Manufacturers face the challenge of meeting growing demand for personalized biopharmaceuticals.

Automation Aids Cell and Gene Therapy Production

Despite many development challenges, stable producer cell lines show real promise.

State of Producer Cell Development for Lentiviral Vector Manufacturing

CPC presents a green alternative to chromatography that supports sustainable operations.

Exploring How CPC Progresses Green Chemistry Practices

Experts outline the benefits of single-use consumables relative to traditional approaches.

Weighing the Benefits of Single-Use Consumables

Considering the differences between small- and large-molecule drug products can help determine analytical testing methods for E&L.

Considerations for Analytical Testing for E&L

Chris Spivey is the editorial director of 

Glass bottles in production in the tray of an automatic liquid dispenser, a line for filling medicines against bacteria and viruses, antibiotics and vaccines. Generative AI | Image credit: © monshtadoid - stock.adobe.com

When aseptic fill/finish is at center stage in biologics manufacturing, financial survival for the company and for the patient is (literally and figuratively) on the line. While monoclonal antibodies (mAbs) were the emergent class of therapeutic proteins in the past decade or two, the spotlight has shifted squarely onto cell and gene therapies, and an array of novel immunologically active agents. The challenge of biologic fragility, complexity, potency, and storage considerations have been mathematically “squared.” However, the rewards for both patients and companies are also rising in lock step, as “the global biologics market size reached US$321 billion in 2022. Looking forward, IMARC Group expects the market to reach US$535.5 billion by 2028, exhibiting a growth rate (CAGR) of 8.1% during 2023–2028” (1).

Extracted from, or produced through, living organisms, biologic therapeutics need special handling during every stage of the manufacturing process and distribution network. In complexity, even the older mAbs were likened to a commercial jetliner, simply because of the sheer number of tightly specified components requiring integration into the final complex product. An otherwise chaotic assemblage needs to be controlled, measured, monitored, and well maintained. Mitigating, and controlling for, at each step for a tendency toward degradation is the central framework. Principle factors to account for are unwanted cross-linking, inherent heat bloom caused by natural growth processes, peptide-bond hydrolysis, deamination, and so on. Care must also be taken to avoid overly concentrating protein solutions because much of the fill/finish success or failure hinges on flow rates. Adjustments are often made to avoid foaming, shear forces, and splashing, which tends to favor the use of peristaltic pumps over piston pumping systems. This method also reduces pressure (being gentler with inherently lower sheer forces) along with “easier cleaning and changeover because their disposable tubing, connectors, and filling needles are the only materials that directly contact the product” (2).

What is most important given the swift changes in types of therapeutics and also the technologies propelling them forward? A presentation by Rick Friedman, deputy director, Office of Manufacturing Quality, FDA/CDER Office of Compliance, emphasized robotic isolators. In his presentation Friedman said, the “use of robotics has potential to profoundly reduce quality and safety risks in aseptic processing. Of course, all technologies have failure modes, and appropriate design, control and maintenance practices are essential.” He added, “basic current good manufacturing practice (CGMP) provides the foundation for a transformational shift to robotics, where design still matters.There is a need to prevent disturbances of first air; ensure size and speed of the arm does not adversely impact airflow.” (3). Overall, FDA appears very bullish on this technological innovation.

There are some trade-offs to the use of robotics in fill/finish to consider, however. Alaina Schlinker, Field Application Specialist, ScaleReady, cautions, “there are situations where their use might not be ideal, due to substantial initial investment and setup costs. For cell and gene therapies in particular, validation, qualification, customization, and adaptation of robotic isolators can be time-consuming and require staff with highly specialized skills. Many in the field also have concerns about their lack of flexibility for rapidly evolving cell and gene therapy processes,” Schlinker concludes. However, despite these trade-offs, the clear trend toward adoption continues to accelerate as can be seen below.

The writing is on the wall for 20th century approaches, as Friedman points to a 2020 International Society for Pharmaceutical engineering (ISPE) survey on notable modernization trends, quoting a survey finding that, “The most notable outcome was the clear trend toward the use of barrier systems and the near extinction of ‘traditional’ cleanroom installations for aseptic filling. Respondents indicated that, in 2020, virtually no systems would be delivered without a form of RABS or isolator, illustrating that the industry is decidedly moving to the use of barrier systems. This is great news for patients and product safety.”

Because biologics are increasingly costly and low volume-based for many emerging therapeutics, it’s constructive to examine what steps are being taken to minimize loss of product while also increasing speed and throughput in the fill/finish stage. Kathie Schneider, director, Global Commercial Lead, Cell Therapy Technologies, at Terumo, states, “The largest risks that customers need to manage in the final manufacturing step of fill and finish is [to] limit any risk of contamination, and ensure that processing time is efficient. Dimethyl sulfoxide (DMSO) is the current gold standard for cryostorage. With accepted formulations for the GMP environment, its inherent toxicity has necessitated observation of minimal contact times, stage of introduction, and removal/wash steps. Hand in hand with this has been to need to document steps for the overall process. While these steps, when controlled and documented, have aided, there has also been a concomitant drive for DMSO free solutions.”In general, industry wide, there are growing calls for such avoidance, or often recycling of solvents, and there is potential environmental-based legislation affecting closely related chemical synthesized therapy solvents, such as acetonitrile in oligonucleotide synthesis in messenger RNA (mRNA) production, and many others. Schlinker points to adoption of closed and automated systems, single-use technologies, process intensification, and advanced analytics to address speed and efficiency bottlenecks. She also believes another key “cross-industry collaboration, which is already at work, [is] to standardize protocols. As innovators in this domain, ScaleReady remains dedicated to advancing these solutions.”

Anand Srinivasan, MS, PhD, executive director, Center for Innovations and Biodesign, Research and Development, BioBridge Global, points out that for cell therapy especially, “there remains a need for ‘miniaturized assays’ to use the small volume of residual product trapped in the tubing, bag, or filters for in-process testing. In the autologous market in particular, there is big push for minimal editing/engineering with less than 24-hour manufacturing time. These measures should be supported with innovations in potency assays, rapid assays for release testing, and logistics with traceability. For allogeneic therapies, the emphasis is not on the manufacturing time, but rather on the unit operations and reliable sensors.”

Srinivasan goes on to conjecture that, “Although automation is hailed as a cure for many obstacles in the space, it may not be necessary for all manufacturing processes and associated analytics. Counterintuitively, it might just increase the cost of goods for small-scale manufacturing process.” But this is currently a somewhat contrarian view. It’s hard to look away from the cell shuttle of Cellares, which calls itself the first Integrated Development and Manufacturing Organization (IDMO), “taking an Industry 4.0 approach to mass manufacturing the living drugs of the 21st century … an entire manufacturing process in a flexible and high-throughput platform that delivers true walk-away, end-to-end automation” (4).

