Aligning mRNA Progress with Regulatory Streamlining

BioPharm Peer Exchange

Technologies to develop and manufacture biopharmaceuticals continue evolving

Streamlining Bioprocesses with Automation

Technological advances are aiding manufacturers to streamline aseptic processes.

Speeding Up Aseptic Processes

Quality Considerations for Using AI in Bio/Pharma

Ask the Expert:

Sterile filtration and use of sterile single-use materials provide the best solutions depending on vector size.

Advances in Sterilization Technologies for Overcoming Viral-Vector Manufacturing Challenges

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

Antibody colored by heavy and light chains against black background; glycosylated immunoglobulin in pink | Image Credit: ©Eric H Cheung-Stock.Adobe.com

Glycan modifications of recombinant proteins and antibodies can impact their function and stability and thus the safety and efficacy of drug products based on these molecules. Understanding these important critical quality attributes (CQAs) is essential, and therefore, regulatory bodies expect a comprehensive understanding of glycans for submission.

Comprehension necessitates overcoming inherent challenges such as positional isomers and heterogeneity, as well as method-induced artifacts that influence sensitivity and specificity, according to Paula Orens, senior global market development manager, Biopharma, at SCIEX.

Multispecifics, including bi-, tri-, and tetraspecific antibodies, present additional challenges to glycan analysis beyond those of traditional monoclonal antibodies (mAbs) because the complexity of these molecules is much greater.

“Site-specific and intact mass analysis may become more challenging for increasingly complex molecules, particularly when the number of N-glycosylation sites, post translational modifications, and potential degradation products increases,” observes Oscar Potter, biological chemistries R&D manager in the Chemistries and Supplies Division at Agilent.

Structurally, multispecific antibodies are a combination of different antibody domains that allow the multispecific to bind at multiple sites. “The various domains will have their own glycosylation patterns (integral to efficacy, safety, and stability) and their own impurities, all of which need to be characterized comprehensively, effectively multiplying the glycosylation analysis complexity by the number of binding sites,” Orens explains. She adds that as the number of binding sites increases, so too does the risk of intra-protein cross talk.

Multispecifics can also include candidates in which an antibody-portion is combined with some other protein-based component. Chimeric proteins, where the domains are not only antibody in origin, are complicated to analyze due to the individual properties of each component, according to Orens. “For example,” she says, “fusion proteins are typically heavily glycosylated with N- and O-linked glycans to improve biological activity, properly stabilize the structure, or improve solubility.”

“Overall,” Orens concludes, “while the complexity of analysis is dependent on the types of domains used in a multispecific, it is certain that the analysis of these molecules will be more complicated than that of a mAb.”

Consequences of incorrect glycosylation analysis

The greater complexity of multispecifics raises the stakes for achieving correct glycan analysis. “Not only can the glycosylation patterns in multispecifics impact the stability, pharmacokinetics, off-target effects, and effector functions such as complement activation and antibody-dependent cellular cytotoxicity, conceivably the additional functions that multispecific antibodies are capable of might result in unique biological consequences from incorrect glycosylation,” Potter comments.

In addition, because glycosylation is a critical and highly regulated quality attribute of all antibodies, misunderstanding or incorrectly characterizing glycosylation patterns could lead to a variety of consequences, depending on the development stage of the candidate, notes Orens. She highlights a couple of examples. “At the hit-identification stage, a mis-characterized but suitable candidate could be eliminated due to poor efficacy or stability. At later development stages, meanwhile, a drug metabolism and pharmacokinetics study could exhibit faster-than-expected clearance or undesired concentration in the target tissue. An incorrect analysis could also result in promotion of a misunderstood candidate through to late-stage toxicology studies, causing unnecessary investment in time and resources for the project only to be shut down later than expected.”

There are several different techniques used for the glycan analysis of antibody-based candidates. “Fundamentally, the same glycan analysis approaches are used for multispecific antibodies as mAbs, most commonly liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry (MS). Each analytical approach offers a plethora of techniques which provide complementary or alternative details about the glycosylation profiles,” Orens observes. She does note, though, that the inherent complexity of the molecule will impart different challenges on each approach.

Typical analytical methods for glycosylation analysis of multispecifics are similar to those used for more traditional therapeutic mAbs, agrees Potter. “Released glycan analysis methods generate a glycan profile, usually based on separation by CE or LC. Intact LC/MS analysis can provide limited information about the major glycoforms, while LC/MS of protease-digested samples can give site-specific information for molecules with more than one glycosylation site,” he outlines.

Released glycan analysis relies on high-resolution separation of fluorescently labeled glycans by hydrophilic interaction liquid chromatography (HILIC) or CE. HILIC is used in conjunction with fluorescence or mass detection to separate and quantify individual glycan species over long gradients, according to Orens. Similarly, CE systems can offer comparable quantitation details with an alternative separation mode, often with faster separation. “These approaches are often used as monitoring techniques to ensure batch consistency during manufacturing,” she says.

Released glycan analysis once suffered from tedious sample prep, according to Potter, but protocols have been greatly simplified in recent years through rapid-release and instant fluorescent labeling sample prep kits. He observes that HILIC methods enable the most exact assignment of glycan structures, either using retention time reference standards or retention libraries, or by hyphenating with MS detection. “HILIC separations can often distinguish between isomeric structures, including potentially harmful alpha-galactose (α-Gal) structures masquerading as beneficial galactosylated complex glycans,” he says.

Measuring the intact mass of multispecific antibodies via LC/MS does not require any sample prep, which can be advantageous. A well-optimized method run on a high-resolution instrument such as a time-of-flight (TOF)-based system is needed, however, Potter observes.

Orens adds that TOF systems can be used as the detection method for several techniques to help identify and localize glycans, as well as quantify, resolve isomers, and structurally elucidate the different species.

