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.

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.

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.

Protocol development to further differentiate and transition stem cell-derived pancreatic progenitors from a monolayer into endocrine cells in suspension culture.

The generation of functional ß-cells from human pluripotent stem cells (hPSCs) for cell replacement therapy and disease modeling of diabetes is being investigated by many groups. We have developed a protocol to harvest and aggregate hPSC-derived pancreatic progenitors generated using a commercially available kit into near uniform spheroids and to further differentiate the cells toward an endocrine cell fate in suspension culture. Using a static suspension culture platform, we could generate a high percentage of insulin-expressing, glucose-responsive cells. We identified FGF7 as a soluble factor promoting aggregate survival with no inhibitory effect on endocrine gene expression. Notch inhibition of pancreatic progenitor cells during aggregation improved endocrine cell induction in vitro and improved graft function following implantation and further differentiation in mice. Thus we provide an approach to promote endocrine formation from kit-derived pancreatic progenitors, either through extended culture or post implant.

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.

Rapid Vector-Based Peak Fitting and Resolution Enhancement for Correlation Analyses of Raman Hyperspectra.

Spectroscopic peak parameters are important since they provide information about the analyte under study. Besides obtaining these parameters, peak fitting also resolves overlapped peaks. Thus, the obtained parameters should permit the construction of a higher-resolution version of the original spectrum. However, peak fitting is not an easy task due to computational reasons and because the true nature of the analyte is often unknown. These difficulties are major impediments when large hyperspectral data sets need to be processed rapidly, such as for manufacturing process control. We have developed a novel and relatively fast two-part algorithm to perform peak fitting and resolution enhancement on such data sets. In the first part of the algorithm, estimates of the total number of bands and their parameters were obtained from a representative spectrum in the data set, using a combination of techniques. Starting with these parameter estimates, all the spectra were then iteratively and rapidly fitted with Gaussian bands, exploiting intrinsic features of the Gaussian distribution with vector operations. The best fits for each spectrum were retained. By reducing the obtained bandwidths and commensurately increasing their amplitudes, high-resolution spectra were constructed that greatly improved correlation-based analyses. We tested the performance of the algorithm on synthetic spectra to confirm that this method could recover the ground truth correlations between highly overlapped peaks. To assess effective peak resolution, the method was applied to low-resolution spectra of glucose and compared to results from high-resolution spectra. We then processed a larger spectral data set from mammalian cells, fixed with methanol or air drying, to demonstrate the resolution enhancement of the algorithm on complex spectra and the effects of resolution-enhanced spectra on two-dimensional correlation spectroscopy and principal component analyses. The results indicated that the algorithm would allow users to obtain high-resolution spectra relatively fast and permit the recovery of important aspects of the data's intrinsic correlation structure.

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.

Two-Dimensional Clustering of Spectral Changes for the Interpretation of Raman Hyperspectra.

Two-dimensional correlation spectroscopy (2D-COS) is a technique that permits the examination of synchronous and asynchronous changes present in hyperspectral data. It produces two-dimensional correlation coefficient maps that represent the mutually correlated changes occurring at all Raman wavenumbers during an implemented perturbation. To focus our analysis on clusters of wavenumbers that tend to change together, we apply a k-means clustering to the wavenumber profiles in the perturbation domain decomposition of the two-dimensional correlation coefficient map. These profiles (or trends) reflect peak intensity changes as a function of the perturbation. We then plot the co-occurrences of cluster members two-dimensionally in a manner analogous to a two-dimensional correlation coefficient map. Because wavenumber profiles are clustered based on their similarity, two-dimensional cluster member spectra reveal which Raman peaks change in a similar manner, rather than how much they are correlated. Furthermore, clustering produces a discrete partitioning of the wavenumbers, thus a two-dimensional cluster member spectrum exhibits a discrete presentation of related Raman peaks as opposed to the more continuous representations in a two-dimensional correlation coefficient map. We demonstrate first the basic principles of the technique with the aid of synthetic data. We then apply it to Raman spectra obtained from a polystyrene perchlorate model system followed by Raman spectra from mammalian cells fixed with different percentages of methanol. Both data sets were designed to produce differential changes in sample components. In both cases, all the peaks pertaining to a given component should then change in a similar manner. We observed that component-based profile clustering did occur for polystyrene and perchlorate in the model system and lipids, nucleic acids, and proteins in the mammalian cell example. This confirmed that the method can translate to "real world" samples. We contrast these results with two-dimensional correlation spectroscopy results. To supplement interpretation, we present the cluster-segmented mean spectrum of the hyperspectral data. Overall, this technique is expected to be a valuable adjunct to two-dimensional correlation spectroscopy to further facilitate hyperspectral data interpretation and analysis.

