Super7 passaging method to improve Chinese hamster ovary cell fed-batch performance.

Chinese hamster ovary (CHO) cells are widely used to manufacture biopharmaceuticals, most of all monoclonal antibodies (mAbs). Some CHO cell lines exhibit production instability, where the productivity of the cells decreases as a function of time in culture. To counter this, we designed a passaging strategy that, rather than maximizing the time spent in log-growth phase, mimics the first 7 days of a fed-batch production process. Cultures passaged using this method had lower net growth rates and were more oxidative throughout 6 weeks of passaging. Fed-batch cultures inoculated by cells passaged using this method had increased net growth rates, oxidative metabolism, and volumetric productivity compared to cells passaged using a conventional strategy. Cells from unstable cell lines passaged by this new method produced 80%-160% more mAbs per unit volume than cells passaged by a conventional method. This new method, named Super7, provides the ability to mitigate the impact of production instability in CHO-K1 cell lines without a need for further cell line creation, genetic engineering, or medium development.

Saline dry fixation for improved cell composition analysis using Raman spectroscopy.

Raman spectroscopy enables the label-free assessment of cellular composition. While live cell analysis is the most accurate approach for cellular Raman spectroscopy, the analysis of fixed cells has proved to be very useful, particularly in collaborative projects where samples need to be serially examined by different laboratories or stored and reanalyzed at a later date. However, many chemicals that are widely used for cell fixation directly affect cellular biomolecules, yielding Raman spectra with missing or altered information. In this article, we compared the suitability of dry-fixation with saline versus chemical fixatives. We compared the Raman spectroscopy of saline dry-fixed cells with the more commonly used formaldehyde and methanol fixation and found that dry-fixed cell spectra preserved more cellular information than either chemical fixative. We also assessed the stability of dry-fixed cells over time and found that they were stable for at least 5 months. Finally, a comparison of dry-fixed and live cell spectra revealed effects due to the hydration state of the cells since they were recovered upon rehydrating dry-fixed samples. Thus, for fixed cell Raman spectroscopy, we recommend dry-fixation with unbuffered saline as a superior method to formaldehyde or methanol fixation.

Second-Generation Human Immunodeficiency Virus Integrase Inhibitors Induce Differentiation Dysregulation and Exert Toxic Effects in Human Embryonic Stem Cell and Mouse Models.

BACKGROUND: Each year, approximately 1.1 million children are exposed in utero to human immunodeficiency virus antiretrovirals, yet their safety is often not well characterized during pregnancy. The Tsepamo study reported a neural tube defect signal in infants exposed to the integrase strand transfer inhibitor (InSTI) dolutegravir from conception, suggesting that exposure during early fetal development may be detrimental. METHODS: The effects of InSTIs on 2 human embryonic stem cell (hESC) lines were characterized with respect to markers of pluripotency, early differentiation, and cellular health. In addition, fetal resorptions after exposure to InSTIs from conception were analyzed in pregnant mice. RESULTS: At subtherapeutic concentrations, second-generation InSTIs bictegravir, cabotegravir, and dolutegravir decreased hESC counts and pluripotency and induced dysregulation of genes involved in early differentiation. At therapeutic concentrations, bictegravir induced substantial hESC death and fetal resorptions. It is notable that first-generation InSTI raltegravir did not induce any hESC toxicity or differentiation, at any concentration tested. CONCLUSIONS: Exposure to some InSTIs, even at subtherapeutic concentrations, can induce adverse effects in hESCs and pregnant mice. Given the increasingly prevalent use of second-generation InSTIs, including in women of reproductive age, it is imperative to further elucidate the effect of InSTIs on embryonic development, as well as their long-term safety after in utero exposure.

Consequences of adjusting cell density and feed frequency on serum-free expansion of thymic regulatory T cells.

BACKGROUND: Given the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents. METHODS: Seeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality. RESULTS: The authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 105 cells/cm2) initially had high growth rates, viability and FOXP3 expression, but these parameters decreased with time and were less stable than those observed in cultures of Tregs restimulated at high cell densities (5 × 105 cells/cm2), which had slower growth rates. High-density expansion was associated with expression of inhibitory molecules and lower intracellular oxygen and extracellular nutrient concentrations as well as extracellular lactate accumulation. Experiments to test the effect of low oxygen revealed that transient exposure to low oxygen levels had little impact on expansion, viability or phenotype. Similarly, blockade of inhibitory molecules had little effect. By contrast, replenishing nutrients by increasing the feeding frequency between 2 days and 4 days after restimulation increased FOXP3, viability and expansion in high-density cultures. CONCLUSION: These data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype.