Potter cautions, though, that while very convenient, measuring the intact mass may only reveal the sum of masses of the two or more glycans on each molecule, which may confound assignment of exact glycan structures. Distinguishing between isomers is also not possible.

The most information can be obtained by first digesting the multispecific with a protease such as IdeS (immunoglobulin G-degrading enzyme of 

) or trypsin into smaller peptides, according to Potter. “Digestion allows for site-specific glycosylation analysis by LC/MS, which may be particularly useful when distinguishing between glycosylation in the fragment antigen-binding (Fab) and fragment crystallizable (Fc) regions, the latter of which greatly impacts the Fc effector functions of the antibody,” Potter contends.

Orens further notes that when peptide mapping is performed using a MS system with specialized fragmentation capabilities such as electron activated dissociation (EAD), it is possible to identify unknown modifications or impurities, then localize the species to a specific domain or amino acid of the protein. “EAD,” she says, “is integral to this process, as it differentiates between isomers and maps specific amino acids, and thus provides a comprehensive understanding of the protein with its modifications.”

Ultimately, Orens emphasizes, the appropriate analytical approach is entirely dependent on the purpose of the analysis.

Given the importance of accurate glycan analysis for multispecifics, the best strategy for ensuring success according to both Orens and Potter is to use a combination of available methods.

“While using top down, sub-unit, or peptide mapping methods and state-of-the-art MS systems enables comprehensive glycan analysis of multispecifics since it provides the most information in a single step, including molecular weight, domain/site localization, species identity, and species quantity, most organizations employ multiple analytical approaches to support their characterization efforts and obtain secondary, confirmatory evidence,” says Orens.

“The best way to ensure accurate and comprehensive characterization,” agrees Potter, “is to use more than one orthogonal method. Released glycan analysis, intact mass LC/MS, and LC/MS after protease digestion needn’t be mutually exclusive—they can be complementary,” he concludes.

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

®
October 2024
Vol. 37, No. 9
Pages: 29-31

When referring to this article, please cite it as Challener, C.A. Glycosylation Analysis for Multispecifics. 

Articles

The best strategy is to use a combination of complementary methods.

Glycosylation Analysis for Multispecifics

Chris Spivey is the editorial director of 

Aaron Cowley, PhD, chief technical officer and co-founder, Captozyme, discussed the advancement in development of microbiome-derived therapeutics at BIO 20204. 

Advancing Development and Manufacturing of Microbiome-Derived Therapeutics (BIO 2024)

® Group’s Chris Spivey sat down with Aaron Cowley, PhD, chief technical officer and co-founder, Captozyme, and chief scientific officer, ReciBioPharm, the biologics unit of Recipharm following a spate of acquisitions (1–3) at BIO 2024 in San Diego, Calif., held on June 3–6. 

Aaron Cowley, PhD, Chief Technical Officer and Co-founder, Captozyme, and Chief Scientific Officer, ReciBioPharm. Image courtesy of ReciBioPharm.

The age of the microbiome, according to urban legend, was ushered into existence when Francis Collins, head of National Institutes of Health (NIH) (on a “nano-sabbatical” to the Broad institute), was discussing with Eric Lander the probable slowdown in funding the sequencing of “simply more organisms” for the sake of keeping infrastructure and technology refinements going. In that discussion, Collins suggested that the hitherto unheralded organism surrounding and living within the already sequenced organisms should provide significantly valuable biological data about both the disease states, as well as the underpinnings of good health, within said organisms.

Fast forward to today, and the field of microbiome research has dramatically matured, alongside the processes, analytical development, and manufacturing of living biological products (LBPs) that have, in many ways, become an end result of this line of inquiry. The potential of this therapeutic class concomitantly gives rise to the need for specialized support to develop and manufacture microbiome-derived therapeutics and has led to a rise in companies providing LBP-related solutions. As stability of product and reproducibility of manufacturing processes have become more standardized, the regulatory support environment and guidance has, of necessity, been playing catchup. This is in large part due to LBPs’ unique mechanisms of action and the absence of well-established precedents—and, also, a deeper understanding of the interaction between well-characterized administered microbial strains and host microbiome.

As a chemist by training Aaron Cowley, PhD, chief scientific officer, ReciBioPharm, became interested in the relationship between enzymes and microbes and the products of those interactions. In the interview, Cowley stated that “As a bio-factory the redox potentiometry of microbes is an important element to microbes andtheir function”. He continues to explain how gut microbes “regulate your body”.

“The microbiome keeps you in homeostasis. If you have too much extra sugar, the microbiome will shift upwards to be able to degrade that sugar. And if you have too much of other food types, it will shift downwards. Most CDMOs [contract manufacturing and development organizations] work with 

] and yeast that love air and need oxygen. There [are] very few [microbe species] [for whom] oxygen is actually a four letter word. So, being able to do tech transfer and train people has blossomed into its own field. There are a few hundred microbiome companies now, and it’s a challenging field because there are trillions of organisms in the gut, and their functions all vary. So, being able to pull out a few organisms to show cause-and-effect in disease is like trying to find life in the universe. It’s quite daunting,” Cowley explained.

Despite this, and while automating and optimizing manufacturing remains a priority, Cowley is enthusiastic about the rapid evolution of advanced therapy medicinal products in general and how, in some ways, the combined and overlapping lessons are helping provide new and more powerful insights. These individual-patient-by-individual-patient data streams, when seen from a pattern-recognition prism, can be combined to reflect back on or give the ability to both understand and deliver a more personalized approach to medicine.

