Compared with small-molecule therapies, which have been in use for more than a century, the field of biological therapies is reasonably nascent. Since the approval of the first recombinant protein in the 1980s, numerous biological therapies have been developed for a variety of indications (1).

“The field of biological therapies has indeed experienced a significant expansion and transformation, largely driven by advancements in molecular and cellular biology,” says Anupreet Kaur, post-doctoral associate in the Brinson Research Group, Institute for Bioscience and Biotechnology Research, National Institute for Standards and Technology, University of Maryland. “One of the key breakthroughs has been the widespread adoption of recombinant DNA technology, which has revolutionized the production of protein therapeutics.”

“[Going back to] the end of the last century, when there were mainly small-molecule drugs on the market, [patients were typically] suffering from major side effects,” notes Peter Timmerman, head of Peptide Science at Biosynth. “[These unwanted effects] paved the way for a new generation of drugs that were more specific and more biologically based.”

A major benefit of biological therapies is their ability to reach targets that have previously been thought of as ‘undruggable’ with more traditional small-molecule therapies (2). Despite being much larger than small-molecule drugs, biological therapies feature a greater affinity and selectivity to a specific target, making them more effective with less off-target toxicity, Timmerman explains.

Specificity to molecular targets is a key attribute of monoclonal antibodies (mAbs), remarks Kaur. “This specificity allows [mAbs] to distinguish between subtle differences, such as single amino acid variation and conformational changes, enabling precise targeting of disease-related molecules,” she says. “Additionally, the bivalency of each antibody molecule contributes to the higher binding strength, which enables mAbs to bind with exceptional affinities, typically ranging from picomolar to nanomolar levels.”

This high binding strength is important for differentiating between the binding partners and for enhancing the therapeutic efficacy of the mAbs, continues Kaur. “Moreover, the Fc [fragment, crystallizable] domains of mAbs play a crucial role in [biological] properties,” she adds. “This region can engage with various components of the immune system leading to functions such as antibody-dependent cellular cytotoxicity, or complete complement dependent cytotoxicity. These effector function-enhanced antibodies have the ability to eliminate the target cells and modulate the immune response, further improving therapeutic potential.”

“As targets for mAb therapies are identified, fewer are agonist targets and more are antagonistic targets,” remarks Scott Rudge, founder of SynerG Biopharma Group. “When an agonist target is found, the antibody can be designed to stimulate a cell or an organ to produce a certain outcome.For example, insulin is a protein agonist, because it causes cells to take up glucose and store it as glucagon.”

However, antagonists, such as check point inhibitors, actually block something from happening, Rudge emphasizes. “If every receptor in the body needs to be inhibited or down-regulated in some way, antagonists are going to require ever increasing doses, which means higher concentration, to be administered subcutaneously or some other way less invasive than intravenous administration,” he adds.

“Subcutaneous and intramuscular delivery, in particular, require highly concentrated formulations, as the maximum volume these routes of administration can accommodate is about 1 mL,” Rudge explains. “Alternatively, other means for delivering high doses of mAbs could be found that deliver more than 1 mL. Research in these two areas is very active.”

Additionally, the development of bispecific antibodies represents a significant milestone for the biopharma industry, Kaur adds. “This platform holds promise for enhancing the efficacy, increasing the half-life, and improving the stability and specificity of biologic therapies, while minimizing the side effects,” she says.

Looking beyond antibody therapeutics, Kaur highlights RNA vaccines as a promising platform that will enable the development of novel vaccines to tackle infectious and other diseases. “Ongoing research aims to further optimise RNA vaccine platforms and expand their application beyond infectious diseases,” she says.

“Also, there are short oligonucleotide therapies, which will present a versatile and promising approach for the treatment of orphan rare diseases by targeting specific genes and other regulatory elements,” Kaur specifies. “Continued research and development efforts are focused on improving the delivery strategies, enhancing the specificity and efficacy, and expanding the clinical application of oligonucleotide-based therapeutics.”

Focusing on potential future trends for the biologics field, Hans de Backer, head of the Peptide Division, Biosynth, emphasizes that there are several key trends that will be ongoing for a long period of time. Some are more industry generalized trends, such as accelerating time-to-market and moving towards more green solutions in manufacturing; however, a more specific trend for biologics will be the drive for increasingly patient-specific solutions instead of generic ‘one-size-fits-all’ type therapies.

A further trend that is shaping the biologics sector is the development of combination therapies, whereby companies are developing these products as combination options from the outset rather than considering a potential combination later on, De Backer specifies. “The expanded range of biological and small-molecule drugs already approved can be being used during development to act on multiple pathways or the same pathways through differing mechanisms. It is therefore increasingly important for researchers to be able to access reagents (as biosimilars or generics) early in development to understand modes and mechanism of action, and routes for improving efficacy,” he says.

“All of the aforementioned trends will be mainly boosted by the introduction of artificial intelligence (AI), which is being seen in all areas playing a key role” De Backer says. In particular, he points out that AI is helping to accelerate time-to-market for drug development, all the way from discovery to production. AI is helping companies to deliver results that in turn are fitting in with funding cycles and ultimately helping them commercialize their drug products.

In terms of personalized medicine, AI will enable further personalized drug manufacture options—but also personalized prescription. AI can be used by clinicians to assess the sort of response they can expect from a therapy, which is allowing them to choose the best therapeutic option for their patients (3). “AI will definitely bring new opportunities and allow for better forecasts [of therapy outcomes], driving personalized medicine to the next level,” De Backer asserts.

Additionally, Timmerman states that he has high expectations of AI as it has the potential to provide information that hasn’t been seen before, particularly for drug discovery. Currently, when trying to look at the drug space, industry is limited to only a fraction of what is available, he emphasizes, whereas AI has the potential to be used to map out the complete drug space, enabling the identification of a greater number of novel chemical entities (4).

There are other areas where AI can provide new information, Timmerman notes. “For example, there is a new modeling program called AlphaFold that basically can predict the structure of all proteins from their primary amino acid sequences,” he says. “[So, now it is not necessary to have] any crystal structure or any other type of 3D structure to know how the protein looks and how to develop a drug against it.”

Recent research has also demonstrated the ability for AI and machine learning to speed up protein engineering (5,6). “Advancements in protein engineering have facilitated the development of novel molecular scaffolds, optimized molecular functions, and improved drug properties,” Kaur adds. “These molecule-level innovations have opened up new possibilities for creating biologics with enhanced therapeutic effects.”

“Gaining an in depth understanding of accepted and multiple antibody interactions will undoubtedly revolutionize the future of biological formulation, as this knowledge can lead to the design of excipients that have beneficial impacts on the protein structure, thereby improving quality and efficacy [of antibody therapeutics] in the future,” summarizes Kaur. “Moreover, understanding the dynamics of antibody and antigen binding properties at the molecule level will help in designing the effective next-level antibodies. Manipulating these binding properties can optimize antibody performance and specificity and affinity for specific application.”

1. Andrews, L; Ralston, S; Blomme, E.; Barnhart, K. A Snapshot of Biologic Drug Development: Challenges and Opportunities. 

, 34 (12), 1279–1285.
2. Leach, M.W.; Rottman, J.B.; Hock, B.; et al. Immunogenicity/Hypersensitivity of Biologics. 