Autophagy-inducing peptide increases CHO cell monoclonal antibody production in batch and fed-batch cultures.

The development of generic biopharmaceuticals is increasing the pressures for enhanced bioprocess productivity and yields. Autophagy ("self-eating") is a cellular process that allows cells to mitigate stresses such as nutrient deprivation. Reputed autophagy inhibitors have also been shown to increase autophagic flux under certain conditions, and enhance recombinant protein productivity in Chinese Hamster Ovary (CHO) cultures. Since peptides are commonly added to bioprocess culture media in hydrolysates, we evaluated the impact on productivity of an autophagy-inducing peptide (AIP), derived from the cellular autophagy protein Beclin 1. This was analyzed in CHO cell batch and fed-batch serum-free cultures producing a human Immunoglobulin G1 (IgG1). Interestingly, the addition of 1-4 µM AIP enhanced productivity in a concentration-dependent manner. Cell-specific productivity increased up to 1.8-fold in batch cultures, while in fed-batch cultures a maximum twofold increase in IgG concentration was observed. An initial drop in cell viability also occurred before cultures recovered normal growth. Overall, these findings strongly support the value of investigating the effects of autophagy pathway modulation, and in particular, the use of this AIP medium additive to increase CHO cell biotherapeutic protein production and yields.

Correction: Applications of Raman spectroscopy in the development of cell therapies: state of the art and future perspectives.

Correction for 'Applications of Raman spectroscopy in the development of cell therapies: state of the art and future perspectives' by Shreyas Rangan et al., Analyst, 2020, DOI: 10.1039/c9an01811e.

Applications of Raman spectroscopy in the development of cell therapies: state of the art and future perspectives.

Therapies based on injecting living cells into patients offer a huge potential to cure many degenerative and deadly diseases, with hundreds of clinical trials ongoing. Due to their complex nature, a basic understanding of their biochemical and functional characteristics, how to manufacture them for safe and efficacious therapy, and how to effectively implement them in clinical settings are very challenging. Raman spectroscopy could provide an information-rich, non-invasive, non-destructive analytical method to complement the use of conventional sample-based, infrequent and destructive biochemical assays typically employed to analyze and validate the quality of therapeutic cells. This article provides an overview of the current state of emerging cell therapies, and then reviews the related Raman spectroscopic state of the art analysis of human cells. This includes spectroscopic data processing considerations, the scope offered by technical variants of Raman spectroscopy, and analytical difficulties encountered by spectroscopists working with therapeutic cells. Finally, we outline a number of salient challenges as cell therapy products are translated from the laboratory to the clinic, and propose how Raman spectroscopy-based solutions could address these challenges.

Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol.

Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing and cryopreservation to develop a Good Manufacturing Practice (GMP)-compatible method to expand and cryopreserve Tregs. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs) and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function and resulted in expansion that was comparable with a single stimulation with aAPCs. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1-3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products.

Types of cell death and apoptotic stages in Chinese Hamster Ovary cells distinguished by Raman spectroscopy.

Cell death is the ultimate cause of productivity loss in bioreactors that are used to produce therapeutic proteins. We investigated the ability of Raman spectroscopy to detect the onset and types of cell death for Chinese Hamster Ovary (CHO) cells-the most widely used cell type for therapeutic protein production. Raman spectroscopy was used to compare apoptotic, necrotic, autophagic, and control CHO cells. Several specific nucleic acid-, protein-, and lipid-associated marker bands within the 650-850 cm-1 spectral region were identified that distinguished among cells undergoing different modes of cell death; supporting evidence was provided by principal component analysis (PCA) of the full spectral data. In addition to comparing the different modes of cell death, normal cells were compared to cells sorted at several stages of apoptosis, in order to explore the potential for early detection of apoptosis. Different stages of apoptosis could be distinguished via Raman spectroscopy, with multiple changes observed in nucleic acid peaks at early stages whereas an increase in lipid signals was a feature of late apoptosis/secondary necrosis.

Stem cells and beta cell replacement therapy: a prospective health technology assessment study.