1. Arranta Bio. Arranta Bio Announces the Acquisition of Captozyme, Creating a Center of Excellence for Microbiome Development and Clinical Supply. Press Release. Nov. 13, 2019.
2. Recipharm. Recipharm Acquires Arranta Bio, a CDMO Leader in Advanced Therapy Medicinal Products (ATMPs), to Expand Its Biologics Offering in the US. Press Release. Feb. 18, 2022.
3. Recipharm. ReciBioPharm: Biologics. 

Videos

During Bio 2024, Aaron Cowley, PhD, chief technical officer and co-founder, Captozyme, discussed the advancement in development of microbiome-derived therapeutics.

Development and Manufacturing Advances for Microbiome-Derived Therapeutics (BIO 2024)

Text sign showing Industry News. Business photo text delivering news to the general public or a target public | Image Credit: © Artur - © Artur - stock.adobe.com

The European Medicines Agency (EMA) announced on Oct. 2, 2024 that the agency is working with the European medicines regulatory network to improve the review and approval process for new medicines in the European Union. To improve efficiency, the agency plans to utilize the network’s resources, streamline drug review processes, and encourage applicants to provide more comprehensive submission applications. EMA hopes these efforts will accelerate the approval process.

The agency identified that the reliability of long-term planning for initial marketing authorization applications (MAAs) has been a recurring problem in need of improvement, which has led to slow approval times. According to EMA, only 35% of MAAs were submitted on time in 2023; only 30–40% of MMAs were submitted on time by the date on the letter of intent from 2018–2022. Requests for more time were made by 42% of applicants in 2023 because they did not have data mature enough at the expected time of submission. “In 2023, the average duration of clock-stops for initial MAAs (198 days) was comparable to the average time of assessment (204 days). In 2022, the average clock-stop was longer (205 days) than the assessment time (196 days),” EMA stated in a press release (1). In September 2024, representatives from EMA, national competent authorities, and industry met at a workshop to discuss submission predictability. A report will be published with recommendations developed from the workshop.

Additional plans to improve sustainability of the EU regulatory network include the reinforcement of best practices for requests for clock-stop extensions, featuring a standard template for requests created by EMA’s Committee for Medicinal Products for Human Use (CHMP) and the Committee for Advanced Therapies (CAT). CHMP and CAT also began strictly applying provisions from the 2009 guideline on extensions in July 2024. “All requests for clock-stop extensions need to be well justified, and the CHMP and the CAT will no longer grant extended clock-stops to solve problems caused by immature application dossiers or issues that were foreseeable prior to the submission of the application,” the agency stated in the press release.

EMA also revamped its two main assessment report templates in 2023. The agency is also planning the launch of a revamped “Overall Template” in January 2024. “With the new templates, there is a clearer differentiation between the evidence submitted by the applicant and EMA’s assessment. Information has been better structured, and duplication deleted,” the agency stated in the press release.

Other plans include the development of better guidance for assessors through report templates, guidance checklists, and training. EMA also piloted an automatic email notification to market authorization holders in 2023 that requests them to submit a list of planned line extensions and submission variations for the following six-month period. “This automatic request will be sent to each company contact point on 1 May and 1 November going forward. This will allow rapporteurs to have better visibility of the volume of post-approval work that they can expect in relation to a product,” EMA said in the release. The agency also plans to improve its dialogue with applicants in 2025.

EMA. Improving Efficiency of Approval Process for New Medicines in the EU. Press Release. Oct. 2, 2024.

The agency is working with the European medicines regulatory network to improve the assessment and approval process for new medications.

EMA to Improve Drug Approval Process

Behind the Headlines: Sickle Cell Therapies; Mpox Vaccines; and ARCH Ventures’ Biotech Fund

This Behind the Headlines news roundup panel discussion on recent news items includes updates regarding sickle cell therapies (e.g,, Pfizer’s withdrawal of Oxbryta and Vertex’s choosing of Lonza as the commercial manufacturer of Casgevy); Emergent BioSolutions $400 million BARDA contract for an mpox vaccine; and the first in a generation novel schizophrenia drug, Cobenfy (xanomeline and trospium chloride). The panel also examines investment revival with ARCH Ventures $3 billion biotech fund raise and the $3.6 billion for Flagship Pioneering. Rounding things out is a discussion on COVID-19-associated childhood myopia and patent thickets artificially keep the price of drugs high.

Pfizer’s withdrawal of Oxbryta, one of a limited number of treatments that won accelerated FDA approval in 2019, surprised doctors, patients, investors, and analysts. CNBC News stated, “Oxbryta was one of the centerpieces of Pfizer’s $5.4 billion acquisition of Global Blood Therapeutics in 2022” (1). Vertex’s gene therapy, Casgevy (exagamglogene autotemcel), was given commercial-scale manufacturing capabilities when Vertex chose Lonza as its contract manufacturing and development organization for global release. This marks a major milestone in gene editing’s future deployment, and much will be learned about what is feasible, and what requires a re-think. Interestingly, Casgevy does not address the root cause of sickle cell but instead suppresses the suppressor of infant hemoglobin production, hence essentially diluting the problems of limited healthy hemoglobin by creating an alternate supply naturally only available to infants (2).

Emergent BioSolutions contract with the Biomedical Advanced Research and Development Authority (BARDA), worth $400 million, for mpox vaccine production comes at a time when we must ask ourselves, was this new strain outbreak controllable had the US government not destroyed its aging stockpiles of the older traditional? In an example of vaccine nationalism, “While waiting on a freeze-dried long shelf-life vaccine to be developed, the United States let its stockpile of liquid monkeypox vaccine languish and expire. By 2017, all 27,993,370 doses in the national Jynneos stockpile had expired, although the US still had a huge stockpile of its other smallpox vaccines. While waste per se is shameful, it’s akin to Greek tragedy when the rise in monkeypox cases internationally is associated with lost opportunities to improve millions of people’s lives in sub-Saharan Africa, where smallpox was eradicated in 1980, and vaccinations halted” (3).