, 42 (1), 293–300.
3. Johnson, K.B.; Wei, W.-Q.; Weeraratne, D.; et al. Precision Medicin, AI, and the Future of Personalized Health Care. 

, 14 (1), 86–93.
4. Duncan, K.K.; Rudnicki, D.D.; Austin, C.P.; Tagle, D.A. Exploring Novel Biologically-Relevant Chemical Space Through Artificial Intelligence: The NCATS ASPIRE Program. 

, 6, 1–6. DOI: 10.3389/frobt.2019.00143
5. Wait, S.J.; Expòsit, M.; Lin, S. Machine Learning-Guided Engineering of Genetically Encoded Fluorescent Calcium Indicators. 

, 4, 224–236.
6. Case, M.; Smith, M.; Vinh, J.; Thurber, G. Machine Learning to Predict Continuous Protein Properties from Binary Cell Sorting Data and Map Unseen Sequence Space. 

Felicity Thomas is the European/senior editor for 

When referring to this article, please cite it as Thomas, F. Looking to the Future of Biologics. 

Articles

Cover Story

Progression in biopharmaceutical drug development and manufacturing is being boosted by technological advances.  

Looking to the Future of Biologics

Article submitted: September 13, 2023.
Article accepted: January 4, 2024.

Pneumococcal infections, stemming from Streptococcus pneumoniae, present a substantial health risk, especially among vulnerable populations. To counter this, vaccines employing bacterial capsular polysaccharide (CPS) have been developed. However, the purification of CPS is a pivotal vaccine manufacturing step, with the clarification process playing a crucial role in achieving optimal product yield and purity. This research focuses on refining the clarification step by assessing the performance of various depth filters. The study explores both conventional cellulose-based and innovative synthetic depth filters, utilizing pneumococcal fermentation harvests from two challenging serotypes. Evaluation criteria include hydraulic performance, throughput, and filtrate turbidity. Employing the constant flow rate sizing method for scale-up, the study determines that synthetic depth filters with suitable pore sizes demonstrate low resistance and high throughput, suggesting their efficiency in impurity removal. Overall, optimizing the clarification step is crucial for improving downstream processing of pneumococcal polysaccharide conjugate vaccines, and this study provides valuable insights into selecting and optimizing depth filters for efficient clarification, ensuring high product quality and process efficiency in vaccine manufacturing.

Swapan Kumar Jana* is director, R & D and Production, PPCV at Serum Institute of India Pvt Ltd, skjana@seruminstitute.com.
Amol Dattatraya Mahajan is manager, R & D and Production, PPCV at Serum Institute of India Pvt Ltd.
Vishal Vijayan is MSAT specialist; Praveen Kumar Ravichandran is MSAT specialist; Subhasis Banerjee is technical consultant; and Sourabh Kumar is manager, MSAT, all at Merck Life Science Pvt Ltd, India.

*To whom all correspondence should be addressed.

When referring to this article, please cite it as Jana, S.K. et al. Optimization of Clarification Step in Pneumococcal Polysaccharide Conjugate Vaccine Manufacturing: Evaluating the Performance of Depth Filters. 

Peer-Review Research

This research focuses on refining the clarification step by assessing the performance of various depth filters. The study explores both conventional cellulose-based and innovative synthetic depth filters, utilizing pneumococcal fermentation harvests from two challenging serotypes.

Optimization of Clarification Step in Pneumococcal Polysaccharide Conjugate Vaccine Manufacturing: Evaluating the Performance of Depth Filters

Industrial manufacture of biotherapeutic drugs and recombinant therapeutic proteins relies predominantly on mammalian cell lines, notably Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293. The HEK293 cell line was originally derived in 1977 by transforming primary human embryonic kidney cells with sheared adenovirus DNA (1). The original cell line has been used to derive several cell lineages over the intervening years (2,3). HEK293 is preferentially used for the manufacture of recombinant adeno-associated viruses (rAAV). Seven rAAV-based gene therapy products, beginning with Glybera in 2012 and Luxturna in 2017, have received regulatory approval for administration to human patients (4). Regulatory agencies recommend that mammalian cell lines expressing recombinant reagents used in commercial manufacturing are derived from single progenitor cells (i.e., these cell lines are monoclonal in origin, “…a collection of cells of uniform composition derived from a single tissue or cell”) (5,6,7). The quality of the biotherapeutic reagent produced by these cells is at the core of this recommendation; any variability in the quality is likely to have an impact not only on the efficacy of the product but may also impact the response in clinical trials. One of the major perceived advantages in using clonal cell lines in manufacturing biotherapeutic reagents is that it facilitates better control of the manufacturing process by reducing background genetic heterogeneity of the cells. This results in more rigorous consistency in the quality of the resultant biotherapeutic reagents.

Historically, clonal cell lines have been derived by the limiting dilution method using cell seeding into 96-well plates to isolate monoclonal cell lines. This is an arduous procedure that, because of its complexity, can be difficult to track and document to the satisfaction of regulatory agencies. Poisson distribution statistics are used to model the likely probability of clonality of the resulting cell lines (8). Typically, two rounds of limiting dilution are required to confirm clonality of the resulting cell lines, a procedural adjustment that is required for regulatory acceptance of clonal origin.

There are several integrated platforms available for cell line development that capture and document what was originally an extremely labor-intensive endeavor. Many of these platforms, include an integrated imaging system which is fundamental to the initial seeding of single cells into 96-well or nanowell plates. The systems enable subsequent tracking and documentation of clonal outgrowth over time and can be used to compile initial data on clonal viability and density. These platforms provide image-based assurance of clonality and facilitate regulatory submissions for filing investigational new drug application (INDs).

Generation of a suspension-adapted, serum-free clonal HEK293 cell line

A high yielding, robust suspension platform for AAV manufacturing (Andelyn Biosciences) demonstrates scalability from 125 mL shaker flasks to stirred tank reactor (STR) 2000L bioreactors using a commercial HEK293 cell line (9–16). A design-of-experiments (DoE) approach was implemented to formulate a model for optimized AAV production and purification. A suspension-adapted HEK293 clonal cell line has been derived and developed to complement this suspension platform for AAV manufacturing. This report highlights critical aspects of the early development work on the clonal suspension cell line.

An adherent HEK293 Good Manufacturing Practice (GMP)-grade Master Cell Bank (MCB) (Andelyn Biosciences) was used as the starting material to derive a series of clonal suspension-adapted HEK293 cell lines. The GMP-MCB was chosen as it had been already subjected to rigorous testing upon its inception for qualification and release. Initially, the adherent HEK293 cell line was adapted to growth in a series of decreasing serum concentrations, from 10% to 5% to 2% to 1% (v/v). Cell lines were then switched to growth in serum-free media, concurrent with growth under suspension conditions. At this point, they were designated as suspension-adapted, serum-free (SASF) cell lines. A total of 15 non-clonal (parental) cell lines were derived from this process.

Isolation of single cell clones from SASF cell lines

Preliminary characterization of early- and mid-passage cells suggested that the cell lines were still adapting to their suspension culture conditions. Growth, viability, and lack of clumping were key criteria in evaluating the suitability of parental SASF cell lines for subsequent clonal derivation. Verified 

 plate seeding (VIPS) was conducted with a cell development platform (Solentim VIPS, Advanced Instruments). This instrument is 

 21 Part 11 compliant and provides Day 0 assurance of cell line clonality. Mid-passage SASF cells at a density of 1.2 E+4 cells/mL were prepared and used to seed 96-well plates with a single cell seeder. Plates were seeded at a final volume of 125 µL suspension media, supplemented with 10% (v/v) conditioned media (InstiGRO HEK, Advanced Instruments).

