipments and supplies to and from sites, Joeckel adds, noting that border closures and reduced air traffic have disrupted the supply chain around the world. “Flight delays are more common, requiring hands-on management and monitoring of the temperatures and hold times of the cells and requiring more robust intervention procedures. Supply shortages have also caused problems—causing some service providers to shut down entirely,” she states.

“Until we have an effective vaccine for COVID-19, the threat of disruptions to the manufacture and delivery of cell and gene therapies remains. The current pandemic is far from over and the threat of new outbreaks is a possibility. Continuing to find ways to ensure business continuity, supply chain resilience, and keeping development programs on track requires ongoing innovation, flexibility, and agility. The overarching reason for urgency is quite simple—there are patients waiting for life-changing, life-saving therapies at the end of every supply chain,” asserts Joeckel.

WuXi ATU, which has made a strategic move by diversifying its suppliers, sees value in conducting extensive risk assessments of its facilities, including reviewing current manufacturing operations and the potential impact from COVID-19 on product safety. “We’ll then take that information and develop and implement mitigation efforts to ensure we can continue to conduct high quality aseptic manufacturing operations. Through our enabling platforms, technology, and security of supply chain, WuXi ATU is ready to accelerate progress and time to market in the years to come,” Chang states.

“COVID-19 has led to many uncertainties, but we have proactively taken precautions to protect and support all our clinical trial participants, trial site staff, and employees, as well as protect the integrity of our clinical trials,” adds Ramsborg.

Automation, digitalization, and supply-chain strategies help mitigate vulnerabilities in both autologous and allogeneic cell therapy manufacturing.

Navigating Cell Therapy Manufacturing Amid Pandemic Woes

Protein aggregation is a particular challenge for biologic drug makers because this can occur at any point in the development or manufacturing process as well as during packaging and storage. Protein aggregates, typically impurities or molecular variants of the therapeutic protein, can change the therapeutic effect of the drug or cause protein degradation. This degradation can have serious implications for patient safety, product stability, potency, and biological activity, among other adverse consequences. Dealing with protein aggregation involves using analytical methods to detect and characterize the aggregates.

Fundamentally, protein aggregation occurs when proteins in their native state combine and aggregate or “clump” together in what is often a non-reversible process. This aggregation can occur either both inter- or intra-cellularly, caused by a change in the native structure of the protein. “The native structure is the most stable form of the protein, which essentially involves folding of the structure such that any hydrophobic regions are contained in the inner portion of the protein and hydrophilic on the exterior,” says Ashleigh Wake, business development director, Intertek Pharmaceutical Services, who explains that native structures are stabilized through a combination of disulfide bonds (formed between two cysteine residues) and non-covalent interactions, such as Van der Waals. “There are numerous ways in which these non-covalent interactions can be disturbed, which causes the protein to misfold or unfold, which ultimately leads to protein aggregations,” Wake states.

The formation of protein aggregates is attributed several main factors, including mutations in either DNA coding or other cellular proteins that are responsible for protein synthesis; variants in amino acid sequences or incorrect sequences that can occur when there is a problem with the translation of messenger RNA (mRNA); and aging of the cellular matrix. This reduces the ability of the matrix to refold protein or break down aggregated regions, according to Wake.

In addition, external factors play a role in protein aggregation, such as the protein being exposed to extremes of heat, pH, or oxidative conditions, says Wake.

Therapeutic proteins, however, require a specific structure for maximum functionality and activity, Jon S. Kauffman, PhD, vice-president, Biopharma Biologics, Eurofins Lancaster Laboratories interjects. “Aggregates can be introduced at any step of the process from production to dosing and can cause variability in potency, safety, and efficacy. Therefore, a thorough understanding of aggregation is critical to the success of biopharmaceutical drug candidates,” Kauffman says.

The mechanism by which aggregation increases immunogenicity is subject to considerable current research, but the inherent resultant particle size is certainly one factor that can be considered in this respect, Wake says.