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.

Critical Evaluation of Spectral Resolution Enhancement Methods for Raman Hyperspectra.

Overlapping peaks in Raman spectra complicate the presentation, interpretation, and analyses of complex samples. This is particularly problematic for methods dependent on sparsity such as multivariate curve resolution and other spectral demixing as well as for two-dimensional correlation spectroscopy (2D-COS), multisource correlation analysis, and principal component analysis. Though software-based resolution enhancement methods can be used to counter such problems, their performances often differ, thereby rendering some more suitable than others for specific tasks. Furthermore, there is a need for automated methods to apply to large numbers of varied hyperspectral data sets containing multiple overlapping peaks, and thus methods ideally suitable for diverse tasks. To investigate these issues, we implemented three novel resolution enhancement methods based on pseudospectra, over-deconvolution, and peak fitting to evaluate them along with three extant methods: node narrowing, blind deconvolution, and the general-purpose peak fitting program Fityk. We first applied the methods to varied synthetic spectra, each consisting of nine overlapping Voigt profile peaks. Improved spectral resolution was evaluated based on several criteria including the separation of overlapping peaks and the preservation of true peak intensities in resolution-enhanced spectra. We then investigated the efficacy of these methods to improve the resolution of measured Raman spectra. High resolution spectra of glucose acquired with a narrow spectrometer slit were compared to ones using a wide slit that degraded the spectral resolution. We also determined the effects of the different resolution enhancement methods on 2D-COS and on chemical contrast image generation from mammalian cell spectra. We conclude with a discussion of the particular benefits, drawbacks, and potential of these methods. Our efforts provided insight into the need for effective resolution enhancement approaches, the feasibility of these methods for automation, the nature of the problems currently limiting their use, and in particular those aspects that need improvement.

Serum free culture for the expansion and study of type 2 innate lymphoid cells.

Type 2 innate lymphoid cells (ILC2s) were discovered approximately ten years ago and their clinical relevance is gaining greater importance. However, their successful isolation from mammalian tissues and in vitro culture and expansion continues to pose challenges. This is partly due to their scarcity compared to other leukocyte populations, but also because our current knowledge of ILC2 biology is incomplete. This study is focused on ST2+ IL-25Rlo lung resident ILC2s and demonstrate for the first time a methodology allowing mouse type 2 innate lymphoid cells to be cultured, and their numbers expanded in serum-free medium supplemented with Interleukins IL-33, IL-2, IL-7 and TSLP. The procedures described methods to isolate ILC2s and support their growth for up to a week while maintaining their phenotype. During this time, they significantly expand from low to high cell concentrations. Furthermore, for the first time, sub-cultures of primary ILC2 purifications in larger 24- and 6-well plates were undertaken in order to compare their growth in other media. In culture, ILC2s had doubling times of 21 h, a growth rate of 0.032 h-1 and could be sub-cultured in early or late phases of exponential growth. These studies form the basis for expanding ILC2 populations that will facilitate the study and potential applications of these rare cells under defined, serum-free conditions.

Two-step sedimentation process for selection of microcapsules containing cell aggregates.

Microencapsulation technologies are being developed to protect transplanted islets from immune rejection, to reduce or even eliminate the need for immunosuppression. However, unencapsulated cells increase the chances of rejection and empty beads increase transplant volumes. Thus, separation processes were investigated to remove these byproducts based on density differences. The densities of islet-sized mouse insulinoma 6 (MIN6) cell aggregates and acellular 5% alginate beads generated via emulsification and internal gelation were ~ 1.065 and 1.042 g/ml, respectively. The separation of empty beads from those containing aggregates was performed by sedimentation under unit gravity in continuous gradients of polysucrose and sodium diatrizoate with density ranges of 1.032-1.045, 1.035-1.044, or 1.039-1.042 g/ml. The 1.039-1.042 g/ml gradient enabled recoveries of ~ 80% of the aggregate-containing beads while the other gradients recovered only ~ 60%. The bottom fraction of the 1.039-1.042 g/ml gradient contained beads with ~ 6% of their volume occupied by cell aggregates. Separation of unencapsulated aggregates from the aggregate-containing beads was then achieved by centrifugation of this purified fraction in a 1.055 g/ml density solution. Thus, these sedimentation-based approaches can effectively remove the byproducts of cell encapsulation.