A representative plate map from the seeding event outcomes is shown in 

. The data generated provide an accurate record of the seeding events as they occurred in real time and thus provide assurance of the clonality of any cell lines which survive to outgrowth. This series of VIPS resulted in a total of 570 seeding procedures. Of these, 395 actions were recorded as single cell seeding events, indicated by green circles on the plate map. If single cells survive, and clonal outgrowth succeeds, then any cell lines created from these events are clonal; they have originated from a single parental cell. Other seeding events in this series resulted in two or more cells being seeded in a single well, 167 events, indicated by a red circle with an X on the plate map which were discarded as they were not clonal in origin.

Assessment of rAAV production by SASF clonal cell lines in 125 mL shake flasks

Verified clonal cell lines were moved from 96-well plates to 24-well plates and then to 125 mL shake flasks. Cell line productivity from the surviving clonal cell lines was evaluated by determining rAAV titers at harvest. This step became the bottleneck in assessing and triaging clones for cell line development. Cell lines were transiently transfected with a combination of three plasmids: rAAV eGFP (enhanced green fluorescent protein) transgene, a common reporter used in process development (PD) internal work, an adenoviral helper plasmid and an AAV helper (serotype D) plasmid. Viral titers were determined by quantitative real-time polymerase chain reaction (qPCR). Analyses of rAAV production for a panel of 32 different clonal SASF cell lines is presented in 

. Harvest titers ranged from 1.21 ± 0.12 E+11 DNase resistant particles (DRP)/mL to 2.99 ± 0.03 E+11 DRP/mL. The commercial (control) cell line, which has been used by PD for internal development, produced a harvest titer of 3.2 ± 0.06 E+11 DRP/mL. The wide range in rAAV titers was consistent with the clonality of these cell lines as, clearly, different monoclonal lines will vary in their capacity for manufacturing rAAV. The differences in virus production, illustrated by 

, demonstrated the phenotypic variation that exists among this panel of cell lines. rAAV titer at harvest was a critical functional parameter for selecting a smaller subset of six clones for further intensive characterization. These selected lines are highlighted on 

rAAV-eGFP serotype D production in SASF clonal lines: scale up and 2L yield assessments

Each of the six candidate clones were batched up to 2000 mL cultures in shake flasks to demonstrate feasibility of flask-scale production and purification. Clones were designated SASF123-5, SASF123-6, SASF124-6, SASF124-7, SASF125-3, and SASF125-4. The optimized manufacturing protocol that had been developed with the PD commercial (control) cell line was used. All cell lines were seeded at 2.50 E+05 cells/mL three days prior to transfection. The cultures were transfected using a transfection reagent (FectoVIR-AAV, Polyplus). All flasks had acceptable cell viability (>97%) on transfection day, indicating that the culture conditions were suitable for all clones.

Flasks were harvested five days post transfection and analyzed for media release of rAAV as well as that retained in the cells. The media was cleared of cells by centrifugation. Analyses of cell pellets revealed that 10–37% of the rAAV (serotype D) remained in the cells. The harvested supernatants were clarified over a 5-inch V100 capsule filter into a membrane (Mini Kleenpak EKV, Pall Corporation), using a standard protocol. The clarified harvests were concentrated and diafiltered using 0.02 m2 tangential flow filtration (TFF) cassettes (Pall Corporation). The recoveries from harvest ranged from 76% to >100%. A >80% recovery from harvest is typical for this TFF process with the PD control cell line. The TFF concentrates were further processed by iodixanol (IDX) ultracentrifugation. The IDX pools were polished by ion exchange chromatography, using 1 mL ion exchange columns.

The initial harvest yields of rAAV-eGFP serotype D ranged between 2.05 E+14 DRP–4.68 E+14 DRP (

). The best-producing clone was SASF125-3. As can be seen in 

, the overall recoveries from harvest to final were in the range of 41–53% as opposed to 20–30% seen with the PD commercial cell line. Qualitative assessment of viral attributes of the purified rAAV from these clones, including drug substance purity, capsid Full:Empty (F:E) ratios, and the in-process recoveries at each step, confirmed that the clones did not present any unusual process characteristics in manufacturing that would have excluded them from further characterization and consideration as final candidates.

Several phenotypic characteristics and process-specific criteria were considered when deciding which SASF cell line would be selected for complete characterization and subsequent transfer for creation of a GMP MCB. Clone SASF125-3 was selected as the final candidate for the clonal suspension cell line. The original designation has been shortened to SASF125; both terms refer to the same cell line. As proof of concept, production with SASF125 was scaled up to a STR 50 bioreactor (Pall Allegro). rAAV-eGFP serotype D was purified to the TFF process stage (

). A transfection reagent was used (FectoVIR-AAV, Polyplus). The rAAV titer at harvest was 4.02 E+11 vg/mL. Average rAAV-eGFP serotype D titers at harvest with the PD commercial cell line, at the same scale, were approximately 1.15 E+11 vg/mL (n=4).

Optimization of rAAV manufacturing with the SASF125 clonal cell line

The original in-house PD suspension manufacturing process for rAAV productions at all scales (125 mL flask to STR 2000 bioreactor) was developed for different serotypes by utilizing the DoE strategy to optimize titers and quality (9,13–16). A commercial cell line was used at these earlier stages of development during the process of deriving the clonal HEK293 lines. It is evident that rAAV manufacturing capabilities vary between different HEK293 lineages (

), a reflection of their different evolutionary trajectories. Recognition of this fact required the use of similar DoE strategies as before to optimize multiple factors critical for rAAV manufacturing. 

 shows representative results from a DoE experiment evaluating the effect of viable cell density at transfection, and the plasmid DNA concentration per 1.0 E+06 cells on rAAV (serotype D) titers. A model was developed using multiple linear regression relating these factors to the vector genome titer, and this model was used to identify optimal ranges for each factor.

Iterative DoEs resulted in an optimized set of conditions for maximizing titers and quality in the SASF125 line. Results from two rounds of DoE are shown in 

 and demonstrate the versatility of the cell line in rAAV manufacturing with different serotypes. This figure summarizes much of the development work that has been accomplished since the isolation of SASF125 and scaling it to the STR 50. As benchmarks, 

 also shows titers obtained from optimized manufacturing conditions with the original commercial cell line. It should be noted that manufacturing conditions and, indeed platform components, are different for the two cell lines. They are different lineages of HEK293 that have been generated under different conditions and thus, as demonstrated, have different manufacturing requirements for optimal rAAV production and purification. There is variability in rAAV yield between different serotypes but the SASF125 outperformed the commercial cell line in five out of the six serotypes investigated to date. Serotypes C and D demonstrate approximately seven-fold increases in rAAV titer.

In summary, a clonal suspension cell line was successfully derived that is suitable for rAAV production. The clonal cell line has been tested with several serotypes, both conventional and engineered. A GMP MCB has been generated, tested, and released for clinical manufacturing of rAAV gene therapy biotherapeutic drugs.