Because protein therapeutics are administered to patients in the form of liquid injections as intravenous (IV), intramuscular (IM), or subcutaneous (SC), they often require high concentrations of proteins, thus making the formulation prone to aggregation. When a protein therapeutic aggregates in the blood stream, it has been shown to invoke immune response, which can result in the patient becoming immune to the monomer molecule, Kauffman elucidates. “The immune response is associated with adverse clinical effects, such as neutralizing the antibody, which inhibits efficacy of the products or causes cross-reactivity and neutralizes an endogenous protein counterpart. This may cause severe hypersensitivity response, such as anaphylaxis. Therefore, ensuring stability of the native molecule and minimizing the formation of higher molecular weight species is vital in reduction of immunogenicity of protein therapeutics,” Kauffman states.

Protein aggregation can potentially occur at almost every stage of a biopharmaceutical process—including development, formulation, and manufacturing—making it critical to have insights for guiding robust processing strategies, adds Lisa J. Graham, PhD, vice-president, Analytics Engineering, Seeq, a Seattle, WA-based software company that specializes in advanced analytics for process manufacturing data. These insights come from combining robust analytical instrument technology, process information with batch context, and advanced analytics to support collaboration, knowledge capture, and workflows, Graham notes.

Characterizing protein aggregates is as important as detecting them. Complete characterization of aggregates requires various pieces of critical information, Kauffman notes. It is key to know at what concentration protein aggregates form, the distribution of aggregate size and the change of aggregation percent and size of aggregates with time and concentration, Kauffman says.

“Particle sizing methods are increasingly being utilized to measure polydispersity and size distribution of the candidates,” Kauffman says. Dynamic light scattering (DLS), analytical size exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (SV–AUC), and transmission electron microscopy (TEM) are some of the analytical methods employed to detect and characterize protein aggregates.

DLS is a non-invasive method typically utilized to measure particle size and distribution. Because it is non-invasive, sample can be recovered at the end of the experiment. DLS can measure a broad range of sample sizes ranging from nanometers to micrometers and allows for fast data acquisition, reproducibility, and high sensitivity, Kauffman states, adding that DLS provides a good starting point to identify the presence of aggregation. The quantification of mass of individual aggregated species can be a limitation with DLS, however.

Analytical SEC is a form of high-performance liquid chromatography (HPLC) that is used to analyze the aggregation state of the sample. SEC uses differing Stokes’ radii to separate various species in a porous stationary phase column, Kauffman explains. “Smaller monomeric species are retained for a longer period in the stationary phase compared to larger aggregated molecules. Retention time of the sample can be compared with the retention of a known protein standard to calculate the molecular weight,” he states.

SEC is limited, however, by the assumption that the sample has the same conformation as the molecular standard and the sample is inert to the stationary phase, Kauffman continues. Furthermore, the effect of dilution while the sample is flowing through the column must be considered, he adds. Multiple angle light scattering (MALS) coupled with SEC (SEC–MALS) is a preferred analytical technique used to calculate the absolute molecular weight of the aggregate. SEC-MALS is effective in evaluating the effect of time and solvent size distribution of the aggregate, says Kauffman.

Meanwhile, the use of SV–AUC to characterize the size-distribution of aggregates has gained popularity in the last decade, Kauffman emphasizes. “Although, SV–AUC was developed in the early twentieth century, the lack of analytical solution to Lamm’s equation limited its application. Samples can be analyzed in their near native condition (buffer and pH) with no interaction with an analytical column like the other methods discussed. Additionally, the allowed range of concentration that can be tested is wider when compared to other methods,” Kauffman says.