While Thomas Heathman, VP of Commercial Operations, Ori Biotech, clearly agrees, emphasizing, “These are living therapies with real risks of cross-contamination, and the end stages of manufacturing can introduce a variety of factors that can threaten product sterility, viability, and potency. This is especially true for autologous therapies, where product sampling is required for every single batch produced and a variety of final product containers are used, such as bags and vials. These can go back to the patient directly or, more typically, be cryopreserved to extend product shelf-life and reduce the logistical burden of returning the product for administration.” Heathman goes on to add that, “By automating closed systems, coupled with digital solutions that can track and trace product from final formulation back the patient for administration, we will enable workflows that can help mitigate these risks and enable patient access to cell therapy products at scale.”

Sterility and contamination avoidance go hand-in-hand

In its Horizons Life Science report, CRB points out that sterile filtration, a well-established tool in drug substance manufacturing, could “unlock huge advantages for gene therapy manufacturers such as smaller and more efficient facilities, lower operating costs, and opportunities for process closure. There’s a significant potential drawback, though: lentiviral and retroviral vectors typically sustain significant yield loss when exposed to a sterile filter” (5).

Natalia Elizalde, PhD, chief business development officer, VIVEbiotech, which specializes in lentiviral vectors, expounds on this point, saying, “Lentiviral vectors are very sensitive to various elements such as pH, temperature, shear forces, and chemical composition—specifically salt. They must be handled very carefully at the time of fill/finish.” She goes on to add, “Performing fill/finish in-house allows us to freeze the vectors as soon as their production is finished; this avoids additional freeze/thaw cycles caused by having to send the final product to an external CDMO to perform this. Moreover, the fact of freezing the lentiviruses in a row has a direct impact on the cost-effectiveness of
the whole process.”

The time and expense that is needed to train and retain operators, and then to re-train them again when increasing molecular diversity for a new therapeutic requires the process to begin anew, is a commonly heard challenge. Christa Myers, senior fellow, Aseptic & Sterile Products, CRB, relates, “There is a lot to learn and be able to speak to within this highly technical side of the industry. Character and personality matter. An operator needs to be able to follow protocols for manufacturing of a quality product. Each also needs to be able to think in a quality improvement mindset in order to plan for corrective actions and preventative actions (CAPAs). Your operators are your most valuable asset and should be trained and treated as such … it is very difficult to retain good staff. There has been a lot of growth and changes, salaries, benefits packages, and growth opportunities are drawing away strong trained operators. The training costs are not small and so keeping operators happy and at your company is important and less expensive than bringing in new staff. These costs are driving the need for automation to increase.”

As a natural consequence, many companies are working to reduce operators with more extensive automation. Myers continues, “The education backgrounds of operations staff are continuing to change. Many are now recruiting from mechatronics programs in community colleges or universities in order to hire operations staff that are well versed in equipment maintenance, robotics, automation, and mechanical systems.”And just as staff need to be constantly updated and refreshed, so too does the machinery itself.“Many think that they can install a fill line and it will easily operate for the next 20 years. A fill line is a complex mix of mechanical parts and automation. In order to manage the lifecycle of the machine, each sensor, each piece of hardware, and each software system needs to be maintained to a point so that the machine does not become obsolete. A strong life cycle management plan will allow the machine to grow older gracefully and not be obsolete quickly,” she concludes.

Jack Wright, vice president Sales & Marketing, Jubilant HollisterStier (JHS), sees equipment growth as integral to industry growth, commenting, “In terms of equipment upgrades, we see that as an important part of growing with the industry and being capable of approaching diverse and complex formulations with confidence. JHS has made significant infrastructure investments—in facilities and equipment—at our Spokane (Wash.) and Montreal locations. Some of these investments were made in particular to expand critical domestic vaccine manufacturing capacity.” And there are digital innovations infiltrating from other spheres of activity. We all became aware of Corning’s newly created Valor glass vials, while a totally new dimension is being pioneered by the company SmartSkin Technologies, offering sensor-enabled containers that measure the damaging forces across pharmaceutical and beverage manufacturing lines. Each biologic product has its own idiosyncrasies and, therefore, requires a fill/finish line in synch with those variables and characteristics. Automation does seem to offer a way out of some of the human errors and inherent inconsistencies, but there does seem to be a price to pay in terms of resource allocation, planning, and system-wide reeducation in how the remainder of the workforce’s role surrounding that automation. Automation therefore is not an unalloyed triumph, but another permutation in an ever-changing manufacturing landscape. The stakes are very high at the fill/finish line, and the benefits of getting things right here, speak volumes about the rest of the products journey.

. August 2023,
The International Market Analysis Research and Consulting Group (IMARC Group).

Lambert P. Dispensing Biopharmaceuticals with Piston
and Peristaltic Pumps. 

Friedman, R. Sustainable CGMP Compliance and Quality Assurance. Presentation at PDA Pharmaceutical Manufacturing & Quality Conference, August 2023.

When referring to this article, please cite it as Spivey, C. Hi-Fidelity Fill/Finish for Biologics. 

Articles

Drug efficacy, reputation, and financial return are all on the line when it comes to fill/finish.   

Hi-Fidelity Fill/Finish for Biologics

Ajay Babu Pazhayattil, DBA, M.Pharm, ajpazha@gmail.com, is a pharmaceutical management consultant for cGMP World

Gyongyi Konyu-Fogel, DBA, D.Ed., gyongyi.konyu-fogel@faculty.umgc.edu, is a graduate professor of business, at the University of Maryland Global Campus.

This survey conducted by the authors examined factors of Roger’s diffusion of innovation theory related to adopting machine learning (ML) and artificial intelligence (AI) in the bio/pharmaceutical manufacturing industry. The sample included industry leaders and bio/pharmaceutical professionals with experience in regulatory affairs, product development, and commercial operations at FDA-regulated bio/pharmaceutical companies. Participants responded to a survey administered with a direct link for confidentiality. Respondents were asked 44 questions on the adoption challenges of ML and AI practices in bio/pharma manufacturing.

According to the results, creating ML and AI solutions catering to manufacturing segments can quickly generate a customer base. The survey also found that the first areas to be affected by AI are likely to be those related to process efficiency because decision making related to manufacturing efficiency tends to be less regulated compared to quality decision making. In addition, the greatest opportunity for return on investment (ROI) in ML and AI implementation in bio/pharma manufacturing maybe waste reduction, batch yields, and faster batch completion, which are well-aligned with existing lean manufacturing principles. In the current scenario, it is essential to provide training courses for bio/pharma professionals to collaborate in identifying opportunities and developing and implementing ML and AI projects. This will help professionals adapt to the digital revolution and stay ahead in the rapidly changing bio/pharma manufacturing industry.

Submitted: May 10, 2023
Accepted: Aug. 9, 2023

Ajay Babu Pazhayattil, DBA, M.Pharm, ajpazha@gmail.com, is a pharmaceutical management consultant for cGMP World, and Gyongyi Konyu-Fogel, DBA, D.Ed., gyongyi.konyu-fogel@faculty.umgc.edu, is a graduate professor of business, at the University of Maryland Global Campus.


Volume 36, No. 10
October 2023
Pages: 24–29

When referring to this article, please cite it as Pazhayattil, A. B.; Konyu-Fogel, G. ML and AI Implementation Insights for Bio/Pharma Manufacturing. 