After a generation of few new treatments for schizophrenia, Bristol Myers Squibb’s $14 billion deal for Karuna Therapeutics has borne fruit with the approval of a highly promising new drug with a novel mechanism of action (4).

Constantino, A. and Capoot, A. Healthy Returns: Pfizer Pulls Sickle Cell Disease Drug from Markets–Here’s Why it Matters. 

Spivey, C. New Medicines, Markets, and Manufacture: CRISPR for Sickle Cell Disease and β-thalassemia. PharmTech.com. Nov. 30, 2023.

BMS. US Food and Drug Administration Approves Bristol Myers Squibb’s COBENFY (xanomeline and trospium chloride), a First-In-Class Muscarinic Agonist for the Treatment of Schizophrenia in Adults. Press Release. Sept. 26, 2024.

Benjamin McCloud is co-founder of Stealth Co.

Chris Spivey is director of Industry Relations and Strategic Partnership at MJH Life Sciences.

This Behind the Headlines news roundup panel discussion on recent news items includes updates regarding sickle cell therapies (e.g,, Pfizer’s withdrawal of Oxbryta and Vertex’s choosing of Lonza as the commercial manufacturer of Casgevy); Emergent BioSolutions $400 million BARDA contract for an mpox vaccine; and the first in a generation novel schizophrenia drug, Cobenfy (xanomeline and trospium chloride). The panel also examines investment revival with ARCH Ventures $3 billion biotech fund raise and the $3.6 billion for Flagship Pioneering. Rounding things out is a discussion on COVID-19-associated childhood myopia and patent thickets artificially keep the price of drugs high.


Feliza Mirasol is the science editor for 

SGD Pharma, a global provider of pharmaceutical glass packaging solutions, announced on Oct. 1, 2024, that it will exhibit an innovative range of products and services at CPHI Milan in Milan, Italy, from Oct. 8–10. The company will introduce its siliconized molded glass vials as part of the presentation. According to a company press release, the siliconization process is an internal treatment that provides additional functionalities to a glass vial, .

The internal silicon coating provides protection for sensitive, aggressive, and viscous drug products. In doing so, the coating reduces interactionwiththepackagingandpreservesdrugproductintegrity, according to the press release. SGD stated in the release that siliconizationisavailable for all glass types and for vials ranging in size from 3 mL to 500 mL, both clear and amber glass. The siliconization processissuited toallroutesofadministration, including parenteral,oral, andnasal administration.

“SGDPharma’s €5 million [US$5.5 million] investment in the siliconization production line at its state-of-the-art Saint-Quentin Lamotte(SQLM)plant [in France]enablescustomerstoreceiveordersofhigh-performancevialsatspeed meeting global demand,” the company stated in its press release.

The growing demand for the approval of biologics as well as new therapeutic approaches and the development of specialty drugs is driving the need for ready-to-use (RTU) packaging, SGD Pharma said in its press release. The company offers a RTU solution in molded glass, which includes its Sterinity EZ-fillvials (aregisteredtrademarkofStevanatoGroupand/oritsaffiliatesinoneormorejurisdictions). The Sterinity EZ-fillvials are designed for the rigorous requirements demanded by the pharmaceutical industry. The increased quality and flexibility of these vials enable the shortening of time-to-market and reduce total cost of ownership, according to the press release. Sterinity is suitable for various applications in veterinary and human health.

“AtCPHI2024,weareshowcasingsolutions developedwithourpharmaandveterinarycustomers’needsinmind,aimedatenablingthem tobringtheirdrugstomarketquicklyandsafely.Thiscommitmenttoinnovationisreflectedby our dedication to sustainability, which is an integral component of our company strategy. As we support theUNGlobalCompact forthefifthconsecutiveyear, we are particularlyproudof our progress in reducing carbon emissions at our global glass manufacturing sites,” said OlivierRousseau,CEO,SGDPharma,in the release. “SGD Pharma’s renewable energy initiatives, including the upgrade of our furnace at Saint Quentin Lamotte (SQLM) in France and the installation of solar panels at our Vemula, India, plant, underscore an overarching focus on decarbonization with the company-wide goal to cut emissions recorded in 2020, by 35% by 2030, and by 65% by 2040. These efforts reflect our responsibility to our customers, partners and the planet as we strive for a safer and healthier environment.”

The company will present an extension to its Sterinity range at CPHI, including the debut of aRTUTypeIImoldedglassvial. Sterinity RTU in Type II is expected to initially be available in size 10H, and an extendedrangeofsizes is expectedto launch during2025. SterinityTypeIiscurrently available in sizes 10 mL, 20 mL, 25 mL, 50 mL, and 100 mL. Type I is available in both clear and amber glass.

In addition, SGD Pharma will present an extension to its IDENCY range that includes 20 mL and 6 mL sizes. The company will also introduce new caps for its Ensiemo syrup bottles. The presentation will include the following:

IDENCY, a Type I glass vial that combines the benefits of both molded and tubular technologies, for parenteral sensitive drugs.

Ensiemofull packaging solution, apharma-grade TypeIII glass vialand pipette, which is now available with polypropylene or polyethylene caps for a tamper evident and child resistant closure.

The company now offers LabServices, an analytical testing service in molded glass for its customersandpartners (1). Through this service, experts at SGD Pharma’s SQLM plant provide analytical testing for pharma and biopharma companies, contract manufacturing organization and contract development and manufacturing organizations, and other glass manufacturers. The company expects to launch more LabServices in 2025.

The showcase will be held at SGD Pharma’s booth, 20B11, during CPHI.