He goes on to explain that TEM is a direct method to visualize the size, shape, curvature, and aggregation state of the protein formulation. The short wavelength of an electron allows for a significantly higher image resolution to be obtained using TEM as compared to other techniques. TEM can provide large size ranges from nanometers to millimeters, but it is limited in its scope of quantitatively evaluating the size distribution of the candidate. “Depending on the case, automated particle detection and size measuring codes need to be written and tested. Additionally, experiments are performed at very low concentration, which might not reflect the true state of the sample. Furthermore, negative staining and freezing also make the visualization condition far from the native condition,” says Kauffman.

Graham agrees that protein aggregation can be detected in several ways, whether visually observed in the solution, detected in the void-volume using SEC, and/or detected using DLS.

“In addition to characterizing the extent of aggregate formation, it is important to understand the conditions under which protein aggregation occurs. Having insight into the aggregation mechanisms and developing mitigating strategies are crucial to ensure the safety and clinical efficacy of the drug product,” she states.

For example, Graham points out, proteins are extremely sensitive to solution conditions and temperature, which are key process variables to consider when trying to gain insight from the aggregation data provided by analytical instruments. “From a process perspective, having an analytics strategy that looks at aggregation data, in context of the physical operation of the system, provides the opportunity to gain insight into the specific process conditions leading to aggregate formation. The occurrence of protein aggregation must be controlled throughout the manufacturing process during bioprocessing, with greater emphasis on purification, formulation, and fill/finish steps, to optimize yield and achieve target quality, while lowering production costs,” she adds.

Characterization of aggregates is potentially one of the key quality attributes to both measure and control, says Wake, who explains that any amount of aggregation, because it has the potential to increase immunogenicity, is therefore critical during quality control. Determining the amounts and sizes of aggregates formed, such that these can be compared to specification limits established during safety studies, can help ensure the continued efficacy and safety of the product, Wake emphasizes.

“More so during product registration, it is critical to establish that the levels of aggregates will remain within this specification through the product lifecycle. This is where formulation of the products plays a key role in stabilization, finding the optimal environment based on, for example, salt/excipient level or pH, to control aggregation,” Wake states.

The methods Wake describes for detecting soluble aggregates include analytical ultracentrifugation (AUC), asymmetrical fast field flow fractionation (FFF or AF4), sodium dodecyl sulfate electrophoresis, microscopy and turbidity in addition to SEC and DLS. “For evaluating secondary or tertiary structural changes, which can be complicit in aggregation this list also includes circular dichroism, Fourier-transform infrared spectroscopy, differential scanning calorimetry, and fluorescence,” she adds.

“For non-soluble aggregates, sub-visible particle or visible particle analysis techniques, such as light obscuration or flow imaging, can be applied. In any characterization, stability, or quality control program it is typical that orthogonal methods be applied to monitor aggregation considering both soluble and non-soluble forms. These methods often complement one another to ensure a comprehensive assessment,” Wake says.

New biologic modalities also pose further challenges to protein aggregate detection and characterization. Graham stresses that protein drug product formulation from varying biologic modalities often outpace monitoring technologies and analytical method development. She also points out that demand is increasing for multi-purpose facilities, not just multi-product facilities, to meet changing market demands for these newer modalities. “As a result, there is a need for an agile approach to continually adapt the evolving analytical methods in concert with the changing manufacturing processes. Coupling these components together into a robust data analytics environment makes it possible to capture the holistic understanding of the process environment necessary for an agile manufacturing strategy,” Graham states.

For example, Graham notes, “advanced analytics used to examine plant time-series data is foundational to developing process understanding, creating models, and developing control strategies to support the delivery of data-based decisions and process improvements. These analytical strategies can be used to create insights from the data supplied by instruments and analyzers to improve the manufacturing process.”

“The challenges for newer modalities are in many ways not that different from those for standard biologics and, where additional complexity does exist, it is more likely the result of the influence of the modality’s biochemistry,” chimes in Wake. “In the case of antibody drug conjugates (ADCs), for example, the inclusion of the linker or drug can increase hydrophobicity given the conjugated molecule a greater tendency to aggregate.”