The authors would like to thank the Analytical Development group of Andelyn Biosciences for their constant support with rAAV analytics, especially PCR. The authors also thank Lenore Giannunzio who was manager of the PD group when the SASF cell line development program was initiated.

Graham, F.L.; Smiley, J.; Russell, W.C.; Nairn, R. Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5. 

. 1977. 36(1):59-74. DOI: 10.1099/0022-1317-36-1-59

Yuan, J.; Xu, W.W.; Jiang, S.; Yu, H.; Poon, H.F. The Scattered Twelve Tribes of HEK293. 

Lin, Y.C.; Boone, M; Meuris, L; Lemmens, I; Van Roy, N; Soete, A; Reumers, J; Moisse, M; Plaisance, S; Drmanac, R; Chen, J; Speleman, F; Lambrechts, D; Van de Peer, Y; Tavernier, J; Callewaert, N. Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. 

Wang, J.H.; Gessler, D.J.; Zhan, W; Gallagher, T.L.; Gao, G. Adeno-associated virus as a delivery vector for gene therapy of human diseases. 

. 2024. 9(1):78. DOI: 10.1038/s41392-024-01780-w

Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological/Biological Products

Guideline on Development, Production, Characterization and Specifications for Monoclonal Antibodies and Related Products

, EMEA/CHMP/BWP/157653, (CHMP, Dec. 2007)

Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals

Chen, C; Le, K; Le, H; Daris, K; Soice, N; Stevens, J; Goudar, C.T. Methods for estimating the Probability of Clonality in cell line development. 

. 2020. 15(2):e1900289. DOI: 10.1002/biot.201900289.

Ketz, J; Bergman, S; Hurley, B; Carper, B; Roodhouse, H; Giannunzio, L; Acharya, S. A DoE Approach to Optimize AAV Production and Purification using FectoVir-AAV. 

Mcilhatton, M; Hurley, B; Bergman, S; Roodhouse, H; Carper, B; Janson, M; Ketz, J; Acharya, S. Derivation of a Clonal HEK293 Suspension Cell Line for High Yield AAV Production. 

Carper, B; Roodhouse, H; Ketz, J; Janson, M; Acharya, S. A Robust Downstream Strategy for High Quality AAV Using an All-column Purification Method. 

Janson, M; Carper, B; Roodhouse, H; Acharya, S. Novel HPLC Applications for Rapid In-process Characterization during AAV Platform Development. 

Acharya, S. Development of a Robust Suspension Platform for Viral Vector Manufacturing. Presentation at the Tools and Technology Forums 2.0. ASGCT 25th Annual Meeting, Washington, DC, May 16–19, 2022.

Ketz, J. A DoE Approach to AAV Production and Scale Up to 500L using FectoVIR-AAV. Presentation at the 

 Gene Therapy Track, BioProcess International, Boston, MA, Sept. 27–30, 2022.

Moreo, A. Development of a Robust Scalable Suspension Platform for AAV Manufacturing up to 2000L. Presentation at the Advanced Therapies Week, Miami, FL, Jan 17–22, 2023.

Acharya, S. A Robust and Scalable Suspension Platform for AAV Manufacturing. Presentation at the Cell & Gene Therapy Manufacturing & Commercialization US conference as part of BioProcess International and Biotech Week Boston, MA, Sept 18–21, 2023.

 is a Senior Scientist in the Process Development lab at Andelyn Biosciences. He has a B.Sc. in Biochemistry & Microbiology, and a Ph.D. in molecular virology, from Queen’s University Belfast. His focus is on upstream production in AAV manufacturing, with emphasis on cell line development. He has many years of experience with generating and characterizing both mouse and human primary, tumor, and organoid cell lines.

is a Senior Scientist in the Process Development lab at Andelyn Biosciences. He has over 6 years of experience on both upstream and downstream optimization of AAV, its scale up from flasks to STR 2000 bioreactors, associated documentation, and tech transfer. He has a B.S. in Biochemistry and a M.S. in Animal Physiology from West Virgina University, WV.

, is a Scientist in the Process Development lab at Andelyn Biosciences. Her focus is on optimization of upstream manufacturing and technical writing. She has over 5 years of experience in AAV manufacturing, its scale up and tech transfer. She has a B.A. in Microbiology from Ohio Wesleyan University, a graduate certificate in Applied Statistics and is on track for Masters of Applied Statistics from Penn State University, PA.

, is a Scientist in the Process Development lab at Andelyn Biosciences. Her focus is on AAV manufacturing from small flask to bioreactor operations, including downstream processing, and associated documentation. She has over 5 years of experience in AAV manufacturing, its scale up and tech transfer. She has a B.S. in Biology from The Ohio State University, Columbus, OH.

, Scientist in Process Development at Andelyn Biosciences, has over 6 years of experience working in downstream purification of standard and novel AAV serotypes. He has also been involved in the development a robust, scalable method for purification of AAV productions from 1L to 500L. He has a B.S. in Biomedical Engineering from The Ohio State University.

 oversees process development as well as preclinical plasmid and viral vector production at Andelyn Biosciences. He leads a diverse organization of interdisciplinary scientists specializing in high throughput manufacturing as well as process development, optimization and scale-up of production platforms. He has over two decades of experience in genetic and molecular research with roles at Syngenta and The Ohio State University. He has a B.Sc. in Biology from Purdue University.

, Associate Director of Process Development at Andelyn Biosciences, is responsible for Process and Platform Development, its optimization, characterization, and technology transfer. Prior to Andelyn Biosciences, he has over 25 years of research experience in mechanisms of genomic instability and pathways of cell survival and proliferation. He has authored numerous peer-reviewed scientific publications and book chapters. He has a B.Sc in Chemistry from St. Stephen’s College, University of Delhi; M.Sc. in Biotechnology from Jawaharlal Nehru University, and a Ph.D. in Biochemistry from Indian institute of Science, Bangalore, India.

When referring to this article, please cite it as Mcilhatton, M; Ketz, J; Bergman, S; Hurley, B; Carper, B; Moreo, A; Acharya, S. Strategy for Derivation and Optimization of a Clonal HEK293 Suspension Cell Line for High Yield AAV Production. 

Upstream Processing

This commentary summarizes the derivation of clonal HEK293 suspension cell lines, selection of clones for rAAV production, and design of experiments-based optimization strategies for characterization of one clonal isolate for high yield rAAV manufacture. Data shown provides proof of concept for the success of this strategy, its applicability for manufacturing different rAAV serotypes and lays the foundation of further clonal cell line characterization for scale up of production.

Strategy for Derivation and Optimization of a Clonal HEK293 Suspension Cell Line for High Yield AAV Production

Sterility must be assured for all biologic drug products. Terminal sterilization achieved via treatment with heat, radiation, or certain chemicals (vaporous hydrogen peroxide, vaporous peracetic acid, or ethylene oxide) is preferred, but it is only applicable to drug substances that remain stable under the harsh conditions involved. For most biologics, therefore, sterility must be assured by using aseptic processing techniques, which involve use of sterile raw materials, equipment, and processes under conditions that prevent microbial contamination and maintain sterility. Viral vectors are no exception.