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.

Augmented Two-Dimensional Correlation Spectroscopy for the Joint Analysis of Correlated Changes in Spectroscopic and Disparate Sources.

Here, we present an augmented form of two-dimensional correlation spectroscopy, that integrates in a single format data from spectroscopic and multiple non-spectroscopic sources for analysis. The integration is affected by augmenting every spectrum in a hyperspectral data set with relevant non-spectroscopic data to permit two-dimensional correlation analysis(2D-COS) of the ensemble of augmented spectra. A k-means clustering is then applied to the results of the perturbation domain decomposition to determine which Raman peaks cluster with any of the non-spectroscopic data. We introduce and explain the method with the aid of synthetic spectra and synthetic non-spectroscopic data. We then demonstrate this approach with data using Raman spectra from human embryonic stem cell aggregates undergoing directed differentiation toward pancreatic endocrine cells and parallel bioassays of hormone mRNA expression and C-peptide levels in spent medium. These pancreatic endocrine cells generally contain insulin or glucagon. Insulin has disulfide bonds that produce Raman scattering near 513 cm-1, but no tryptophan. For insulin-positive cells, we found that the application of multisource correlation analysis revealed a high correlation between insulin mRNA and Raman scattering in the disulfide region. In contrast, glucagon has no disulfide bonds but does contain tryptophan. For glucagon-positive cells, we also observed a high correlation between glucagon mRNA and tryptophan Raman scattering (∼757 cm-1). We conclude with a discussion of methods to enhance spectral resolution and its effects on the performance of multisource correlation analysis.

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.

Effects of cysteine, asparagine, or glutamine limitations in Chinese hamster ovary cell batch and fed-batch cultures.

Amino acid availability is a key factor that can be controlled to optimize the productivity of fed-batch cultures. To study amino acid limitation effects, a serum-free chemically defined basal medium was formulated to exclude the amino acids that became depleted in batch culture. The effect of limiting glutamine, asparagine, and cysteine on the cell growth, metabolism, antibody productivity, and product glycosylation was investigated in three Chinese hamster ovary (CHO) cell lines (CHO-DXB11, CHO-K1SV, and CHO-S). Cysteine limitation was detrimental to both cell proliferation and productivity for all three CHO cell lines. Glutamine limitation reduced growth but not cell specific productivity, whereas asparagine limitation had no significant effect on either growth or cell specific productivity. Neither glutamine nor asparagine limitation significantly affected antibody glycosylation. Replenishing the CHO-DXB11 culture with cysteine after 1 day of cysteine limitation allowed the cells to partially recover their growth and productivity. This recovery was not observed after 2 days of cysteine limitation. Based on these findings, a fed-batch protocol was developed using single or mixed amino acid supplementation. Although cell density and antibody concentration were lower compared to a commercial feed, the feeds based on cysteine supplementation yielded comparable cell specific productivity. Overall, this study showed that different amino acid limitations have varied effects on the performance of CHO cell cultures and that maintaining cysteine availability is a critical process parameter for the three cell lines investigated.

Defocused Spatially Offset Raman Spectroscopy in Media of Different Optical Properties for Biomedical Applications Using a Commercial Spatially Offset Raman Spectroscopy Device.

In this study, we show how defocused spatially offset Raman spectroscopy (SORS) can be employed to recover chemical information from media of biomedical significance within sealed plastic transfusion and culture bags using a commercial SORS instrument. We demonstrate a simple approach to recover subsurface spectral information through a transparent barrier by optimizing the spatial offset of the defocused beam. The efficiency of the measurements is assessed in terms of the SORS ratio and signal-to-noise ratio (S/N) through a simple manual approach and an ordinary least squares model. By comparing the results for three different biological samples (red blood cell concentrate, pooled red cell supernatant and a suspension of Jurkat cells), we show that there is an optimum value of the offset parameter which yields the maximum S/N depending on the barrier material and optical properties of the ensemble contents. The approach was developed in the context of biomedical applications but is generally applicable to any three-layer system consisting of turbid content between transparent thin plastic barriers (i.e., front and back bag surfaces), particularly where the analyte of interest is dilute or not a strong scatterer.

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.