A survey was conducted to examine the adoption of machine learning and artificial intelligence in the bio/pharma manufacturing industry.

ML and AI Implementation Insights for Bio/Pharma Manufacturing

Editor of Pharmaceutical Technology Europe

A maze with a human figure navigating through it, displayed against a challenging, problem-solving background, concept of Complexity, created with Generative AI technology | Image credit: © koldunova - stock.adobe.com

Biologics are becoming more established in the development pipeline as a result of their ability to treat a variety of conditions and diseases in a targeted and effective way. The increasing prevalence of diseases, such as cancer, autoimmune diseases, and genetic diseases, globally is driving continued growth for biologic therapies (1).

However, the inherent complexities associated with biologics makes formulation and development uniquely challenging when compared with small-molecule drug products. To learn more about the formulation challenges associated with biologics, potential solutions and approaches that are helping to overcome these challenges, the regulatory landscape, and how outsourcing partners can be beneficial, 

® spoke with Aaron Frimel, manager, Process Development at Thermo Fisher Scientific.

 Could you highlight the main challenges associated with biologics formulation development?

 With early phase development, the hardest challenge can be getting enough representative material early enough in the development lifecycle to support a thorough formulation study (time and size) to achieve the target product profile. Another challenge is having methods ready for the formulation studies so that method performance is understood well enough to know whether a change in product quality is due to assay performance or formulation performance.

With late-phase development, routes of administration, dosing, and degradation products on long-term stability are better understood and may necessitate changes that can sometimes be difficult to accommodate and may even require altering the target product profile.

: What strategies can companies employ to overcome these challenges?

: High-throughput, characterization-type assays [can be employed] to uncouple the start of formulation work from method development, as well as reduce material per sample in order to screen larger design spaces, which sets the project up for earlier success with less potential for re-development. [Additionally, with] in-house cell line development, the material requirements and representativeness are dealt with by using high-titer cell lines and targeted integration. Process development groups can then perform material generation runs using the center-point of their platform processes to generate material early in the project lifecycle so that the final formulation is selected at or before the conclusion of process development activities.

With late-phase projects, material representativeness and availability are much less of a problem, but clients may choose to go to a higher concentration for dosing purposes or to change the route of administration. With an existing formulation, and known degradation patterns, [it is possible to] stage a small-scale robustness study with the critical stability indicating assays to achieve the target concentration while mitigating the most significant liabilities.

If the client is trying to blunt the formation of a degradant, [then it is beneficial] to have an analytical suite that is larger than just release analytics. [Thermo] had a program recently that saw some charge heterogeneity issues and [by using a] multi-attribute-method (MAM) [it was possible] to understand that the shift in the acidic shift in a cIEF [capilliary isoelectric focusing] profile was a trade-off between deamidation and oxidative modifications, and [it was possible] to reduce both degradation patterns by raising the pH 0.5 units while adding methionine—something [that] would have never been [possible without] such a powerful analytic to tease apart these modifications on the amino acid level.

 Have there been any technological innovations or novel approaches implemented that have helped with biologics formulation?

 With biologics formulation, the range of available excipients is limited, and a lot of the advancements come from the development of more sensitive, stability-indicating analytics that are precise and trend with a high degree of linearity. [Because] the range of excipients that are available (and not patent protected, if possible) is small, being able to achieve the maximum potential of what is available is the best option.

Increases in throughput or decreases in material requirement and touch time are the things that allow a formulator to screen wider ranges and are what gives a project a good chance of finding a global optimum, rather than a local optimum, when screening formulations.

 Has the regulatory landscape changed for biologics formulation? If so, how can companies navigate these regulations with ease?

 The most common thing [being seen] since the pandemic is an increase in the amount of early phase projects targeting a subcutaneous format, many times using a pre-filled syringe. The benefits are obviously easier self-administration, and opinions on self-administration have rapidly changed both as a result of the pandemic and as a result of the Type 2 diabetes drugs that are being used to treat obesity.

Pre-filled syringes are regulated as combination products. While this isn’t a new regulatory landscape, FDA has clarified its guidance on combination products and streamlined the process for triaging whether the primary mode of action of the product classifies it as either a drug or a device while eliminating the need to get approvals for both.

 How can partnerships benefit in biologics formulation development? Are there any best practices you can share?

 A great thing about outsourcing biologics formulation work is that the teams at these outsource facilities see a wide range of molecules and molecule behavior and can act on this breadth of experience. What separates a good partnership from a great one is how well the outsource partner works to understand the client’s goals and mentality when it comes to risk in order to customize the approach to meet the customers goals while incorporating the technical knowledge gleaned from previous projects into the design. Some of the most productive conversations we have with clients are the informal sidebar discussions where a project member casually mentions the long-term goal for the project. It is surprising how many times pieces of information from informal conversations can be incorporated into the existing work and eliminate the need for future studies.

Both the technical experience and establishing rapport come down to the same thing, listening. Both listening to the data during study execution and actively listening and questioning the client to understand their long-term goals so that they can be addressed in the project design.

Being on the outsource side, [Thermo encounters] a range of client mentalities, both in terms of clients shopping projects as well as clients that commit to a workscope with [a particular] team. The clients who put in the work to understanding a CDMO’s [contract development and manufacturing organization’s] technical capabilities, resourcing, and any client-specific considerations prior to committing to a CDMO tend to be the more satisfied clients. Specifically, the customer that takes the time to evaluate, understand, and articulate their internal needs as best as possible tend to be more skilled at evaluating whether a particular CDMO will match these needs as closely as possible.

Biologics Market Size, Share, and Trends Analysis Report by Source (Microbial, Mammalian), by Product (MABs, Recombinant Proteins, Antisense & RNAi

), by Disease Category, by Manufacturing, by Region, and Segment Forecasts, 2023–2030. Market Report, May 2023.

Felicity Thomas is the European/senior editor for 

When referring to this article, please cite it as Thomas, F. Navigating Biologics Development. 

Biologics formulation comes with a unique set of challenges, which can be overcome through innovative strategies and good partnerships. 

Navigating Biologics Development

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

Lab technician examining a petri dish containing a precision fermentation sample, representing advanced biotechnology research, generative ai | © InputUX - stock.adobe.com

The recent resurgence in microbial fermentation for biologics production can be attributed to many factors. Advances in biochemistry and a growing understanding of when glycosylation is not needed, improved capabilities for modification of naked proteins, the emergence of new protein formats and scaffolds well-suited to this production technology, and its applicability to DNA plasmid manufacturing are among them. Successful optimization efforts result in scalable fermentation processes that not only afford high titers and productivity, but also exhibit a high level of robustness and reliability.

Technical challenges to large-scale fermentation

The cost-effectiveness of microbial fermentation compared to cell culture makes it attractive when suitable, but there are numerous challenges that must be overcome. Bacteria and yeast typically do not have the cellular machinery capable of coping with complex biological structures, so it is of utmost importance to spend some initial time and effort to identify the best production system for each molecule of interest, according to Jonas Müller, team manager for microbial upstream processing at Lonza.