There are two main potential sources of contamination, according to Cyrill Kellerhals, COO at Andelyn Biosciences: operators and the facility design. “Proper training for operators on proper procedures and immersing them in the quality mindset is foundational,” he insists. “Investment in employee satisfaction and career development can help keep turnover low and thereby enable continuity in operational excellence,” he adds. A fit-for-purpose facility design leveraging state-of-the-art technologies such as centralized building sensors, monitoring, and control for greater connectivity and single-pass-through air, meanwhile, can mitigate both microbial and cross-contamination risks, Kellerhals notes.

The actual sterilization process for viral vectors involves sterilizing-grade final filtration followed by aseptic filling. For these steps, the risks for microbial and cross-contamination are largely associated with manual errors that lead to batch failure or batch record deviations, according to Nigel Jackson, principal engineer, bioprocess at Cytiva.

Additional challenges for the sterilizing-grade filtration step include the need to perform a pre-use post-sterilization integrity test (PUPSIT) and a post-use integrity test in situ, the potential for leak development within single-use manifolds during installation or processing, and the need to minimize hold-up volumes, Jackson comments.

In addition, viral-vector final formulations are very concentrated, and filtration of these solutions is challenging, notes Holger Laux, director of viral vector process development at CSL Behring.

Challenges to sterility can also arise during other unit operations required for production of viral vectors. “Multiple steps are involved in upstream (e.g., seed train, infection/transfection, harvest), downstream (e.g., cell lysis, chromatography, filtration, and formulation), and fill/finish (e.g., filling and freeze drying) processes, with each step bearing the risk of losing sterility,” says Karl Heller, vice-president CMC development with Ascend Advanced Therapies. Cell-stack production involving several manual manipulation steps is a prime example of operations that introduce contamination risk, says Laux.

“Sterile manufacturing of live viral vectors used to be the ultimate challenge in the biopharmaceutical industry,” Heller continues. Today, however, the variable nature of viruses (DNA/RNA, enveloped/non-enveloped, size range from 20 nm to 400 nm) and their associated properties (e.g., chemical, physical, and thermal stability) remain to some extent the biggest challenges for sterile manufacturing, he believes. “All these properties must be considered and evaluated when developing upstream and downstream unit operations to obtain high-quality, stable, and sterile drug substances and drug products in high yield,” Heller remarks.

Lentiviral (LV) vectors tend to present a greater challenge for sterilizing-grade filters than adeno-associated viral (AAV) vectors, according to Jackson. “LV vectors at 0.1–0.12 µm in diameter are far closer to the pore size of the filters, and therefore, developing a robust filtration process is more difficult, especially if aggregation occurs,” he explains. The high concentration of viral vector final formulations increases the challenges to process development and scaleup, adds Laux. “Effective control of LV formulation and a strong toolbox of various sterilizing-grade filter options are required to find a solution,” Jackson notes.

AAV vectors, which are much smaller (approximately 0.02 µm), can readily undergo a 0.2-µm or even a 0.1-µm filtration to obtain a sterile product, adds Heller. For some large viral vectors, such as poxvirus vectors including vaccinia and modified vaccinia virus Ankara (MVA) with a size of approximately 350 nm, that type of filtration is simply not possible.

Whether a virus is enveloped or nonenveloped also impacts the sterile filtration process, according to Heller. “Enveloped viruses are usually [much] more susceptible to thermal or chemical influences, so no extreme conditions (e.g., low pH) can be applied, particularly during downstream processing. These limitations may increase the risk of losing sterility,” he observes.

Regardless of the indication or program, Kellerhals emphasizes that quality systems and regulations are universal and overarching, and adherence to strict quality standards is essential to ensure consistency and safety for patients.

Scaling itself does not necessarily increase the risk of losing sterility during viral-vector manufacturing, particularly for high-volume, low-titer products such as vaccines. Maintaining sterility can be more burdensome, however, for low-volume/high-titer advanced-therapy products, notes Melanie Langhauser, team lead downstream processing at Ascend Advanced Therapies.

“The challenge in scale comes in adapting the GMP [good manufacturing practice] systems to the smaller batch volumes in viral-vector processes compared to traditional products,” agrees Jackson. “Experience in delivering adaptable systems to suit specific product needs becomes vital to overcome this challenge,” he adds.

As an example, Langhauser points to the need to find appropriate single-use equipment suitable for GMP manufacturing at smaller scale. “For a 50-L bioreactor run, the intermediate downstream processing volumes can already be below 2 L after the capture steps, but single-use mixers currently on the market start at 50 L,” she explains. Chromatography systems have presented challenges as well, as most were developed for monoclonal antibody processes with starting volumes of 1000 L. Suppliers are, Langhauser observes, working to address these gaps and meet the specific needs of advanced therapy manufacturers.

As a final note on scaling viral-vector sterile filtration processes, Laux points out that at CSL, it has been observed that not the volume, but the number of virus particles per filter area is the critical factor for upscaling.

Several strategies for overcoming sterilization challenges

“Consistency and quality in any type of biological manufacturing process, including [those for] viral vectors, can be ensured by using quality-by-design and design-of-experiment techniques to address questions of optimization and reduce variability before implementing a drug substance/drug product sterile manufacturing regime,” states Kellerhals. Indeed, conducting a screening study to identify filters that perform best at small-lab scale followed by their evolution at larger-lab scale is recommended before deciding on the best options for upscaling sterile filtration processes, according to Laux.

“For LV vectors, titer losses of 30–50% during sterile filtration have been reported in several publications, often due to excessive aggregation and vector loss. Through our filter screening efforts, however, CSL has been able to develop a sterile filtration process that affords infectious titer yields above 90% at scales relevant for clinical trial and commercial manufacturing,” Laux says. The increase in titer is important because higher yields reduce the cost of goods for these expensive products. “It is therefore worth evaluating several sterile filters based on the process and formulation conditions, as it is possible to achieve both higher yields and increased patient safety,” he observes.

In addition, training of operators in aseptic techniques, use of certified and controlled manufacturing spaces (e.g., biosafety cabinets), single-pass-through air supply, continuous environmental monitoring, and placement of quality team members in manufacturing suites for additional oversight are other steps that can be taken to ensure sterility, according to Kellerhals.

Logical design and physical layout of single-use manifolds with a focus on user experience can also facilitate the design of standard operating procedures that minimize operator errors during complex processing steps such as PUPSIT and in-situ post-use integrity testing, adds Jackson. “The ultimate solution, though, is implementation of fully automated, good-manufacturing-practice-compliant systems which minimize the risk of operator error,” he contends.

The choice between the two, Jackson comments, is a cost-and-benefit risk assessment. He does note that separate pre-sterilized, single-use manifolds with sterile connectors can improve ease of installation and prevent the risk of damage during installation, and integration of effective leak testers into the production system can confirm a leak-free system at the point of use.

While single-use equipment and materials have facilitated sterile manufacturing of viral vectors, Heller notes that these tools come with a price. “The cost differences between single- and multi-use equipment and accessories can be significant, which can be an issue especially for smaller companies in the early stages of development at smaller scales, where cleaning validation is not a top priority,” he explains.

Hold-up volumes also become increasingly important as the process scale shrinks. “For low-volume/high-titer products, every single drop of dead volume in a system can be a massive product loss,” Heller states. Minimizing hold-up volumes and maximizing yield can be achieved through effective custom design of the filtration system and manifolds to optimize them for the specific production scale, according to Jackson. “Optimizing the sterilizing-grade filtration performance through process optimization or selection of higher throughput products can lead to a smaller final filter, reduced hold-up, and higher yield,” he adds.