1. SGD Pharma. New Labservices for Pharmaceutical Molded and Tubular Glass, Available Exclusively to SGD Pharma Customers and Partners. 

SGD Pharma plans to showcase its siliconized molded glass vials at CPHI Milan as part of its range of sustainable products and services.

SGD Pharma to Showcase Sustainable Packaging with Siliconized Molded Glass Vials at CPHI Milan

Sexy Science in Pharmaceuticals: Hot-Melt Extrusion

Traditionally, hot-melt extrusion (HME) is co-located with downstream processing and solid-dosage form manufacturing. Regulatory bodies continue to encourage quality-by-design (QbD) approaches, quality management maturity (QMM), and process analytical technology (PAT) improvements. These are all essential tools in a well-organized HME platform and work together to enhance product outcomes, but also to improve process understanding. HME, as an approach, is aptly suited to this framework.

As molecular diversity ramps up, the total number of difficult-to-formulate drugs poses severe manufacturing challenges.Creativity, innovation, and bringing in material science practices from other fields seems a fruitful approach.

“Approximately 30–40% of all marketed drugs have low solubility (BCS Class II and IV). Overall, it is estimated that up to 90% of small-molecule APIs in development face solubility challenges; as such, solubility enhancement approaches are critical for the successful advancement of therapeutics that have yet to reach the market” (1).

This episode of Sexy Science in Pharmaceuticals brings together two world-class scientists to discuss one of the top solutions for low solubility. For bioprocessing and manufacturing larger molecules, low solubility and protein aggregation also needs addressing, as does dedicated storage and handling throughout. We discuss these challenges and cite several paths around or through these issues.

As an opening introduction to this discussion, we examine the general aspects of HME supporting bioavailability enhancements for poorly soluble BCS class 2 and class 4 compounds. Examples discussed include Noxafil, an oral suspension used in adults and children to help prevent or treat fungal infections from widespread dissemination throughout the body (also used to treat a fungal infection called “thrush” caused by 

in the mouth or throat area), and Belsomra for insomnia.Both products are part of the Merck portfolio.

In this part of the discussion, a robust processing space to obliterate crystalline structures to create a single-phase homogenous solid amorphous dispersion is defined. Also, we draw up a more green sustainable future without high solvent use and disposal—HME not requiring large volumes of acetone, in contrast to spray drying dispersion processing. In HME, as the temperature rises beyond the glass transition temperature for that material the polymer becomes a liquid. The drug solubilizes into that molten phase. So, we have now a liquid phase that has dispersed drug crystals, which over time break down in the extruder, dissolve, and diffuse into that molten matrix to create a single molecularly dispersed phase. We then use X ray diffraction to look for (hopefully) the absence of crystalline peaks associated with the API. 

In another quality check to see if we’ve been successful, we use differential scanning calorimetry where we want to see a single glass temperature observed in the material, thus showing we’ve also eliminated drug rich domains.

In this part, we discuss HME biological macromolecules for slow release subcutaneously implantable vaccine delivery and injection molded microneedle patches. Aggregation in proteins and frailties of virus like particles leading to inhomogeneity needs to be addressed.

The commercial philosophy of spray dying in early R&D stages, then switching to HME for commercialization is outlined in this discussion. In general, looking at mixing and quenching.Dealing with whole bacterial spore cells extruded into polymer matrices to degrade polyurethanes (in environmentally friendly and sustainable ways), in collaboration with BASF, is also described.

1. Merck. Enhancing API Solubility During API Processing and Formulation Development. eBook, PharmTech.com Oct. 26, 2023.

Professor Jonathan Pokorski, Department of Chemical and Nano Engineering, UC San Diego Jacobs School of Engineering

Professor Pokorski’s laboratory works to bridge chemical synthesis, molecular biology, and materials science to make new materials for biomedical applications. The Pokorski lab is particularly interested in marrying protein and polymer science to generate new materials for drug delivery, imaging, and vaccination. Pokorski received his doctoral degree in organic chemistry from Northwestern University in 2007, where he designed, synthesized, and tested diverse peptidomimetic systems for use in medical diagnostics and therapeutics. Following his PhD studies, Pokorski moved to The Scripps Research Institute, where he used both chemical and genetic engineering of viral nanoparticles to synthesize novel drug delivery systems. During his postdoctoral training, Pokorski first earned an NIH Ruth Kirschstein fellowship and later secured an NIH Pathway to Independence Award.

Prior to his appointment at UC San Diego in 2018, Pokorski began his independent career as an Assistant Professor at Case Western Reserve University in the department of Macromolecular Science and Engineering. Pokorski was a finalist for the prestigious John Diekhoff graduate mentoring award at CWRU from 2013 to 2017, was named an ACS PRF new investigator, and is a Kavli fellow. Research in the Pokorski lab is funded through grants from the National Institutes of Health, National Science Foundation, and the American Chemical Society.

Dr. James DiNunzio, Senior Principal Scientist, Merck

Dr. James DiNunzio is currently a Senior Principal Scientist at Merck & Co., Inc. in Rahway, NJ where he heads the Hot-Melt Extrusion Subject Matter Expert Network Team and co-leads the Continuous Processing Technology Development Team.Since joining Merck in 2013, he has contributed to multiple programs in the disease areas of oncology, virology, and CNS (central nervous system) disorders.Prior to Merck, he served as a Senior Scientist in the Pharmaceutical & Analytical R&D group at Roche where he was responsible for the formulation design of poorly soluble compounds using enabled technologies.He has also held positions of increasing responsibility at PharmaForm and Forest Laboratories.