In terms of detection and characterization, however, challenges with all aggregation analysis often involve ensuring that the system or systems are sensitive enough in terms of traditional limit of quantitation (LOQ) as well as size range, Wake continues. Other challenges include the notion that the analytics are not affected by the components of formulation, or the process of analysis is not inducing the aggregation that is being measured.

“SEC, for example, is perhaps the most widely used technique for assessment of aggregation with many benefits, such as ease of use and the ability, through use of external standards or MALS detection, to predict molecular weight of each observed species. Adversely the process of separation can induce aggregation, and its application is limited only to the assessment of soluble aggregates over a specified mass range, that is, large soluble aggregates can elute outside the size exclusion range and has limited resolution,” Wake says.

As the complexity of modalities increase, these challenges increase, such as, for example, in ensuring that the system can achieve the required LOQ or resolve aggregates of similar overall mass. “The best approach in all instances, therefore, is to include orthogonal assessment,” Wake emphasizes. “For example, adding an AUC assessment alongside SEC can help confirm that the process of separation is not causing aggregation and that the mass range being assessed is fully inclusive.”

A modality such as ADCs tend to have aggregation issues, notes Kauffman, who explains that these issues stem from the fact that some of the payloads employed in ADCs are highly hydrophobic and increase the potential for aggregation. “The analytical procedures need to be robust and specific enough to handle the more complex structure since ADCs are composed of monoclonal antibodies, a wide range of linkers, and payloads that are typically highly cytotoxic,” Kauffman states.

Meanwhile, there are a significant number of advanced therapy medicinal products (ATMPs) in the pipeline, Kauffman also stresses. Many of these ATMP candidates use a viral vector, such as an adeno-associated virus (AAV) or lentivirus to deliver RNA/DNA to its target. “By nature, the outer coat or capsid of these viruses is made up essentially of protein aggregates. Therefore, many of the analytical techniques discussed here are also used to study higher order structure of these gene therapy products,” Kauffman says.

With the growing challenges posed by new biologic modalities, there is a need for further advancement in analytical techniques or methods to improve aggregate detection and characterization. The ultimate aggregation tool, for example, would potentially allow highly sensitive and reproducible quantitative and qualitative assessment of soluble and insoluble aggregates in a single analysis without pre-treatment of the sample, that is, direct analysis of the drug substance or drug product, Wake highlights. This poses a considerable task, however, given the diversity of potential aggregates.

“There are, however, numerous new technologies under evaluation that aim to introduce additional orthogonal approaches, overcoming some limitations of current approaches and importantly improvements to existing technology,” Wake says. “Several new approaches have been reported in recent years, including differential mass analysis, which can be applied to low concentration proteins to provide data on mass as well as re-application of existing analytical technology, such as surface plasmon resonance and flow cytometry. These have not typically been associated with aggregation analysis.”

In terms of existing approaches, in the meantime, improvements in software for AUC and AF4 analysis continue to make these options much more viable for routine quality control applications, Wake adds.

“The techniques described [DLS, SEC, SV–AUC, TEM, etc.] can be quite complex and require a high degree of expertise,” Kauffman interjects. “Advances will need to be made to the software required to perform data acquisition and analysis so that they can be more widely used. Further, the software must be developed to be Part 11 (1) compliant should the technique be required for stability and/or release testing of clinical and commercial material.”

Graham adds that innovations in analytical instrument technology are needed to drive faster, more accurate measurement of protein aggregation. Advanced analytics are also needed that put data into the hands of process engineers and scientists. “These subject matter experts are invaluable for getting the insights needed at the pace required to keep up with the operational demands,” Graham states.

Furthermore, Graham makes the case that subject matter experts use experience based on what they have seen in the past and weigh that against what the data says to further apply critical knowledge and intuition. This balanced strategy creates a vital collaboration between human and machine, Graham emphasizes.