Another fundamental challenge has been the lack of a good understanding of expression systems and processes, observes Christopher Lennon, director of microbial upstream processing with FUJIFILM Diosynth Biotechnologies, Billingham, United Kingdom. “Without this understanding, it can be difficult to produce the right amount of protein at the required product quality,” he remarks. He does point out, however, that increased access to more sensitive technologies, particularly mass spectrometry, has provided greater insight into product quality at a much earlier stage in development than was previously available. This requires more iterative process development to ensure product quality attributes are met and at earlier stages in the process.

Process control can be an issue when scaling to large volumes, particularly with respect to the oxygen transfer rate and heat management. “For fast-growing bacterial cultures, it is necessary to ensure that sufficient oxygen transfer occurs throughout the entire culture volume to maximize growth, which in turn requires sufficient mixing and airflow,” says Cameron Graham, senior process engineer with BIOVECTRA.

Heat management is important because microbial fermentations typically have tight temperature ranges of +/- 1 or 2 °C of setpoint, according to Graham. “Every millimole of oxygen consumed produces heat, and agitation produces heat, all of which must be removed by the equipment to ensure there are no uncontrolled temperature spikes. This issue becomes a major concern when scaling up production, particularly beyond the 1000-liter scale,” he explains.

Options include replacing chilled water-cooling systems with ultra-low temperature glycol in the vessel jacket, but Graham observes that above 1000 liters, increases to the internal heat transfer area may be required. Surface area enhancements such as baffles that have internal cooling coils have been used to address this challenge.

The use of antibiotics to select for bacteria that maintain high expression levels of the desired product is an issue specific to bacterial fermentation. During downstream processing, the antibiotic must be removed, and regulatory agencies expect demonstration of removal, according to Gregor Awang, a specialist in biologics development and innovation at BIOVECTRA. Typically, this goal is achieved by using very low (10s of ug/mL) starting concentrations of specific, preferred antibiotics and validating the removal of the antibiotic using specific assays and the multiplicative dilution effect of several purification steps. “The development of cell lines that do not require antibiotics but maintain stable expression has consequently become a major goal in recombinant protein and plasmid production,” he notes.

Increasing plasmid expression is another challenge, Awang says. “Producing more plasmid is not as straightforward as simply increasing the number of cells. Retaining plasmids can impact cell growth, thus it is essential to find a balance that optimizes plasmid production and growth rates, which can be tricky to achieve,” he explains.

Another feature of microbial fermentation that dictates its applicability for biologics production is the glycosylation capability of the specific microbes involved. 

) can perform very few post-translational modifications, while yeast do glycosylate proteins, but not in the same manner as mammalian cells. For these reasons, 

) are perfect and complementary production systems for biomolecules that do not need human glycosylation, Müller states.

While the glycans made by yeast are simpler than those generated during mammalian cell culture and typically not an issue for product consistency, they are mannose-rich, and thus protein products generated in yeast may potentially be immunogenic in humans, according to Awang. He notes that a few companies have worked on humanizing the glycosylation process by inserting specific enzymes into the yeast genome, making yeast capable of making a truncated human glycosylation pattern, but he is not aware of any of these cell lines being used for commercial products.

The types of post-translational modifications required for a product will therefore determine the optimum host organism, according to Müller. While some modifications might be done ex vivo, he emphasizes that the most elegant approach is to leverage the host organism’s cellular machinery. It may be possible to improve results through process parameter optimization, but the existing pathways cannot be replaced completely.

Post-translation modifications, says Lennon, therefore first need to be identified. How they will be controlled then becomes specific to the types of modifications involved. “Some can be controlled by using different expression strains of the same organism, while others may require a more wholesale change to the process, host or cell line,” he concludes.

An important development in commercial-scale microbial fermentation is the development of single-use technologies (SUTs) designed to address the specific processing needs of fermentation reactions. “Single-use systems have historically struggled with heat exchange and have been of limited use for more intensive recombinant protein processes; however, new systems are coming out that close this gap,” Lennon says.

SUTs are advantageous because they eliminate the need for cleaning between batches and simplify the setup and qualification process. BIOVECTRA has shown that single-use units are suitable at the 100- and 1000-liter scales when using high-growth bacteria and yeast. “We’ve used our 100-liter vessel successfully in a bacterial process with an oxygen transfer rate over 400 millimoles per liter per hour, supplying the necessary oxygen transfer rate and cooling capacity. We’re currently using our 1000-liter single-use unit in a similarly demanding process with high-growth yeast and seeing performance comparable to what is expected from a stainless-steel unit,” states Graham.

Currently, single-use microbial fermentation technology is generally limited to a capacity of around 1000 liters, mainly due to cooling capacity constraints. However, as technology continues to advance, Graham thinks it likely that there will be growing demand within the industry to see larger single-use volumes—at least up to 10,000 liters—that take advantage of single-use technology.

For larger stainless-steel vessels, meanwhile, Graham notes that easily cleanable modifications that increase internal heat transfer surface area can simplify the process and reduce downtime between runs. BIOVECTRA has, for instance, modified a 17,000-liter stainless steel vessel with internal cooling baffles that are cleanable to ASME BPE standards and improve consistency and reproducibility between runs.

It is also worth noting, according to Awang, that SUTs do create the opportunity to more easily produce smaller product quantities, such as those required for DNA-based personalized medicines. “By running a small single-use fermentation from 10 to 100 liters, enough DNA can be obtained to create the therapeutic. When larger amounts are needed, large stainless-steel vessels can produce kilograms or tens of kilograms of DNA; having both options available gives the best of both worlds,” he comments. He goes on to observe that because SUTs make bacterial fermentation much simpler, it may be possible to allow installation of a fermentation plant on site or nearby a hospital for faster delivery of personalized medicines for which one-batch-per-patient targeted, small-scale production of highly potent autologous therapeutics (e.g., DNA and mRNA vaccines and gene-modified cell therapies) is involved.

Real-time monitoring of biologics manufacturing to enable improved process control has become an important goal in the biopharmaceutical industry. Production via microbial fermentation is no exception. “Use of PAT [process analytical technologies] during a fermentation run enables detection of potentially problematic variations and allows for manual or automatic corrections to bring the process back to the center of the validated operating space,” Awang says. “By using PAT to ensure success and consistency, you can save time and resources while maintaining the highest quality standards,” he adds.

Optimization of microbial fermentation processes for protein production requires consideration of the type of organism being used. Yeast effectively secretes proteins into the fermentation medium, making product recovery relatively easy using tangential flow filtration (TFF) and/or centrifugation, according to Awang. E. coli, however, produces proteins in the cytoplasm either as soluble proteins or insoluble inclusion bodies, although some may be secreted into the periplasmic space between the inner and the outer membranes.

“These different means of production require different harvest strategies depending on the specific product being made,” Awang says. Products secreted into the periplasm can be recovered by removing the outer membrane via osmotic shock, while proteins secreted into the periplasm will require full lysis of the cells, which is a more complicated process.