BACKGROUND: Although current beta cell replacement therapy is effective in stabilizing glycemic control in highly selected patients with refractory type 1 diabetes, many hurdles are inherent to this and other donor-based transplantation methods. One solution could be moving to stem cell-derived transplant tissue. This study investigates a novel stem cell-derived graft and implant technology and explores the circumstances of its cost-effectiveness compared to intensive insulin therapy. METHODS: We used a manufacturing optimization model based on work by Simaria et al. to model cost of the stem cell-based transplant doses and integrated its results into a cost-effectiveness model of diabetes treatments. The disease model simulated marginal differences in clinical effects and costs between the new technology and our comparator intensive insulin therapy. The form of beta cell replacement therapy was as a series of retrievable subcutaneous implant devices which protect the enclosed pancreatic progenitors cells from the immune system. This approach was presumed to be as effective as state of the art islet transplantation, aside from immunosuppression drawbacks. We investigated two different cell culture methods and several production and delivery scenarios. RESULTS: We found the likely range of treatment costs for this form of graft tissue for beta cell replacement therapy. Additionally our results show this technology could be cost-effective compared to intensive insulin therapy, at a willingness-to-pay threshold of $100,000 per quality-adjusted life year. However, results also indicate that mass production has by far the best chance of providing affordable graft tissue, while overall there seems to be considerable room for cost reductions. CONCLUSIONS: Such a technology can improve treatment access and quality of life for patients through increased graft supply and protection. Stem cell-based implants can be a feasible way of treating a wide range of patients with type 1 diabetes.

Process Analytical Utility of Raman Microspectroscopy in the Directed Differentiation of Human Pancreatic Insulin-Positive Cells.

Continued advances toward cell-based therapies for human disease generate a growing need for unbiased and label-free monitoring of cellular characteristics. We used Raman microspectroscopy to characterize four important stages in the 26-day directed differentiation of human embryonic stem cells (hESCs) to insulin-positive cells. The extent to which the cells retained spectroscopic features of pluripotent cells or developed spectroscopic features suggestive of pancreatic endocrine cells, as well as assessing the homogeneity of the cell populations at these developmental stages, were of particular interest. Such information could have implications for the utility of Raman microspectroscopy process analysis for the generation of insulin-positive cells from hESCs. Because hESC seeding density influences the subsequent pancreatic development, three different seeding density cultures were analyzed. Transcription factor and other marker analyses assessed the progress of the cells through the relevant developmental stages. Increases in the Raman protein-to-nucleic acid band ratios were observed at the final endocrine stage analyzed, but this increase was less than expected. Also, high glycogen band intensities, somewhat unexpected in pancreatic endocrine cells, suggested the presence of a substantial number of glycogen containing cells. We discuss the potential process analytical technology application of these findings and their importance for cell manufacturing.

High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays.

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.

Effects of insulin on human pancreatic cancer progression modeled in vitro.

BACKGROUND: Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined. METHODS: Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model. RESULTS: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling. CONCLUSIONS: Our data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired during pancreatic cancer progression.

Metabolic switching, growth kinetics and cell yields in the scalable manufacture of stem cell-derived insulin-producing cells.

BACKGROUND: Diabetes is a disease affecting over 500 million people globally due to insulin insufficiency or insensitivity. For individuals with type 1 diabetes, pancreatic islet transplantation can help regulate their blood glucose levels. However, the scarcity of cadaveric donor islets limits the number of people that could receive this therapy. To address this issue, human pluripotent stem cells offer a potentially unlimited source for generating insulin-producing cells through directed differentiation. Several protocols have been developed to make stem cell-derived insulin-producing cells. However, there is a lack of knowledge regarding the bioprocess parameters associated with these differentiation protocols and how they can be utilized to increase the cell yield. METHODS: We investigated various bioprocess parameters and quality target product profiles that may influence the differentiation pipeline using a seven-stage protocol in a scalable manner with CellSTACKs and vertical wheel bioreactors (PBS-Minis). RESULTS: Cells maintained > 80% viability through all stages of differentiation and appropriately expressed stage-specific markers. During the initial four stages leading up to the development of pancreatic progenitors, there was an increase in cell numbers. Following pancreatic progenitor stage, there was a gradual decrease in the percentage of proliferative cells, as determined by Ki67 positivity, and a significant loss of cells during the period of endocrine differentiation. By minimizing the occurrence of aggregate fusion, we were able to enhance cell yield during the later stages of differentiation. We suggest that glucose utilization and lactate production are cell quality attributes that should be considered during the characterization of insulin-producing cells derived from stem cells. Our findings also revealed a gradual metabolic shift from glycolysis, during the initial four stages of pancreatic progenitor formation, to oxidative phosphorylation later on during endocrine differentiation. Furthermore, the resulting insulin-producing cells exhibited a response to several secretagogues, including high glucose. CONCLUSION: This study demonstrates process parameters such as glucose consumption and lactate production rates that may be used to facilitate the scalable manufacture of stem cell-derived insulin-producing cells.