Dr. DiNunzio received his PhD in Pharmacy (Pharmaceutics) from The University of Texas in 2009 specializing in thermal manufacturing of amorphous dispersions.He also holds M.S. and B.S. degrees in Chemical Engineering from Columbia University and The State University of New York at Buffalo.He currently serves on the Editorial Advisory Boards of AAPS 

.He has authored over 25 research papers and book chapters in the field of hot-melt extrusion and formulation design for bioavailability enhancement.His current research interests include formulation design for bioavailability enhancement, melt extrusion process design for challenging systems, and the development of continuous manufacturing technologies.

This premiere episode of Sexy Science in Pharmaceuticals features Professor Jonathan Pokorski, UC San Diego Jacobs School of Engineering, and Dr. James DiNunzio, Senior Principal Scientist at Merck & Co., Inc. in Rahway, NJ, discussing the benefits of hot-melt extrusion.

Do you Suffer from Product Loss?

Do you Suffer from Product Loss in Fluid & Cold Chain Management?

Cheryl Barton is director of PharmaVision, 

European union DNA | Image Credit: ©Kestutis - Stock.adobe.com

Over the past two decades, 32 cell and gene therapies (CGTs) have been approved by the regulatory authorities worldwide, and more than 2000 novel products are under clinical investigation (1). However, many patients do not have access to these life-saving therapies, partly due to the complexity and high cost of manufacture (2). But how can CGTs be made more cost-efficient given the high labor and facility costs?

Frontrunners in Automated Cell and Gene Therapy Manufacturing

According to Miguel Forte, CEO, of Kiji Therapeutics, France, and president of the International Society for Cell & Gene Therapy, “It is clear that CGTs have the opportunity to deliver enormous value to patients; nonetheless, manufacturing is complex, and it has been challenging to deliver the product. Innovative technologies and services have enabled the industry to move towards an automated, closed-system approach that is easier and safer to operate and is less costly and closer to the patient.”

 from autologous cell therapies (patient to patient), allogenic cell therapies (donor to patient), or 

 cell therapies (cell line to patient). Each approach requires manufacturers to tailor the biomanufacturing process to generate specific cell types (and/or vectors), safely, efficiently, and cost-effectively. Manufacturing tools have tended to lag behind scientific advances in CGTs leading to bottlenecks in cell production. Currently, most CGT manufacturers use disconnected single-step/modular or semi-automated solutions; however, the industry is migrating towards fully automated cell therapy manufacturing processes (3). And this approach could significantly reduce manual intervention, risk of contamination, and human error; improve product quality and speed of delivery; and reduce production costs (4).

According to David Smith, vice president of Development, BioCentriq, “Labor costs, and facility costs are sky high, which have led to high price points, particularly in autologous cell therapies where production is 1:1. Automation is a key part of improving costs, reducing labor both in and outside the clean room, and implementing release by exception can help to reduce bottlenecks. Companies are trying to integrate unit operations whether it’s an ‘all-in-one-box’ approach or a ‘unit-operator’ approach where units can be plugged in or out and are integrated within robotic systems to offer greater flexibility.”

For instance, Lonza’s Cocoon Platform and Miltenyi Biotec CliniMACS Prodigy offer an ‘all-in-one-box’ approach providing a closed, automated system for patient-scale cell therapy manufacturing from cell isolation, activation, transduction/transfection, expansion, and harvest within a validated cell manufacturing system (5,6). CliniMACS Prodigy modular Instruments can also be run in parallel to provide large-scale batch production.

UK-based Ori Biotech launched its IRO platform developed in 2024 in collaboration with CTMC (a joint venture between Resilience and MD Anderson Cancer Center). This platform automates the activation, transduction, expansion, and harvest of a wide range of cell types to rapidly scale up manufacturing from the clinic to the commercial setting (7).

Paris-based Astraveus SAS has developed the Lakhesys Benchtop Cell Factory, an end-to-end solution for the manufacture of adherent, non-adherent, and inducible pluripotent stem cells (iPSCs) (8). The company has raised €16.5 million in seed financing and received a €10.4 million grant from the French government as part of its “Innovation in Biotherapies and Bioproduction” initiative, to advance the development of its technology (9).

Read the article in the Emerging Therapies eBook.

https://www.e-digitaleditions.com/i/1526741-biopharm-international-september-2024/4?

https://www.e-digitaleditions.com/i/1526741-biopharm-international-september-2024/0?

Cheryl Barton, PhD is director of PharmaVision.

Article details
BioPharm International®
eBook: Emerging Therapies
September 2024
Pages: 4–7

When referring to this article, please cite it as Barton, C. Frontrunners in Automated Cell and Gene Therapy Manufacturing. 

BioPharm International Emerging Therapies

Innovative solutions are making personalized cell and gene therapies accessible to all.

CPHI Milan announced its annual survey results on Sept. 27, 2024. The results showed that growth of business for contract development and manufacturing organizations (CDMOs) is expected to accelerate in 2025. These predictions come from new research findings from 280 pharmaceutical companies; the findings will be released at CPHI Milan as part of the CPHI Annual Report and Survey. The research findings also suggest that the industry does not feel favorable toward the recently passed BIOSECURE bill (1).

CPHI Milan noted in a press release that most analysts assessed the first half of 2024 as having slower-than-expected recovery. The press release quoted Gil Roth, president of the Pharma & Biopharma Outsourcing Association (PBOA), as having said, “While reports indicate that dollars are flowing back to late-stage assets [at CDMOs], there is little evidence that early stage pipeline products are receiving funding [yet]. This means that the lifeblood of CDMOs—projects moving through the development pipeline and smaller customers sticking with key service providers—remains hobbled.”

In the upcoming 2024 CPHI annual report, the organization takes note that the wider pharma industry is more optimistic with a forward-looking outlook for contract services companies’ growth in the next 18 months. The report shows that 49% of industry respondents are “highly positive” and 44% are “neutral”, while only 7% are predicting “negative” prospects.