Some cytoplasm-located proteins are produced as insoluble inclusion bodies, which require recovery from the lysed culture supernatant by high-speed centrifugation, denaturation, and renaturation to produce soluble, properly folded product, according to Awang. The notes that the additional steps may introduce more impurities or potentially lead to damage to the product.

In fact, while inclusion bodies historically served as a means for pre-purifying protein products, the subsequent refolding process requires dedicated unit operations with large buffer volume capacity and handling of a large amount of chaotropes (typically urea), according to Müller. “These steps are usually the bottleneck of many current facilities and are quite cost-intensive, especially if we consider the waste management aspect. A promising approach to address this challenge is cell engineering, which drives the formation of correctly folded molecules in the cell,” he observes.

Cell line engineering is just one aspect to consider when planning a strategy for microbial fermentation of biologics. The diversity of processes and potential biomolecules that can be made using this technology necessitates the development of a specific strategy and tailor-made process for each product, according to Müller. “We recommend investing time and effort in host selection and early process development,” he stresses. “To achieve the highest possible titer/yield, it is essential to ensure right from the project start that the most suiting organism, expression system, sequence of unit operations and operating conditions are selected,” Müller adds.

The diversity of fermentation processes and molecules produced by them makes establishing a universal microbial fermentation platform process nearly impossible. “Developing a platform or library of cell lines and plasmids that can be effectively utilized depending on the type of protein or product would be a great solution to a big problem in fermentation,” Awang notes. Moving toward a platform-like approach for the downstream processing steps could, meanwhile, provide one means for standardizing some aspects of these manufacturing processes, according to Müller.

Rather than seek to establish one overall platform process, FUJIFILM Diosynth Biotechnologies has developed individual platform solutions optimized for different product accumulation routes (soluble/insoluble, cytoplasmic, or secreted). The company’s pAVEway expression workflow starts with identifying the best expression host/expression vector/gene sequence using automated bioreactor and downstream analysis processes. “This enhanced workflow enables optimal strain selection based on both product titer and product quality. Once the starting conditions are selected, we can improve yield by evaluating the effects of factors such as media and feed composition, induction temperature, and pH,” Lennon comments.

It is also possible to establish a platform process for a particular type of product. Platform solutions for the manufacture of plasmid DNA are increasingly common, but even these approaches require flexibility to accommodate different plasmids and formats.

While process optimization often focuses on product yield for microbial fermentation, consistency from run to run is most important. “Regulatory agencies have provided clear guidelines for developing a fermentation process that meets quality requirements. This involves understanding the system and its capabilities and identifying the design space in which the fermentation can operate, especially with E. coli,” explains Awang.

“It is best,” Awang contends, “to use a systematic approach to development and optimization that includes design-of-experiment studies to understand how the cell line and the culture conditions interact to influence growth and product yield.” He adds that most of the process development and characterization work can be performed at lab scale using small single-use fermenters for model building.

Consistency must then be achieved between lab- and plant-scale models, which is realized by fine-tuning the process until agreement in performance at both scales is reached. Maintaining consistency in multiple parameters to optimize comparability can be a challenge, though, particularly for certain parameters, according to Graham. He points to the concentration of dissolved oxygen as being particularly problematic and notes that it is crucial to carefully consider the type of vessel being used to optimize agreement between the models.

Having consistent small and large-scale models makes it possible to investigate deviations from the norm in plant runs at lab scale, which accelerates the analysis of process consistency and deviations while reducing the cost, Awang observes. In addition, lab-scale models are used to more rapidly qualify raw materials and assess the risk that certain raw materials may be potential sources of variability in plant-scale manufacturing runs.

Optimization of fermentation reactions should not focus on one aspect such as yield. It should include process robustness and consistency, among other attributes such as the oxygen demand and oxygen transfer rate. In addition, optimization is influenced by the ultimate goal for the fermentation process, according to Awang.

“If the goal is to supply hundreds of tons of material to the global market, that expectation must be considered when creating the cell line and fermentation process. Jumping into process development without thinking about the end goal often leads to problems with process scalability later on,” Awang comments. He also notes that careful documentation of cell lines (where they came from, how they were stored, confirmation of identity) is essential to ensure both product consistency and regulatory compliance.

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


Vol. 36, No. 10
October 2023
Pages: 15–18

When referring to this article, please cite it as Challener, C. A. Optimizing Fermentation Processes for Biologics Manufacturing. 

Using a systematic approach and achieving run-to-run consistency are essential.

Optimizing Fermentation Processes for Biologics Manufacturing

Feliza Mirasol is the science editor for 

Affinity chromatography, which has been used for nearly 40 years, is the preferred method for separating and purifying large-molecule therapeutics in downstream processing. For the most part, affinity chromatography makes purifying biological molecules simple and straightforward because of the method’s selectivity, scalability, and ease of process development (1).

The advancement of increasingly complex biomolecules in the drug development pipeline, however, drives the continual push to improve downstream processing steps. Innovations in chromatography technology, such as the development and enhancement of affinity ligands, has enabled the improvement and optimization of affinity chromatography.

The preference for affinity chromatography

With decades of use under its belt, affinity chromatography has been shown to be a preferred downstream processing technique. This preference is due to the fact that affinity chromatography offers high selectivity, resolution, and capacity in most protein purification schemes, emphasizes Khaled S. Mriziq, PhD, senior manager, Marketing, Process Chromatography, Protein Purification Business, Bio-Rad Laboratories. Mriziq explains that, compared to other downstream purification methods, affinity chromatography has the advantage of utilizing a protein’s biological structure or function for purification, and, as a result, purifications that would otherwise be time consuming and complicated can often be easily achieved. “Affinity chromatography is increasingly used in platform monoclonal antibody (mAb) processes,” he notes.

Affinity chromatography can reduce a multi-step downstream process to a process where the number of steps is significantly reduced, which minimizes cumulative yield losses, asserts Brittany Crocco, Product Manager—Chromatography Resins, Repligen. This advantage makes affinity chromatography, and the significant process economic improvements it delivers, an obvious technology choice from process simplification and improved process economy perspective, she states.

Although resins are the workhorse of chromatography methods, ligands were developed to increase the specificity with which the resins can capture target molecules.

“Beyond the high selectivity required, [the ligands] must also be easily conjugated to the base matrix, enable efficient elution in conditions mild enough to ensure the target stability, and be chemically stable to enable robust operation. This is especially important when the resin needs to be cleaned-in-place, for instance using sodium hydroxide at concentrations even as high as 0.5 M,” Crocco explains.

While stability and the ability to easily reuse resins may not be of high importance early in the drug lifecycle, the cost considerations for such maintenance at the commercial level will become significant. Thus, in the further development of innovative ligands for these applications, stability must be a key feature consciously designed from the start, Crocco concludes.

Beyond high specificity, affinity resins for new modalities must have the same basic characteristics as those expected in the more mature recombinant protein (e.g., mAb) field: capacity and stability, stresses Crocco.