Label-free determination of the cell cycle phase in human embryonic stem cells by Raman microspectroscopy.

The cell cycle is a series of integrated and coordinated physiological events that results in cell growth and replication. Besides observing the event of cell division it is not feasible to determine the cell cycle phase without fatal and/or perturbing invasive procedures such as cell staining, fixing, and/or dissociation. Raman microspectroscopy (RMS) is a chemical imaging technique that exploits molecular vibrations as a contrast mechanism; it can be applied to single living cells noninvasively to allow unperturbed analysis over time. We used RMS to determine the cell cycle phase based on integrating the composite 783 cm(-1) nucleic acid band intensities across individual cell nuclei. After correcting for RNA contributions using the RNA 811 cm(-1) band, the measured intensities essentially reflected DNA content. When quantifying Raman images from single cells in a population of methanol-fixed human embryonic stem cells, the histogram of corrected 783 cm(-1) band intensities exhibited a profile analogous to that obtained using flow-cytometry with nuclear stains. The two population peaks in the histogram occur at Raman intensities corresponding to a 1-fold and 2-fold diploid DNA complement per cell, consistent with a distribution of cells with a population peak due to cells at the end of G1 phase (1-fold) and a peak due to cells entering M phase (2-fold). When treated with EdU to label the replicating DNA and block cell division, cells with higher EdU-related fluorescence generally had higher integrated Raman intensities. This provides proof-of-principle of an analytical method for label-free RMS determination in situ of cell cycle phase in adherent monolayers or even single adherent cells.

Ontogeny stage-independent and high-level clonal expansion in vitro of mouse hematopoietic stem cells stimulated by an engineered NUP98-HOX fusion transcription factor.

Achieving high-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Here, we couple the ability of engineered NUP98-HOXA10hd expression to stimulate > 1000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin(-)Sca-1(+)c-kit(+) cells, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. We find that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at ∼ 60% to 90% unit efficiency in cultures initiated with single cells. Clonally expanded HSCs consistently show balanced long-term contributions to the lymphoid and myeloid lineages without evidence of leukemogenic activity. Although effects on fetal and adult HSCs were indistinguishable, NUP98-HOXA10hd-transduced adult HSCs did not thereby gain a competitive advantage in vivo over freshly isolated fetal HSCs. Live-cell image tracking of single transduced HSCs cultured in a microfluidic device indicates that NUP98-HOXA10hd does not affect their proliferation kinetics, and flow cytometry confirmed the phenotype of normal proliferating HSCs and allowed reisolation of large numbers of expanded HSCs at a purity of 25%. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process.

A human embryonic stem cell line adapted for high throughput screening.

Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However, optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs, including being karyotypically normal and their ability to generate teratomas containing all three germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20, they remain highly useful. In particular, CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus, 10(8) CA1S cells can be generated within one week from 10(6) cells to seed 10(6) wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm, a finding that was confirmed with H1 hESCs, although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines, these cells should ease the development of optimized hESC growth and differentiation protocols. In particular, they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses.

Label-free imaging of mammalian cell nucleoli by Raman microspectroscopy.

The nucleolus is a prominent subnuclear structure whose major function is the transcription and assembly of ribosome subunits. The size of the nucleolus varies with the cell cycle, proliferation rate and stress. Changes in nucleolar size, number, chemical composition, and shape can be used to characterize malignant cells. We used spontaneous Raman microscopy as a label-free technique to examine nucleolar spatial and chemical features. Raman images of the 1003 cm(-1) phenylalanine band revealed large, well-defined subnuclear protein structures in MFC-7 breast cancer cells. The 783 cm(-1) images showed that nucleic acids were similarly distributed, but varied more in intensity, forming observable high-intensity regions. High subnuclear RNA concentrations were observed within some of these regions as shown by 809 cm(-1) Raman band images. Principal component analyses of sub-images and library spectra validated the subnuclear presence of RNA. They also revealed that an actin-like protein covaried with DNA within the nucleolus, a combination that accounted for 64% or more of the spectral variance. Embryonic stem cells are another rapidly proliferating cell type, but their nucleoli were not as large or well defined. Estimating the size of the larger MCF-7 nucleolus was used to show the utility of Raman microscopy for morphometric analyses. It was concluded that imaging based on Raman microscopy provides a promising new method for the study of nucleolar function and organization, in the evaluation of drug and experimental effects on the nucleolus, and in clinical diagnostics and prognostics.

Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture.

Introduction: Human pluripotent stem cells (hPSCs) provide many opportunities for application in regenerative medicine due to their ability to differentiate into cells from all three germ layers, proliferate indefinitely, and replace damaged or dysfunctional cells. However, such cell replacement therapies require the economical generation of clinically relevant cell numbers. Whereas culturing hPSCs as a two-dimensional monolayer is widely used and relatively simple to perform, their culture as suspended three-dimensional aggregates may enable more economical production in large-scale stirred tank bioreactors. To be more relevant to this biomanufacturing, bench-scale differentiation studies should be initiated from aggregated hPSC cultures. Methods: We compared five available bench-scale platforms for generating undifferentiated cell aggregates of human embryonic stem cells (hESCs) using AggreWell™ plates, low attachment plates on an orbital shaker, roller bottles, spinner flasks, and vertical-wheel bioreactors (PBS-Minis). Thereafter, we demonstrated the incorporation of an hPSC aggregation step prior to directed differentiation to pancreatic progenitors and endocrine cells. Results and discussion: The AggreWell™ system had the highest aggregation yield. The initial cell concentrations had an impact on the size of aggregates generated when using AggreWell™ plates as well as in roller bottles. However, aggregates made with low attachment plates, spinner flasks and PBS-Minis were similar regardless of the initial cell number. Aggregate morphology was compact and relatively homogenously distributed in all platforms except for the roller bottles. The size of aggregates formed in PBS-Minis was modulated by the agitation rate during the aggregation. In all cell culture platforms, the net growth rate of cells in 3D aggregates was lower (range: -0.01-0.022 h-1) than cells growing as a monolayer (range: 0.039-0.045 h-1). Overall, this study describes operating ranges that yield high-quality undifferentiated hESC aggregates using several of the most commonly used bench-scale cell culture platforms. In all of these systems, methods were identified to obtain PSC aggregates with greater than 70% viability, and mean diameters between 60 and 260 mm. Finally, we showed the capacity of hPSC aggregates formed with PBS-Minis to differentiate into viable pancreatic progenitors and endocrine cell types.

Engineering protein glycosylation in CHO cells to be highly similar to murine host cells.

Since 2015 more than 34 biosimilars have been approved by the FDA. This new era of biosimilar competition has stimulated renewed technology development focused on therapeutic protein or biologic manufacturing. One challenge in biosimilar development is the genetic differences in the host cell lines used to manufacture the biologics. For example, many biologics approved between 1994 and 2011 were expressed in murine NS0 and SP2/0 cell lines. Chinese Hamster ovary (CHO) cells, however, have since become the preferred hosts for production due to their increased productivity, ease of use, and stability. Differences between murine and hamster glycosylation have been identified in biologics produced using murine and CHO cells. In the case of monoclonal antibodies (mAbs), glycan structure can significantly affect critical antibody effector function, binding activity, stability, efficacy, and in vivo half-life. In an attempt to leverage the intrinsic advantages of the CHO expression system and match the reference biologic murine glycosylation, we engineered a CHO cell expressing an antibody that was originally produced in a murine cell line to produce murine-like glycans. Specifically, we overexpressed cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) and N-acetyllactosaminide alpha-1,3-galactosyltransferase (GGTA) to obtain glycans with N-glycolylneuraminic acid (Neu5Gc) and galactose-α-1,3-galactose (alpha gal). The resulting CHO cells were shown to produce mAbs with murine glycans, and they were then analyzed by the spectrum of analytical methods typically used to demonstrate analytical similarity as a part of demonstrating biosimilarity. This included high-resolution mass spectrometry, biochemical, as well as cell-based assays. Through selection and optimization in fed-batch cultures, two CHO cell clones were identified with similar growth and productivity criteria to the original cell line. They maintained stable production for 65 population doubling times while matching the glycosylation profile and function of the reference product expressed in murine cells. This study demonstrates the feasibility of engineering CHO cells to express mAbs with murine glycans to facilitate the development of biosimilars that are highly similar to marketed reference products expressed in murine cells. Furthermore, this technology can potentially reduce the residual uncertainty regarding biosimilarity, resulting in a higher probability of regulatory approval and potentially reduced costs and time in development.