To achieve connectivity, innovations in advanced analytics need to address sevaral issues, including data connectivity and access, where time series and contextual data are typically stored in disparate sources. Access to all potential data sources must be provided while complying with data governance regulations and practices, Graham notes.

Advanced analytics must also address the need for iterative and agile processes. Production optimization is an iterative process, for example, so advanced analytics must be able to support fluidity in organizational priorities and context, rather than delivering a one-time snapshot of operational status. Further, advanced analytics must support organizational goals, such as providing easy-access (and enough) data, quickly generating insights, and enabling data-based decision making, though this does not always equal execution, Graham highlights. She adds that leadership and employee objectives must support a move to an analytics-centric culture.

Thus, while a variety of current analytical methods can be used for protein aggregation detection and characterization, changing market needs require innovative approaches and strategies to provide meaningful data and analysis for better quality control.

Guidance for Industry: Part 11, Electronic Records; Electronic Signatures—Scope and Application


Vol. 33, No. 9
September 2020
Pages: 35–39

When referring to this article, please cite is as F. Mirasol, “Risky Business: Assessing Protein Aggregation Grows Increasingly Challenging,” 

Current and newer biologic modalities pose increasingly complex challenges to the detection and characterization of protein aggregates.

When Assessing Protein Aggregation Grows Increasingly Challenging

Dissolution testing is an important aspect of drug development, allowing drug developers to determine the stability of a drug as well as providing 

This article originally appeared in the August 2020 issue of 

For solid oral dosage forms, dissolution testing is a requirement, necessary for detecting physical changes in an API as well as in the formulated product (1). Furthermore, including this analytical test early in the drug development lifecycle can allow drugmakers to optimize the drug’s release in a particular formulation.

With oral administration, drug absorption from a solid dosage form depends on the release of the active substance and its ability to be absorbed through the gastrointestinal (GI) tract of the patient. These two critical steps, solubilization and absorption, make 

 dissolution studies relevant predictors of a drug’s 

 dissolution tests for solid oral dosage forms (e.g., tablets, capsules) are thus used to assess lot-to-lot drug product quality, guide the development of new formulations, and ensure continuing product quality and performance after changes in formulation, manufacturing processes, manufacturing scale up, and manufacturing site (1).

A major issue in drug development is the need to enhance the level of drug in the patient and the availability of the drug to be absorbed by the patient’s body to have the intended therapeutic affect for which it was made. The challenge has always been that, without correct optimization of drug levels and solubility, a drug may not have sufficient therapeutic effect. Whereas, too high drug levels and solubility can conversely lead to adverse events or toxicity in the patient. These factors make dissolution testing crucial to the drug development process.

For most dosage forms to be therapeutically effective, APIs must be absorbed into a body’s circulatory system, normally through the GI tract as mentioned earlier, for the drug to be transported to the targeted site. The dissolution and absorption process, therefore, contribute to the drug’s bioavailability, and it is in understanding this multi-step process that a proper 

 dissolution testing method can be developed.

 conditions, including media (e.g., solvent) and hydrodynamics, can simulate 

 conditions. However, conditions that are optimal for quality control purposes may not be applicable for establishing 

 correlation (IVIVC) (relationship between 

 response). This may make it necessary to establish two dissolution tests, one to meet development objectives and another to meet regulatory demands (3).

Dissolution tests should be robust and reproducible and should have the ability to discern changes in product performance. Typically, the characteristics of a drug’s dosage form dictate what specific dissolution testing technique should be used; however, industry testing standards are given by the United States Pharmacopeia (USP) (2).

USP describes four apparatus setups for dissolution testing techniques:

Apparatus 1 comprises a vessel (can be covered), a motor, a metallic drive shaft, and a cylindrical basket.

Apparatus 2 comprises the assembly in Apparatus 1 but adds a paddle element (blade).