Discussion in the contract services sector also revolves around the shortage of available contract capacity for glucagon-like peptide 1 (GLP-1) drugs. The impact of the BIOSECURE Act and how it may affect contract service industry prospects is also a major concern. According to the report, a consensus among survey respondents suggest that the Act has further accelerated the post-COVID trend for greater supply chain diversity; both US-based and particularly India-based CDMOs are reporting greater interest in establishing strategic partnerships.

A significant finding in the report is that a majority of the industry does not support the BIOSECURE Act’s stated goals. Nineteen percent view China based-CDMOs as “a threat and should be removed from Western supply chains”, whereas 38% of respondents argue that BIOSECURE is a “politically motivated issue that sets a concerning precedent for the industry.” The remaining 43% of industry respondents, in other words, the largest grouping, have a balanced view, where they both support “WuXi’s tremendous contribution to the industry,” but at the same time want to err on the side of caution, believing it is sensible for the industry to “diversify supply partners.”

“The contract services zone at CPHI continues to grow year on year, in large part driven by the industry’s need for trusted partners who can provide capacity, help navigate the nuances of complex therapeutics and support the road to commercialization. CPHI Milan serves as a vital platform for connecting existing and prospective partners, and we look forward to continuing to serve this growing industry segment in 2025, and beyond. We also see a desire among our attendees [i.e., the people awarding contracts] to meet current partners and advance projects, but also to significantly widen their networks as we head into 2025,” said Tara Dougal, Brand & Content director at CPHI Milan, in the press release.

The research findings show that global innovator and generic-drug pharma companies are actively looking for wider supply chain customers and key development partners. With more than 600 contract services providers expected to attend CPHI Milan on Oct. 8–10, partnering discussions are expected to increase as biotech funding motivates greater deal volumes for 2025 and beyond.

CPHI Milan will host 150 speakers and hold more than 100 sessions across multiple facets of pharmaceutical discovery, development, manufacturing, and production. Approximately 62,000 professionals in the pharmaceutical industry are expected to attend.

Results from the CPHI annual survey show that 49% of industry respondents are “highly positive” on the contract services sector seeing growth over the next 18 months.

CPHI Milan Survey Predicts Faster CDMO Growth in 2025 While Finding No Industry Favor for BIOSECURE Act

Bolstered by a pilot program launched by the Biden administration, ReciBioPharm’s chief scientific officer, Aaron Cowley, discusses ongoing progress in developing continuous manufacturing for mRNA.

Biopharma’s Progress with Continuous Manufacturing in mRNA (BIO 2024)

US government investment in manufacturing advances for messenger RNA (mRNA) hit a high note in the summer of 2023 with the launch of the “Curing the Uncurable via RNA-Encoded Immunogene Tuning” (CUREIT) project. The CUREIT project was launched by the Advanced Research Projects Agency for Health (ARPA-H) as part of the Biden administration's moonshot initiative. With up to $24 million in new funding, this effort, led by a team at Emory University in Atlanta, Ga., has the goal of developing “… generalizable mRNA platforms that can be harnessed to train the immune system to more effectively fight cancer, … other applications could extend to autoimmune disorders, transplants, and infectious diseases” (1). 

A month prior to the Biden administration’s announcement, Peter Marks, director of the Center for Biologics Evaluation and Research (CBER) at FDA, allocated an award of $82 million to Massachusetts Institute of Technology (MIT) researchers to create a center for continuous mRNA manufacture (2). The main manufacturing subcontractor for this three-year pilot program is ReciBioPharm. The over-arching goal is to establish the first biologic drug using a fully continuous manufacturing integrated process, from 

Aaron Cowley, PhD, Chief Scientific Officer, ReciBioPharm. Image courtesy of ReciBioPharm.

At the BIO 2024 conference held in San Diego, Calif., on June 3–6, Pharmaceutical Technology Group’s Chris Spivey spoke with Aaron Cowley, PhD, chief scientific officer at ReciBioPharm to check in on the pilot program’s progress. Cowely stated that “being able to have eyes on the process, and the product, as it goes along the full train of manufacturing, and being able to release that data in real-time, that is the overall goal.” When addressing potential scale-up bottlenecks or other obstacles, particularly on the analytical side, Cowely went on to say, “the beauty of this grant is that we are able to take the best technologies, regardless of company source, and incorporate them, to see what’s working and what’s not work[ing] [for continuos manufacture]. After a year and a half, we already have a process development system that’s capable of going from [IVT] through [to] fill/finish, fully operational, so we’re collecting data. We have the process analytical technology (PAT) that’s actually driving the process already. We can see the kinetics of the process. We can see the impurity profile. We can take the material and send it to waste, if it's not meeting criteria, or move it ahead to the next unit operation … we’ve been using off-the-shelf PAT equipment, but not in the way it’s been traditionally deployed.”

The pilot project has placed a heavy emphasis on process modeling with the help of machine learning to repeatedly evolve the system performance and product outcomes. Cowley said they’ve hit paydirt by now being able to remove system variability and are now “able to control what others currently cannot control. It’s like manufacturing in a vacuum. So, it’s much easier to track your product, and the quality of your product.”

1. The White House. As Part of President Biden’s Unity Agenda, Biden Cancer Moonshot Announces Launch of ARPA-H’s CUREIT Project Led by Emory University to Develop New Tools to Strengthen the Immune System and Save Lives. Press Release. Aug. 23, 2023.
2, Winn, Z. MIT Researchers to Lead a New Center for Continuous mRNA Manufacturing. 

Under a pilot program initiative introduced by the Biden administration, Aaron Cowley, chief scientific officer, ReciBioPharm, talked about the ongoing progress in developing continuous manufacturing for mRNA.