Finally, Langhauser highlights the increased availability today of GMP-compliant small tangential-flow filtration (TFF) systems with peristaltic pumps for performing buffer exchange at extremely low volumes with single-use tubing, especially at the end of a manufacturing process.

While successful production of viral-vector drug substance and drug products is achievable today, there are always opportunities to optimize and improve existing processes and systems to enhance yields and ease-of-use without compromising on critical patient safety, according to Jackson.

Suppliers and manufacturers have already made progress in addressing many of the challenges to ensuring the sterility of viral-vector products, adds Langhauser. She points to the development of smaller single-use mixers for small-volume applications, single-use filters and TFF membranes at various scales that are available or under development, and irradiated single-use biocontainers with integrated mixers and pre-calibrated pH probes as useful solutions for advanced therapy manufacturing.

One of the keys to successful development of new technologies, Langhauser adds, is close collaboration between suppliers and manufacturers with regards to design and suitability testing of potential new solutions, as working together helps facilitate the introduction of new solutions that can dramatically improve efficiency, productivity, quality, and safety.

Sterile filtration will remain the standard, but with more automation

The fundamental issue with sterile viral-vector manufacturing is the need to keep out or remove undesired organisms such as bacteria or viruses from processes that are intended to generate live viral vectors in sterile formulations. “Most likely, sterile filtration for sufficiently small viruses will remain the method of choice, whereas for larger viruses, use of sterile single-use materials, stringent control of starting materials, and aseptic processing will be hard to replace,” concludes Heller. Advances are likely to occur, therefore, in automation, which, according to Jackson, will further drive down the risk of batch failure.

Kellerhals agrees. “Balancing human ingenuity and adaptability against reducing opportunities for human error is a natural outcome of the scale up process, so opportunities for contamination are very limited at the manufacturing scale. Still, human error, including shedding of contaminants from a human operator into the manufacturing environment, is something we limit today with automation and procedural rigor. Future innovations will reduce these risks even further and as close to zero as possible,” he says.

Finally, Kellerhals observes that technology and human ingenuity go hand in hand when striving for continuous improvement. He also emphasizes that “the evolution of sterility standards over time to where they are today should reassure us all that our medicines are safely manufactured for our patients.” In addition, he believes that future iterations of technology will only further this assurance, but that well-trained operators and well-honed and followed procedures will always be the foundation for aseptic manufacturing.

As for many other technologies in biopharmaceutical manufacturing, there are certain basic principles that should be followed to ensure sterility, according to Heller. “The best way to protect your product from undesired organisms is to strictly obey the famous KISS principle: Keep It Simple Stupid,” he says.

That includes careful selection of equipment and starting materials with scalability and manufacturability under current GMPs taken into consideration and being guided by data, not perception.

“The fewer the unit operations in a process, the fewer the interventions—manual or automated—that will be required for it to run properly. That makes it easier to maintain sterility throughout all steps and produce sterile, high-quality viral-vector products,” Heller concludes.

Sterility in viral vector therapies is a critical and mandatory aspect in delivering overall patient safety, adds Jackson. “Driving improvements in the safety profiles of viral vectors is key to the industry expanding the application of this technology to a wider variety of diseases, which,” he states, “will enable the industry to make a difference for more varied patient populations.”

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

®
Vol. 37, No. 5
May 2024
Pages: 24-26, 33

When referring to this article, please cite it as Challener, C.A. Advances in Sterilization Technologies for Overcoming Viral-Vector Manufacturing Challenges. 

Manufacturing

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

Data are the biopharma industry’s most valuable asset today, which is prompting the industry to step up its measures to not only gather these data, but to protect them as well. In a conversation with Nathalie Batoux, product manager, Research Innovation, IDBS, 

® delved into the approaches the biopharma industry has taken to shore up data integrity protection.

 Explain why data are the biopharma industry’s most valuable asset. The analogy is that there’s a gold rush for data (1); what does that mean?

 With the current digital transformation happening in the biopharma industry, the industry has also seen the explosion of [tools such as] artificial intelligence (AI) and machine learning (ML). There is also a significant amount of data that exists now based on the products already on the market. With the explosion of AI and ML, people want to reuse the data that they have. So, instead of running new experiments, already existing data are being used; these historical data are very important. There are some new initiatives underway to review and repurpose existing drugs for other applications, thus, AI and ML are being used to review these already existing data. Not only is the biopharma industry producing a vast amount of new data, it also has its hands on a significant amount of historical data.

 Describe the impact of this perception—this ‘gold rush’ for data—on data integrity. Has it led to a flurry of activity?

Yes, there is a flurry of activity. There are many startups that are proposing AI and ML to the biopharma industry, amongst other industries. There are initiatives, as I mentioned, around the repurposing of existing drugs using historical data at hand.

 Can you discuss where data integrity practices in the biopharma industry are the strongest and where they are the weakest?

 My perspective is to look at the evolution within the industry, and where we are now with digitalization, data integrity is getting much stronger. Looking at the practice of manually recording, or manually transcribing, data from one system to another, this is very much error prone. Whether intentionally or accidentally, errors are highly likely. If there is an error in the data, then any analysis conducted later on will be incorrect, and the decision-making using that data will not be good and will thus be counterproductive. This will cost money at the end of the day.

Good data integrity involves the use of more automated systems, using digital transformation where the system records the context around the results. For example, in an experiment, scientists—even though they are very well trained—are still human. There’s only a finite number of parameters that they can deal with. While a computer, or a series of computers, can record all sorts of things around the context. It’s as simple as the date and time of an experiment that might change the outcome, whether [the experiment] is done in the summer or winter. That is important as temperature and humidity conditions may affect the outcome.

The other advantage is that, with data digitally recorded, there is traceability, and most of the modern systems now have audit capabilities. [This makes it easier] to understand what has happened to generate that data, but also what has happened after the data has been generated—such as, has the data been transferred from one system to another? Having all that context, having this history, ensures that, in the first place, the data—that is, the data that will be used to train the models for ML or analysis, or for use byAI—will be of quality. [And that assurance comes from] all that context, all that history behind the data.

Data that are digitally recorded [are] easier to find. [In the laboratory], finding an experiment on paper or in a notebook is not easy. [Digitally recorded data] offer interoperability and [better] accessibility. If [the data are] on paper, for example, [the lab tech] needs to have that piece of paper in front of [them] to be able to see it. This hinders interoperability [because] that piece of paper, if the handwriting is, as they say, like a doctor’s handwriting, not many people will be able to understand it. Being ‘reusable’ as a whole is very, very important for data, so having FAIR [findable, accessible, interoperable, and reusable] data is one of the principles of data integrity. This will enable the digital transformation and all those initiatives that are happening with ML and AI.

 Let’s talk about the data itself. Good data versus bad data; how is that determined?

 As mentioned earlier, having all the context around the data recorded is one of the attributes of good data. The other attributes, as I mentioned, are the FAIR principles; however, one needs to make sure that they are recording everything. It’s human nature to try to record things that are happening and [what is] working, but from a learning perspective—and for ML, in particular—one needs to also record what didn’t work because [there is] much more to learn from things that didn’t work. So, the ‘how to’ recognize good data versus bad data requires having rigor, using well documented SOPs [standard operating procedures], and having controls in place, all of which are quite important because these help with the reproducibility of that data.