Over the past few decades, affinity ligands have been improved, with the main advances seen in immobilization chemistry, ligand engineering, and base matrix design. “IMAC-based ligand resins have seen several improvements in recent years, including the development of smaller base beads for higher resolution and better ligand stability to minimize ionic leachables in chromatography steps,” notes Mriziq, who adds that modifications of the ligand have also improved dynamic binding capacity, allowing for the accommodation of higher titers in the feedstream.

“Chromatographers are wiser now, and they understand the cost pressures to cycle capture resins extensively to improve process economics,” Crocco observes.

Crocco points out that the more a resin is cycled, the more drug each liter of resin produces, and hence the cost contribution of the affinity resin towards each dose of the drug is lower. But to be able to cycle the resin more frequently requires an effective cleaning process. “The industry standard cleaning agent is sodium hydroxide, which is very effective, hence the affinity resin needs to be stable in sodium hydroxide, minimally 0.1 molar, but preferably 0.5 molar. Not all classes of affinity ligands have the required stability, however, and the past 10 years have seen the emergence of new affinity ligand types that are inherently much more stable and able to withstand the rigors of bioprocessing,” Crocco states.

While modern Protein A resins have set the bar high, chromatographers expect and require similar sodium hydroxide stability and long resin lifetime in affinity resins for other modalities as well, Crocco also emphasizes. “The evolution of the newer, more stable ligands means that, now, chromatographers don’t need to compromise and can always seek a resin that can be cleaned with hydroxide,” she concludes.

“Diversity of ligand libraries used for ligand screening significantly increases chances for discovery of unique ligands that, when put on the right solid support, can address very unique purification challenges,” says Crocco. “For example, Repligen starts with more than 50 ligand scaffolds to identify the best ligands with predefined requisite characteristics. This procedure sometimes includes unique selectivity requirements. For instance, where a unique selectivity is required that is not attainable with conventional resins (such as separation of isoforms, truncations, active vs non-active forms of enzymes, etc.), affinity chromatography can be applied as a polishing step with properly selective ligands,” Crocco explains.

Current innovations include ions and ligands that are stable, as described earlier. Low levels of ligand leaching as well as the ability to remain stable against harsh cleaning reagents are critical for operators during manufacturing, Mriziq confirms. “Voice of the customer has driven these innovations,” he states.

Looking toward the future of downstream purification, the complexity of emerging biologics is making affinity purification attractive. “Due to their selectivity, scalability, and ease of process development, affinity ligands enable a simpler platform purification process, resulting in a faster time to market,” says Mriziq.

Meanwhile, adeno-associated virus purification workflows or non-mAb biologics are driving the innovation of newer affinity chromatography approaches, Mriziq adds.

1. Łącki, K.M.; Riske, F.J. Affinity Chromatography: An Enabling Technology for Large-Scale Bioprocessing. 

 (1), e1800397. DOI: 10.1002/biot.201800397


Volume 36, No. 10
October 2023
Pages: 19–20

When referring to this article, please cite it as Mirasol, F. Meeting Complexity Demands with Innovative Affinity Ligands. 

The innovation of affinity ligands must take stability into consideration for present and future purification applications.

Meeting Complexity Demands with Innovative Affinity Ligands

Brian Greven is senior director of Quality & Validation at Matica Bio.

Medical vials on production line at pharmaceutical factory, Pharmaceutical machine working pharmaceutical glass bottles production line, | Image credit: © Prasanth - stock.adobe.com

In 2022, the number of newly approved biologic drugs outpaced that of small molecules for the first time (1). Notably, almost a third of the new approvals comprised novel modalities, such as cell and gene therapies, antibody–drug conjugates, and bispecific proteins. In fact, a report forecasted biologics sales to exceed that of small molecules drugs by 2027 (2). The steady rise in popularity of gene therapies and other biologics is the underlying reason for the growing importance of aseptic manufacturing processes in the biopharmaceutical industry.

Traditional small-molecule drugs are chemically derived and manufactured using terminal sterilization, in which the drug product in its primary packing is sterilized using high heat, radiation, or filtration. In contrast, biologics—which include gene therapies, transplant tissue, recombinant proteins, and monoclonal antibodies—are composed of tissue, cells, nucleic acids, proteins, sugars, and more, which degrade and lose potency when subjected to the harsh conditions of terminal sterilization.

Gene therapies deliver DNA, RNA, or ribonucleic proteins into the living cells of a patient to correct a specific genetic variation or introduce a therapeutic gene. Delivery of genetic material can be achieved in several ways, but is commonly achieved by using viral vectors, such as adeno-associated viruses or lentiviruses, which cannot withstand terminal sterilization.

At the same time, gene therapies are administered parenterally (i.e., injected into the body intravenously, intramuscularly, or subcutaneously). Parenteral drug delivery bypasses the body’s natural defenses against pathogens, thereby increasing the risk of infection to the patient, which necessitates the more complex process of aseptic manufacturing of viral vector-based gene therapies to ensure their efficacy, while being safe to inject into patients.

Gene therapies also pose an additional challenge. Traditional biopharmaceutical manufacturing is built around the “one product, one facility” paradigm. Here, high-volume stainless steel bioreactors produce a single drug product on a large scale at a dedicated manufacturing facility. However, due to the personalized nature of gene therapy, as well as the size of the patient population for which they are intended, this landscape is undergoing a transformation as smaller, adaptable, multi-product manufacturing gains prominence. Here, the author discusses innovations and trends in aseptic manufacturing processes, with a focus on considerations to produce viral vectors for gene therapy.

According to the guidance issued by FDA (3), in an aseptic process, the drug product, any excipients, the container, and closure are individually sterilized using appropriate methods, and then put together in a high-quality, sterile environment. This step, referred to as sterile fill/finish, is done in a cleanroom and often uses special equipment that is self-contained in a sterile environment. Because it is impossible to sterilize the drug product in its final container, it is crucial that the containers be filled and sealed in a highly controlled environment, which is constantly monitored for the presence of microbes and particulate matter, and ensures the appropriate temperature, humidity, air pressure, and ventilation.

Despite these measures, it is well established that operators are the predominant source of microbial contamination in aseptic processes, accounting for an estimated 80–90% of common contamination sources (4). Aseptic control is especially critical in gene therapy production, because there are more manual manipulations in these processes compared to traditional biologics (often left over from processes developed in an academic lab), making them more vulnerable to microbial ingress. Developing safe, reliable, and scalable manufacturing processes for gene therapy remains a key limitation to their clinical application. Central to such processes are the use of closed systems that incorporate single-use technologies (SUTs).

Closing the loop on stainless steel bioreactors with SUTs

As the name suggests, a closed system is one in which the product is physically isolated from the surrounding environment and personnel. Materials may be added into, or removed from, the system at designated control points in a way that does not expose the product to the environment.

Achieving 100% closure has long been a coveted goal for biomanufacturing. A fully closed aseptic manufacturing process typically includes:

—including sealed and sterile chambers, preventing any contact between the cell culture and the external environment

Closed media and reagent delivery systems

—including the use of sterile and disposable bags or containers with integrated tubing and connectors that allow for aseptic transfer of fluids

—including sterile connectors or sampling ports that enable aseptic sampling, as well as integrated sensors and probes for in-process monitoring of parameters such as pH, dissolved oxygen, and temperature

—including closed filtration systems, centrifuges, or other media separation techniques

Closed formulation and fill/finish systems

—including sterile, disposable, and closed filling equipment with integrated sealing mechanisms

Integrated control and automation systems

—sterile, disposable, ready-to-use equipment and consumables such as media and buffer bags, filters, tubing and connectors, chromatography columns, and bioreactors.