Much work has been going into developing alternative routes of drug delivery for biologic therapeutics. The biopharmaceutical industry has been focusing on enhancing ease-of-use and patient compliance. Biologics today are mostly administered parenterally, which can be painful and undesirable for patients. Furthermore, parenteral formulations of biologics require a strict temperature-controlled environment. 

Researchers have long been exploring means to formulate biologic-based molecules that can ensure their molecular stability and mitigate some of the stringent manufacturing requirements. In this episode of the Drug Solutions podcast, Feliza Mirasol, science editor of 

® explores the issues challenging biologic formulation and the progress made so far in moving toward alternative routes of biologic drug delivery with Nigel Theobald, CEO of N4 Pharma, a specialist pharmaceutical company developing novel delivery systems for oncology, gene therapy and vaccines.

Nigel Theobald is CEO of N4 Pharma, a specialist pharmaceutical company developing novel delivery systems for oncology, gene therapy and vaccines. Theobald has over 25 years’ experience in healthcare, in building businesses, strategy development and implementation, and in a strong network covering all aspects of pharmaceutical product development and commercialization. He was the head of healthcare brands at Boots Group plc in 2002 before leaving to set up a series of successful businesses, including Oxford Pharmascience Group plc, which he grew over five years into an AIM-quoted company with a market capitalization of £40 million (US$53 million) upon departure. Theobald formed N4 Pharma in 2014.

 presents the Drug Solutions podcast, where the editors will chat with industry experts from across the pharmaceutical and biopharmaceutical supply chain. Join us as experts share insights into your biggest questions—from the technologies, to strategies, to regulations related to the development and manufacture of drug products.

Podcasts

This podcast explores the challenges of and the progress made so far by the biopharma industry toward alternative drug delivering methods for biologic drugs.

Podcast: Alternative Drug Delivery Methods for Biologics: Where Are We At?

Roxana McCloskey is senior global marketing manager, Protein Therapeutics, at SCIEX.

Zoe Zhang is manager, Biopharma Applications, at SCIEX.

Cell line development (CLD) plays a pivotal role in the production of biotherapeutic molecules. The primary goal during CLD is to generate and identify the most stable cell lines capable of robust production of high-quality therapeutic molecules. To achieve this, screening for both product quality and productivity is essential. Identifying and optimizing stable high-yield cell lines early contributes to accelerating the entire drug development process and the success of the pipeline. 

Ensuring Cell Line Stability for Protein Therapeutics

In addition to classical biologic drugs such as monoclonal antibodies (mAbs), new therapeutic modalities are being developed at a rapid pace. These include bi-, tri-, and multi-specific antibodies, antibody-drug conjugates (ADCs), and fusion proteins such as bispecific T-cell engagers (BiTEs) and peptide-Fc proteins or peptibodies (1,2). The increasing complexity and diversity of these new modalities require innovative analytical approaches that integrate technologies such as capillary electrophoresis (CE) and mass spectrometry (MS). Integrated CE–MS technologies provide the necessary sensitivity, accuracy, and precision to monitor key product quality attributes (PQAs), detect data inconsistencies, and thus characterize, screen, and select optimal cell line clones early in the CLD process.

Given the vast number of potential clones entering the development pipeline, methods to rapidly prioritize candidates are crucial. These methods must be able to quickly and precisely assess the critical quality attributes (CQAs) of the protein products to narrow down the best candidates. Chemical post-translational modifications (PTMs), such as glycosylation, deamidation, and oxidation, are considered CQAs as they often result in charge variants that affect the product’s stability, efficacy, potency, and safety. As such, PTMs must be characterized and controlled throughout the drug development pipeline.

Traditional methods such as enzyme-linked immunosorbent assays (ELISAs) have limitations in sensitivity, specificity, and coverage, which can delay the identification of inconsistencies. Identifying and mitigating data inconsistencies early in the development process is paramount during CLD to ensure the stability and efficacy of the final biotherapeutic product. The integration of CE- and MS-based technologies offers a robust solution that can significantly enhance the detection of data inconsistencies, facilitating the development of stable cell lines for bioproduction.

CE provides rapid assays that can assess key factors affecting final product stability, such as charge and glycan profiles, while MS offers precise information about key factors affecting product quality, such as molecular modifications and their localization and protein structure.

During the manufacturing of mAbs and other complex therapeutics, heterogeneity can arise from enzymatic cleavage and PTMs. Ensuring stability and consistency in therapeutic protein production necessitates meticulous monitoring and control of PTMs and genetic mutations. Moreover, the early detection and characterization of these PTMs during CLD can prevent costly and time-consuming issues later in the production process.

The final therapeutic product can be affected by changes induced by various cell processing and growth conditions, which, therefore, must be comprehensively understood. Alterations in titer, cell culture media, and other process conditions can induce PTMs that affect the stability and activity of the final protein product. Therefore, gathering comprehensive information about key quality attributes, including PTMs, is crucial to understanding the stability of proteins produced by cell lines.

1. SCIEX. Improving the mAb Characterization Process. Whitepaper. Feb. 8, 2024.
2. Zhang, Z.; McCloskey, R. Untangling the Identity of Charge Variants in Antibody-Drug Conjugates. 

Roxana McCloskey is senior global marketing manager, Protein Therapeutics, and Zoe Zhang is manager, Biopharma Applications; both at SCIEX.

When referring to this article, please cite it as McCloskey, R; Zhang, Z. Ensuring Cell Line Stability for Protein Therapeutics. 

Stable cell line development can benefit from applying integrated analytical technologies.

Featured Content

Streamlining Bioprocesses with Automation

Speeding Up Aseptic Processes

Ask the Expert:

Advances in Sterilization Technologies for Overcoming Viral-Vector Manufacturing Challenges