Apparatus 3 comprises a set of cylindrical glass vessels flat at the bottom, a set of glass reciprocating glass cylinders, inert fittings, nonsorbing, nonreacting screens, that cover the tops and bottoms of the glass cylinders, and a motor and drive assembly to move the cylinders.

Apparatus 4 comprises a reservoir and pump for dissolution medium, a flow-through cell, and water bath (2).

Dissolution testing has also historically been used for quality control, in R&D applications to detect the effects of critical variables in the manufacturing process, and for comparative 

Poorly soluble drugs are another challenge to dissolution testing, particularly when the dosage form is meant to be an immediate-release formulation. Challenges to testing the dissolution of poorly soluble drugs include developing an appropriate test method, validating the method, demonstrating that the method is appropriately discriminatory, and dealing with the potential of an 

With poorly soluble drugs, the extent of drug release tends to be low, and it is common that the dosage from cannot be fully dissolved. Additionally, the rate at which drug is released is too slow, which hinders attainment of meaningful data from dissolution testing. Some approaches to improving the dissolution of poorly soluble drugs have been studied (4).

To start, increasing the volume of the dissolution solution helps to optimize the maximum dissolvable dose. Other approaches include changing the dissolution medium to another solution/compound that increases the solubility of the compound or reducing dissolution sink requirements. In addition, nonaqueous solvents can be used if appropriate. Media that has been known to improve saturation solubility include biorelevant media, surfactants with mixed micelles, and certain nonaqueous media, in addition to water.

Dissolution data for regulatory consideration

Dissolution testing has increasingly become an important tool in regulatory decision-making over a drug, especially generic drugs. The testing can be used to waive requirements for 

 bioequivalence studies, for example, in addition to its role in determining formulation development, in monitoring manufacturing processes, and its use as a quality control test. It also plays a role in predicting the potential for dose-dumping in the case of a modified-release drug product if taken with alcoholic beverages. 

 dissolution testing can reduce the regulatory burden on FDA as well as alleviate the need for human studies that may be unnecessary for generic drug development. At the same time, the quality of the generic drug product would not be impacted (5).

FDA’s guidance (1) on dissolution testing discusses the Biopharmaceutics Classification System (BCS), which is based on API solubility and permeability. According to the guidance, highly permeable drugs are classified as Case 1 for high solubility and Case 2 for low solubility APIs. APIs that have low permeability are classified as Case 3 for high solubility and Case 4 for low solubility. The classification serves to provide a basis for setting up 

 dissolution specifications for the test and can also provide a basis for predicting 

The development of dissolution tests for a generic drug product must take into consideration the official methods and standards described by USP, while ensuring that the testing method is robust and can be reproduced in daily operations. The method should also be transferrable between laboratories. Finally, the test method must have the capability of distinguishing any changes in solubility behavior or drug properties that may affect the end product’s performance 

If there is an absence of an available USP method appropriate for the generic drug in question, a dissolution testing method can be developed following FDA guidelines or recommendations (6). In its guidance, FDA explains three categories under which dissolution specifications for generic products should be established:

A USP drug product dissolution test is available

A USP drug product dissolution test is not available, but a dissolution test for the reference listed new drug application (NDA) drug product is publicly available

A USP drug product dissolution test is not available, and a dissolution test for the reference-listed NDA drug product is not publicly available (1).

As shown, setting dissolution specifications for generic drugs depends on whether or not an official compendial test exists for the drug product and what dissolution test was employed for the reference drug.

Even if an FDA-recommended method is not sufficient or fitting for a drugmaker’s specific API/product, then the generic drugmaker can develop its own dissolution method (5). In the end, FDA requires comprehensive dissolution testing data in the drug’s application as part of the generic drug review process.