In addition, if using a digital system with controls in place, then [the technician] will receive an alert, and [this allows] them to record an exception. It is very important to record all those things, not only for good data in general, but also for the data that regulatory bodies require. [Keep in mind], all the compliance requirements are very important. Another point [to highlight] is that for accessibility and interoperability, more than one system will need to be used; likely, it will require several systems. And, data needs to be connected easily, so having silos that are not talking to each other, that don’t represent each other, that is not good data. Having the ability to connect the data—to connect to the structure of that data in order to understand them—that is a symptom of good data.

 How is the biopharma industry handling this flow of data; how is it preparing for this gold rush that was mentioned earlier?

 [With the] digitalization that is happening, less companies are using paper, and [it seems that] more and more companies have been investing in data scientists to be able to mine the data. However, there is a difference between having all that data mined by specialists and the people who are actually using the data at the bench, so having democratization of data is extremely important. Empowering [bench] scientists with decision-making based on large data sets, including past data, at the click of a button is very valuable. Rather than having to wait for a few weeks for data scientists’ analyses—although the data scientists’skills are still important for more complex analyses—a more diverse set of scientists or end users can have accessibility to data with [the goal] of helping to put algorithms in place for the analyses, and so on.

1. Batoux, N.; Rayner, D. In a Digital Gold Rush, Data Integrity is Priceless. 

Feliza Mirasol is the science editor for 

®
Vol. 37, No. 5
May 2024
Pages: 27–28

When referring to this article, please cite it as Mirasol, F. Shoring Up Data Integrity Practices. 

Analytics

Digital transformation is allowing for better handling, analysis, and protection of vast data collection.

Shoring Up Data Integrity Practices

"Primary engines of value creation”—this is the role that leading market commentators see biologic drugs playing in the evolving biopharmaceuticals market (1). Demand for protein-based, large-molecule therapies has been growing steadily for decades, but few could have predicted the boom which followed in the wake of COVID-19. Significantly accelerated by the pandemic, sales of biologic drugs are predicted to exceed those of traditional small-molecule medications by 2027 (1). Runaway demand, exciting scientific breakthroughs, and robust interest from governments and the public: the outlook is bright for biologics. To fulfill this growing potential, producers will need to make drug stability a priority, and this could mean reconsidering the use of a ubiquitous processing ingredient.

Polysorbates (PS 20 and PS 80) are the surfactants most used in biopharmaceutical formulations to protect proteins from denaturation, aggregation, and surface adsorption; these surfactants are present in as many as 90% of marketed therapeutic antibodies (2). Despite their popularity, polysorbates are prone to a number of issues, with serious implications for drug safety, stability, and efficacy. As pressures mount, could it be time for the industry to adopt a new favorite excipient solution for biologics? This article will explore the findings of two recent studies comparing the performance of polysorbates with hydroxypropyl -cyclodextrin (HPβCD)—an alternative biologic stabilizer.

Reliable, multifunctional, and safe, is HPβCD the answer to the question: how do you solve a problem like polysorbates?

To fully understand the “problem” with polysorbates, the industry must examine the essential role ultrafiltration/diafiltration (UF/DF) and protein stabilization during the final formulation stage play in biologic drug manufacturing, and why processing aids are crucial to their success. As 

 shows, UF/DF comes relatively late in downstream biopharma processing, helping achieve the desired concentration and formulation composition of biologics, such as monoclonal antibodies (mAbs) and recombinant proteins. Though an indispensable processing step, the shear stress and continuous adsorption/desorption interactions with the filtration membrane during UF/DF subject active ingredients to harsh conditions. This typically results in protein aggregation and the generation of visible and subvisible particles, jeopardizing the safety and efficacy of the drug. It is here that surface active molecules, such as polysorbates, enter the frame. During high-stress processes, polysorbates act as a chaperone, allowing aggregation-prone hydrophobic sites on the surface of the biologic to pass more easily through membranes to preserve their folded structure and reduce interface exposure (3). 

With decades of evidence pointing to their effectiveness in preventing protein adsorption and aggregation, as well as their demonstrated safety, polysorbate 80 (PS 80) and polysorbate 20 (PS 20) are considered the surfactants of choice in more than 90% of approved mAb products (2). Due to their amphiphilic (both hydrophilic and hydrophobic) properties, mAb proteins have a high propensity for surface-mediated unfolding and adsorbing into interfaces (3). Polysorbates compete with proteins at interface points, reducing the interfacial exposure of the proteins. Despite their widespread use, there are several serious questions surrounding the effectiveness of polysorbates in biologic processing. Polysorbate degradation via oxidation and hydrolysis can produce peroxides and fatty acids that could negatively impact the quality and stability of a medication’s therapeutic protein. Polysorbates can also be nonspecifically absorbed onto UF/DF filter membranes, leading to the formation of micelles bigger than the membrane’s molecular weight cut-off, which in turn causes inconsistencies and membrane blockage. As the biopharma industry moves to make efficiency its central priority, researchers begin to wonder, could an alternative excipient help flip the script?

UF/DF is an integral part of downstream biotherapeutic processing, though it’s not without its challenges. A typical UF/DF process involves several passes through a pump and membrane cassette, often taking several hours to complete (4). During this process, active proteins are exposed to a range of physical stresses, including interfacial interaction, which, as discussed previously, can lead to the formation of higher order aggregates and particulates (4). Therefore, to survive the UF/DF process intact, proteins need an accompanying surfactant to smooth their multiple passes through the membrane cassette and protect them from interfacial stresses (4). For the majority of biologic processing steps, polysorbates are the solution of choice; however, their usefulness runs out by the UF/DF stage. Not only do polysorbate molecules tend to bind nonspecifically with filter membranes, but their propensity to form micelles bigger than the membrane’s molecular weight cut-off can cause filters to become blocked during the UF/DF process (4). Researchers at Roquette postulated that HPβCD could offer biopharma producers an alternative excipient solution, better suited to the requirements of the UF/DF process.

The focus of this study into HPβCD’s potential as an alternative to polysorbates was two-fold: assess its permeability through commonly used filtration membrane types and capacity to reduce particle formation during the UF/DF of human plasma immunoglobulin G (IgG) and adalimumab solutions (4). In the first instance, varying concentrations of HPβCD were observed in the retentate and permeate of solutions passed through a regenerated cellulose-type membrane following centrifugation (4). This indicated that HPβCD may be unsuitable for regenerated cellulose membranes, as it tends to be retained during ultrafiltration (4). With another commonly used polyethersulfone (PES) membrane, however, the results were a complete contrast. In all four variations tested, concentrations of HPβCD remained constant, providing convincing evidence that unlike polysorbates, it is able to pass freely through PES membranes (4). What’s more, these results remained largely consistent when the excipient was tested via an alternative tangential flow filtration (TFF) setup, adding credence to HPβCD’s suitability for UF/DF processes (4).