Beyond preventing the potential loss of product due to contamination, closed systems that use SUTs offer several other advantages that are extremely beneficial in the context of gene therapy manufacturing, such as:

—Closed systems can be operated in various environments, including aseptic cleanrooms, as well as controlled-not-classified (CNC) environments. CNCs, in particular, eliminate the need for airlocks and extensive gowning, enabling a less segregated facility that can support the manufacture of smaller batches of multiple products in parallel. Small-batch aseptic processes are integral to the manufacture of personalized treatments such as gene therapies.

—Construction of manufacturing facilities employing reusable stainless-steel equipment can take four to five years, with additional time required for plant validation (5), making them slow to scale. In contrast, closed systems using SUTs offer a plug-and-play solution, which allows a facility to be up and running in a matter of months, especially when leveraging prefabricated, modular cleanrooms. Modular manufacturing facilities offer the flexibility to scale as needed to meet clinical trial needs or market demands.

—A closed system minimizes the need for extensive operator involvement and streamlines the overall procedure. If the closed system is implemented using SUTs, time savings are further amplified due to reductions in setup, product changeover, line clearance, cleaning, and validation. In fact, the elimination of time-consuming, clean-in-place and steam-in-place operations needed with stainless-steel equipment is a key driver for the rapid uptake of SUTs (6).

—A traditional biomanufacturing facility typically requires an upfront capital investment of more than $450 million. This poses significant risk during clinical trials and initial commercial production and can be especially daunting for startups and small biopharmaceutical companies. Closed systems that utilize SUTs have comparatively lower operating costs through energy savings from the elimination of airlocks, environmental monitoring and cleaning, reduction in gowning, and more. Case studies comparing the costs of facilities that use SUTs with those that use stainless-steel equipment demonstrate approximate capital and operating expenditure savings of up to 70–25%, respectively (7).

Implementing a fully closed system is, however, technically challenging, and many biopharmaceutical companies partner with a contract development and manufacturing organization (CDMO). However, many CDMOs, such as FUJIFILM Diosynth, BioCentriq and others, often opt for a functionally, rather than fully, closed system, which has limited exposure to the processing environment. A functionally closed system may be routinely opened for manual intervention and returned to a closed state by sterilization before process use. Such systems are easier to engineer, but more prone to contamination due to its reliance on the operator to ensure sterility.

In contrast, Matica Bio’s facility in College Station, Texas is a GMP-certified, 100% fully (physically and functionally) closed manufacturing system that was built using modular, prefabricated cleanrooms.

While gene therapies hold immense promise in revolutionizing healthcare by addressing the underlying genetic causes of diseases, their successful translation to widespread clinical application hinges on the ability to produce them in a manner that is reliable, scalable, and economically viable. The integration of modular facilities and closed processing have emerged as a compelling choice for CDMOs in the gene therapy arena, minimizing the risk of contamination, and enabling efficient workflows, rapid scaling, and greater flexibility.

Senior, M. Fresh from the Biotech Pipeline: Fewer Approvals, but Biologics Gain Share. 

. Jan. 9, 2023. 41 (2) 174–182. https://doi.org/10.1038/s41587-022-01630-6.

Looking Ahead to 2022 – The Future of Pharma

Guideline on Sterile Drug Products Produced by Aseptic Processing

PDA. PDA Technical Report No. 22 (2011 Revision). Process Simulation Testing for Aseptically Filled Products. 2011 (accessed Sep 1, 2023).

Samaras, J.; Micheletti, M.; Ding, W. Transformation of Biopharmaceutical Manufacturing Through Single-Use Technologies: Current State, Remaining Challenges, and Future Development, 

Annual Review of Chemical and Biomolecular Engineering

Morrow J.K.; Langer E.S.; Rise of Single-use Bioprocessing Technologies: Dominating Most R&D and Clinical Manufacture. 

Ravisé A.; Cameau E.; De Abreu G. et. al. Hybrid and Disposable Facilities for Manufacturing of Biopharmaceuticals: Pros and Cons. 

. 2009, 115, 185-219. doi: 10.1007/10_2008_24.

When referring to this article, please cite it as Greven, B. Innovations and Trends in Aseptic Manufacturing Processes. 

The steady rise in popularity of gene therapies and other biologics is the underlying reason for the growing importance of aseptic manufacturing processes in the biopharmaceutical industry.

Innovations and Trends in Aseptic Manufacturing Processes

MilliporeSigma, the US and Canada Life Science business of Merck KGaA, Darmstadt, Germany, announced on Sept. 26, 2023 that it has opened two new good manufacturing practice (GMP)-grade messenger RNA (mRNA) drug substance manufacturing sites in Darmstadt and Hamburg, Germany. The addition of these sites makes the company the first contract testing, development, and manufacturing organization (CTDMO) to offer integrated services for all stages of mRNA development, manufacturing, and commercialization, including products and testing, according to a company press release.

Under an ongoing €1 billion (US$1.06 billion) investment, the new sites are part of the company’s goal to advance mRNA technologies and build its global mRNA network and capabilities. The acquisitions of AmpTec in January 2021 (1) and Exelead in February 2022 (2) were also part of this investment.

The company has invested €28 million (US$30 million) into the new Darmstadt and Hamburg GMP mRNA drug substance manufacturing sites. The new facilities add 75 new jobs and provide mRNA services at all scales and applications, from pre-clinical to commercial scale. Services include analytical development and biosafety testing specifically designed for mRNA technologies. In addition, MilliporeSigma offers differentiated polymerase chain reaction-based technology for clinical and commercial mRNA manufacturing, which results in high-quality mRNA.

“mRNA has emerged as the breakthrough technology of this century, providing great promise to not only treat, but potentially cure, a wide array of diseases such as cancer, heart disease, and muscular dystrophy,” said Dirk Lange, head of Life Science Services, Life Science business sector of Merck KGaA, Darmstadt, Germany, in the press release. “We are now the first CTDMO to streamline the entire mRNA process for our clients.”

“As a researcher working on finding cures with mRNA technology and as a believer in its curative promise, collaborating with the Life Science business of Merck KGaA, Darmstadt, Germany has helped to advance my research in finding a cure by leveraging mRNA’s promise for muscular dystrophy,” Simone Spuler, head of the Myology Lab at the Experimental and Clinical Research Center (ECRC), a joint institution of the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and Charité—Universitätsmedizin Berlin, in the press release.

1. Merck KGaA, Merck KGaA, Darmstadt, Germany, Announces Acquisition of AmpTec to Expand mRNA Capabilities for Vaccines, Treatments and Diagnostics. Press Release, Jan. 7, 2021.