Guidance for Industry, Dissolution Testing of Immediate Release Solid Oral Dosage Forms

 (CDER, August 1997).
2. USP, General Chapter <711>, “

,” USP29–NF24 (US Pharmacopeial Convention, Rockville, 2011).
3. Lubrizol Life Science Health, “

 Dissolution Testing for Solid Oral Dosage Forms,” 

, March 9, 2020.
4. K. Gowthamarajan and S.K. Singh, 

 17 (3) 24–32 (2010).
5. O. Anand, et al., 


Vol. 44, No. 8
August 2020
Pages: 47–48

When referring to this article, please cite it as F. Mirasol, “Defining Drug Stability with Dissolution Testing,” 

Dissolution testing is an important aspect of drug development as it provides stability parameters and helps predict drug behavior in-vivo. 

Outlining Drug Stability and Solubility with Dissolution Testing

Determining product quality is a critical measure of a proposed biosimilar product. Appropriate analytical methods are needed to assess quality as well as for applying quality control (QC). One of the key challenges in developing or selecting an assay to determine biosimilar product quality is determining which critical quality attributes (CQAs) are the most important for a given stage of the drug development lifecycle, notes Stephen Shakespeare, product manager, Sartorius.

“For example, a binding assay may be more relevant during clone selection than a mechanism-of-action-bioassay, which would be more suited for the final stages of development,” Shakespeare says.

Titer is another critical process parameter, and to ensure final product quality, it is necessary to measure the product concentration throughout a bioreactor run, adds Laura Madia, sales and marketing manager at IDEX Health & Science.

Another point of consideration is the kind of information that is required from the assay. “Is it selective and specific for the product? Does it address the CQAs that have been identified from analysis of the innovator?” says Shakespeare, who points out that the use of orthogonal methods provides a host of information on the product that is critical for ensuring the quality.

“There are a number of challenges facing biopharma production, and their impact (overall production, development timelines, cost, etc.) can be more significant for companies developing biosimilars,” includes Madia. “One challenge in particular is the drive for improving process efficiencies. Biosimilars are considered to be cost-effective alternatives to expensive biologics. There is a significant pressure placed on reducing production costs through improved manufacturing processes to improve yields without introducing product variation that might require additional processing or testing.”

The tradeoffs between time and cost are particularly important when developing biosimilars given the limited development time, Madia continues, saying that “biosimilars do not benefit from the same patent protection as novel biologics so there is typically more pressure on prices immediately after approval.”

Another question to consider is what are the most effective assays currently used to test for biosimilar product quality? When assessing a biosimilar, Shakespeare explains, it is critical to take what he calls an orthogonal approach to looking at antibody fragment (Fab) binding and fragment crystallization (Fc) binding using a variety of techniques.

“In Fc functional assays, for example, antibody-dependent cellular cytotoxicity (ADCC) can be addressed in numerous ways using peripheral blood mononuclear cells (PBMCs) or natural killer (NK) cells isolated from blood. Meanwhile, reporter gene assays can be validated for submission to the regulators. The sensitivity of each of these methods provides key information on the quality of the product at different stages of development,” Shakespeare notes.

He adds that there is an array of methods, from enzyme-linked immunosorbent assay (ELISA) and flow cytometry to surface plasmon resonance (SPR), that can be used to determine Fab binding.

Another example of an orthogonal approach is the use of size exclusion and ion exchange analytical methods, which can show differences in aggregation/fragmentation and charge profile, respectively. These attributes can be important when assessing the stability and safety of a biosimilar, Shakespeare says.

Understanding the innovator biologic is also critical to understanding the CQAs of the biosimilar product, Shakespeare also notes. Understanding the innovator CQAs allows the biosimilar developer to determine the quality target product profile (QTPP) design space. Design-of-experiment software (e.g., Umetrics software, Sartorius) enables biomanufacturers to monitor the production process and track whether certain biosimilar CQAs are drifting, allowing them to respond accordingly.

“Being able to monitor different aspects of a biosimilar production in real-time, such as binding and function, enables you to control CQAs and minimize any potential downstream issues with delivering consistent quality product,” Shakespeare states.