Regarding the goal of mitigating particle formation, results were similarly promising. Using the same TFF setup, researchers conducted UF/DF experiments using the proteins human plasma IgG and adalimumab in the presence of different concentrations of HPβCD. In the diafiltration setup, both the retentate and permeate were recirculated for four hours to mimic the duration of a typical commercial drug production process. The results at the end of the recirculation process suggested that HPβCD effectively modulated the rate of turbidity (measure of clarity in liquids) increase in the human plasma IgG solution (4). In fact, the turbidity of the sample was reduced by more than two times in the presence of just 10 mM of HPβCD, while dynamic light scattering (DLS) data also showed that it had lowered the relative intensity of larger particles (> 0.1 m) compared to the control sample (4). The addition of a larger, 25 mM concentration of HPβCD reduced the rate of turbidity increase in the adalimumab solution yet further, meaning that the final turbidity was more than four times lower in the sample containing HPβCD than in the control group.

Researchers were able to observe HPβCD’s effectiveness in reducing UF/DF-induced particle formation in both the human plasma IgG and adalimumab solutions too. Looking at the human plasma IgG specifically, a 10% protein loss was observed in the control, whereas all samples containing HPβCD achieved almost complete protein recovery at the end of the four-hour recirculation (4). Not only then is HPβCD suitable for UF/DF where polysorbates are not, but its presence could actually boost efficiencies in biologic production lines—no small point in a time of rising demand for the industry.

The potential benefits HPβCD can bring to the biologic manufacturing process are not exclusive to the UF/DF process. Indeed, this multifunctional excipient exhibits continuous functionality into the final stages of formulation, particularly protein stabilization.

Polysorbates vs. HPβCD: Protein stabilization

The presence of protein aggregates and visible/sub-visible particles within a biologic formulation can compromise product quality and long-term stability, as well as lead to an unwanted immune response in the patient. As such, numerous health regulators around the world require biopharmaceutical companies to closely monitor visible and subvisible particle formation within formulations by comprehensive characterization, as outlined in 

 <789> (5). With scrutiny mounting on the issue of particle formation, recent research has sought to investigate the potential of alternative surfactants that could offer a more stable proposition than traditional polysorbates, with the prime candidate being HPβCD.

Due to its regulatory status as an approved excipient for oral and parenteral administration, HPβCD has been considered as a functional biologic surfactant for more than a decade (6). There is robust evidence to support HPβCD’s effectiveness in preventing thermal or agitation- and lyophilization-induced protein aggregation, in addition to its capacity to reduce interfacial stress-induced particle formation during UF/DF (5). This stabilizing effect has been attributed to the excipient’s ability to reduce protein–protein hydrophobic interactions and displace proteins from interfaces thanks to its weak surface activity (5). Unique properties such as those listed above formed the foundation of a recent investigation into HPβCD as a potential replacement for conventional polysorbates in biologic formulations.

The resultant study compared the chemical stability of HPβCD against that of PS 20 and PS 80 under various stress conditions. This included heat and chemical stressors, as well as its capacity to stabilize proteins within a formulation. When subjected to heat stress, HPβCD showed little change in product recovery (90.7–100.7% recovery depending on the HPβCD grade), while PS 20 and PS 80 both underwent significant degradation, with only 11.5% and 7.3% undegraded product remaining, respectively (5). Similar results were recorded when the surfactants were subject to autoclave, light, and oxidative stresses, with the HPβCDs remaining almost completely stable, while both polysorbates displayed some level of degradation (95.5%–98.8% usable product remaining for PS 20 and 85.5%–97.4% for PS 80) (5).On closer analysis, researchers found the HPβCDs’ chemical structure remained consistently stable, compared to the significant hydrolytic degradation and oxidation seen with the polysorbate molecules (5).

Finally, attention turned to HPβCD’s performance as a functional surfactant in a test formulation. Researchers evaluated the stability of various model mAbs when subjected to light stresses. Compared with the more traditional PS 80 preparation, the mAb formulated in the presence of HPβCD showed a notable decrease in protein aggregation, a superior monomer, and total protein recovery while reducing both protein aggregation and subvisible particle formation (5). Evidently, HPβCD excipients hold immense potential as biologic stabilizers.

At the end of this compare-and-contrast exercise, it is important to acknowledge the real value polysorbates bring to biologics processing. In a wide array of applications, they act as effective buffers and mediating agents, and their years of evidence as safe and nontoxic excipients cannot be discounted—but neither can the clear potential of HPβCD. In the studies discussed above, it can be seen what a significant impact stabilizing cyclodextrins could have on protein stability, mitigating particle formation, and streamlining vital processes. In some instances, such as UF/DF, substituting traditional polysorbates for HPβCD appears to be the logical choice, but this will not be so clear cut in every scenario. The future of biologics processing then could be one where these two surfactant groups are used interchangeably or even in tandem, to produce the very best outcomes for patients. That is, after all, the ultimate problem every drug producer is seeking to solve.

Biologic Sales to Pass Innovative Small Molecules in Next Five Years, Says GlobalData

, May 10, 2022.
2. Bollenbach, L.; Buske, J.; Mader, K.; Garidel, P. Poloxamer 188 as Surfactant in Biological Formulations—An Alternative for Polysorbate 20/80? 

, 121706.
3. Grabarek, A. D.; Bozic, U.; Rousel, J.; et al. What Makes Polysorbate Functional? Impact of Polysorbate 80 Grade and Quality on IgG Stability During Mechanical Stress. 

 (1), 871–880. DOI: 10.1016/j.xphs.2019.10.015
4. Hong, S.; Peng, T.; Gokhale, R. Mitigating Particle Formation During Ultrafiltration/Diafiltration of Biologics with KLEPTOSE HPβCD (Hydroxypropyl Beta-Cyclodextrin). Whitepaper, 2023.
5. Zhang, H.; Hong, S.; Tan, S. S. K.; et al. Polysorbates versus Hydroxypropyl Beta-Cyclodextrin (HPβCD): Comparative Study on Excipient Stability and Stabilization Benefits on Monoclonal Antibodies. 

, 6497. DOI: 10.3390/molecules27196497
6. Serno, T.; Carpenter, J. F.; Randolph, T. W.; Winter, G. Inhibition of Agitation-Induced Aggregation of an IgG-Antibody by Hydroxypropyl-Beta-Cyclodextrin. 

Tao Peng, PhD, is biopharma research manager at Roquette.

®
Vol. 37, No. 5
May 2024
Pages: 29–33

When referring to this article, please cite it as Peng, T. Polysorbates: Part(icle) of the Problem? 

Outsourcing

A comparison between polysorbates and HPβCD determines the better stabilizer for biologics formulation.

Polysorbates: Part(icle) of the Problem?

Research efforts are continuing to expand in the area of biological therapies. These large-molecule drugs are proving to be significant revenue drivers for bio/pharma companies and are providing therapeutic options for disease areas that have previously suffered from unmet medical needs (1).

Additionally, biologics are useful in providing more targeted disease treatment when compared with small-molecule drugs and lessening the risk of side effects for patients. The targeted nature of these large-molecule therapies is explored in this issue’s cover story, featured on pages 7–11, along with the potential that technological advances hold in pushing future boundaries and some of the challenges facing both the development and manufacture of biologic drugs (2).

Advanced therapy medicinal products (ATMPs), which are promising and often curative biologic therapies, face their own specific development and manufacturing challenges. Scalability of manufacturing for these emerging therapies is a particular bottleneck and something that San Francisco-based Cellares, a self-titled integrated development and manufacturing organization (IDMO), has sought to change. In an interview with 

Technological advances are helping advance biologics development and manufacturing and reduce bottlenecks.

BioPharm International, May 2024