2. Merck KGaA, Acquisition of Exelead Completed, Plans to Invest More Than €500 Million in Technology Scale-Up. Press Release, Feb. 23, 2022.

With the two new GMP-grade mRNA manufacturing sites in Germany, MilliporeSigma can now offer fully integrated mRNA services.

MilliporeSigma Opens Two New GMP Manufacturing Sites in Germany for mRNA Production

Pamela James, PhD, is vice-president, Product, Vector Laboratories.

In the continued quest for truly personalized medicine, biomarker discovery has expanded far beyond genomics and proteomics. Scientists are increasingly interested in glycans, the carbohydrates that link to proteins and lipids in a process known as glycosylation. Glycosylation is a fundamental regulator of countless biological activities, including immune cell function in health and disease. Research seeking to characterize dysregulated glycosylation and its effects on immune regulation will be vital to the development of new diagnostics and therapeutics. Despite progress in recent years, research has only begun to scratch the surface of this broad topic.

Glycosylation analysis can provide a deeper and more nuanced understanding of immune cell function, going beyond typical biomarkers to establish more detailed phenotypic subcategories of B and T cells and offer insight into disease etiology and progression. The continued study of immune cell glycosylation will undoubtedly drive progress in the identification of methods for early detection and characterization of disease states. However, further advances in analytical technology will be instrumental in helping this field reach its full potential.

The diverse assortment of cell surface glycans, known collectively as the glycome, offers a rich and dynamic tapestry of insights into biological processes. Glycosylation is a ubiquitous post-translational modification (PTM), with approximately 50–70% of all proteins bearing glycans (1). As cell types, including immune cells, become more extensively characterized and subdivided into categories based on biomarkers, glycosylation status has become an increasingly important variable within these analyses. Glycosylation is incredibly dynamic—unlike proteins, the structures of glycans are not coded by a set genetic template. Rather, it is regulated by the milieu of enzymes surrounding the glycoform, meaning that the population of glycans found on any given cell is highly diverse and constantly changing (2). This dynamic diversity, coupled with the powerful role of glycans in regulating downstream functions, means that the glycosylation profile of a cell may provide a deeper insight into real-time functionality than biomarker expression can.

Interactions between glycans and glycan-binding proteins play a vital role in controlling and fine-tuning immune cell development, activation, and function. For example, the presence of specific O-glycan modifications to P-selectin binding ligands dictates the migration of T cell precursors to the thymus. Further glycosylation also mediates the maturation of thymocytes into CD4+ and CD8+ T cells (3). O-glycan remodeling is also an important feature of B cell development, such that distinct glycoforms can serve as markers of different phases of differentiation (4).

The glycan signatures on healthy, dysregulated, or malignant cells, as well as those on pathogens, serve as signals for immune recognition of “self” vs. “other.” The glycome is thus indispensable in regulating immunity vs. tolerance. Changes in immune cell glycosylation are a useful indicator of the cells’ activity throughout cell differentiation, activation, and apoptosis that can be used to characterize subsets of B and T cells in various disease states. These alterations have been associated with various immune cell functions, such as immune cell trafficking, antigen and cytokine receptor activation, cellular signaling, and more.

For example, glycosylation plays a key role in modulating tumor immunogenicity and immune evasion. Tumor cells secrete galectins that can bind to glycans present on effector T cells and attenuate their activity, leading to an immunosuppressive effect. Specific tumor-associated galectins restrain T cell activation, limit cytokine production, or even induce apoptosis (5). Growing evidence also implicates dysregulated glycosylation in autoimmunity. One study examined B cell markers in samples from individuals with primary Sjögren’s syndrome and systemic lupus erythematosus, two chronic autoimmune conditions (6). Researchers discovered notable changes in glycosylation within specific B cell subsets that were altered in both diseases, as well as similar alterations in certain serum immunoglobulins. These and other findings suggest that any dysfunction in normal B and T cell glycosylation can result in the development of pathogenic autoinflammation and contribute to a general perturbation of immune system homeostasis (7).

Lectin-based flow cytometry is among the most important tools for profiling glycosylation in heterogeneous cell populations. Using fluorophore-conjugated lectins that bind to specific glycan structures, researchers can analyze and quantify glycans present throughout a sample of cells in suspension. Flow cytometry offers a number of advantages that make it ideal for profiling immune cell glycosylation, including its high throughput. Researchers can quickly analyze hundreds of thousands of cells for multiple glycan biomarkers within phenotypic subsets identified by more traditional surface markers. Flow cytometry is ideal for assessing liquid samples such as serum; cells extracted from tissue samples such as the thymus, spleen, and lymph nodes; or bone marrow. However, the nature of the flow cytometry method precludes the ability to gain useful spatial information about the distribution of glycans in solid tumors or tissues using this method alone.

Nonetheless, using flow cytometry to profile glycosylation can still help healthcare providers and researchers characterize important immune mechanisms in hematological malignancies. An example of the utility of flow cytometry was shown in a 2023 study that assessed glycan markers on leukocytes in patients with myelodysplastic neoplasms (MDS) (8). MDS are age-related disorders of white blood cell overproliferation, the course of which can range from relatively benign conditions to acute myeloid leukemia (AML). In the study, the researchers identified distinct glycosylation profiles associated with cell maturation and lineage differentiation. They also characterized unique cellular glycosignatures associated with disease course, differentiating low-risk MDS from AML. These findings suggest that lectin-based flow cytometry of leukocytes could be used to identify distinct glycosylation profiles for MDS risk stratification.

Liquid chromatography and mass spectrometry are two methods used separately and in tandem to characterize glycans on a more specific level. Liquid chromatography separates distinct compounds contained within a liquid sample by running it through a solid column. The different components within the sample are then separated based on their speed passing through the column. Mass spectrometry measures the mass-to-charge ratio of ions in an analyte of interest to create a mass spectrum that can be used to determine the mass or chemical composition of the analytes.

When used together, liquid chromatography–mass spectrometry (LC–MS) allows researchers to separate and identify the components of a complex biological mixture. This combination is particularly useful in instances where liquid chromatography would not provide sufficient resolution or when analyte masses would overlap when assessed with mass spectrometry alone. Liquid chromatography, mass spectrometry, and LC–MS are valuable methods for profiling immune cell glycosylation in many applications.

Researchers have leveraged liquid chromatography and mass spectrometry to examine the relationship between immunoglobulin glycosylation status and aging, cancer, autoimmune disorders, and more. A 2020 study of inflammatory biomarkers in multiple sclerosis used hydrophilic interaction liquid chromatography with mass spectrometry to characterize glycans on immunoglobulin G (IgG) (9). When comparing serum IgG from multiple sclerosis patients to age- and sex-matched controls, the researchers identified significant differences in four of 24 measured IgG glycan traits. Additionally, the specific structural alterations observed in IgG glycans were associated with increased proinflammatory potential, suggesting the utility of glycomic changes as possible biomarkers in multiple sclerosis. The results of this study illustrate the ability of LC–MS to provide detailed structural information about glycans in a sample.

Glycosylation analysis can provide a deep and nuanced understanding of immune cell phenotype and function.

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