There are situations where a process depends on a quick measurement to move forward, Madia remarks. “Measuring titer is not considered simple nor quick. Titer is often run on a high-performance liquid chromatography (HPLC) in the QC lab due to the high level of skill required for method development and day-to-day operation. It is not uncommon for upstream process development or manufacturing teams to wait 24 hours or longer to see the results of a HPLC titer analysis because they had to send it to a separate lab,” Madia explains. As a result, titer is often underutilized or batched for retrospective insights, rather than using titer for real-time decision making, Madia points out.

Since the manufacture of the first biosimilars, there have been many innovations in analytical techniques and practices. In recent years, for example, high throughput (HTP) flow cytometers (e.g., iQue screener, Sartorius) have enhanced the speed with which data can be generated, Shakespeare highlights. HTP flow cytometry, coupled with live cell imaging (e.g., Incucyte, Sartorius), enhances the breadth of information that is collected and allows for more informed decisions to be made faster, he explains.

Other recent analytical innovations include capillary electrophoresis techniques, which have replaced slab gel techniques, resulting in assays that have high resolution, are highly reproducible, and are applicable to a wide range of protein molecules—not just immunoglobulins and biosimilars. “The use of capillary isoelectric focusing (cIEF) and capillary electrophoresis sodium dodecyl sulfate (CE–SDS) techniques are now almost universally applied for CQA determination, stability studies, and lot release testing,” Shakespeare says.

Manufacturers, meanwhile, are continuing to implement liquid chromatography–mass spectrometry (LC–MS) techniques for investigating glycan structure, verifying sequences, analyzing intact mass, and analyzing post-translational modifications of the innovator and of biosimilar candidates, Shakespeare adds. “By introducing simple and affordable kits, manufacturers are getting greater instrument resolution, improvements in software processing and databases, and fast sample preparations,” he explains.

“In a similar vein, the ongoing reduction in particle size to < 3 µm in ultra-high-performance liquid chromatography (UHPLC) columns continues to enable reduced run times and higher throughput while maintaining peak resolution,” Shakespeare also adds.

Having an innovative new product that measures titer on demand is also a beneficial advancement, Madia adds. Drawing on experiences with the Tridex Analyzer (IDEX), Madia highlights how innovative technology can enable development and facilitate experimentation by providing trend data quickly and easily. With the trend data, it may be possible to determine product loss during the evaluation of filtration options or to monitor breakthrough with a new piece of equipment. “Ultimately, a full analysis will be required as part of the product acceptance, but by eliminating options early, it is only necessary to run a full analytical evaluation on the strongest candidates,” Madia says.

The most common application for antibody titer is in determining concentration in the bioreactor tank at the end of a cell culture run, Madia emphasizes. Once a process moves to production, current analytical tools are too large or complex to be co-located near the tank or in the manufacturing suite, she explains.

“During the crucial transition from upstream to downstream purification, manufacturing teams require the final titer data in order to load the purification columns. Once the tank is emptied and the filtration step is complete, production stops. Product is put in a holding tank while the team waits for results. Eliminating downtime while waiting for titer could shave hours or days and can also reduce the risk of moving product between areas. Using real-time titer to streamline this transition can make a real impact at manufacturing scale,” Madia says.

In May 2019, FDA proposed a draft guidance for the development of biosimilars (1) in which it outlines comparative analytical studies for determining quality criteria. The guidance itself recommends using orthogonal techniques and not relying on one analytical method when analyzing biosimilars, notes Shakespeare.

“For example, consider analyzing the charge profile of a sample using ion exchange on a UHPLC as well as by using cIEF on a capillary electrophoresis instrument. Both techniques will show the difference in charge profiles between the innovator and biosimilar, but when a manufacturer is gathering evidence of the biosimilarity of their molecule compared to the innovator, there is strength in using multiple techniques that incorporate different instruments,” Shakespeare says.

The use of analytical assays is crucial for determining that biosimilar critical quality attributes remain on point.