Systematic evaluation of genome-wide metabolic landscapes in lactic acid bacteria reveals diet- and strain-specific probiotic idiosyncrasies.

Lactic acid bacteria (LAB) are well known to elicit health benefits in humans, but their functional metabolic landscapes remain unexplored. Here, we analyze differences in growth, intestinal persistence, and postbiotic biosynthesis of six representative LAB and their interactions with 15 gut bacteria under 11 dietary regimes by combining multi-omics and in silico modeling. We confirmed predictions on short-term persistence of LAB and their interactions with commensals using cecal microbiome abundance and spent-medium experiments. Our analyses indicate that probiotic attributes are both diet and species specific and cannot be solely explained using genomics. For example, although both Lacticaseibacillus casei and Lactiplantibacillus plantarum encode similarly sized genomes with diverse capabilities, L. casei exhibits a more desirable phenotype. In addition, "high-fat/low-carb" diets more likely lead to detrimental outcomes for most LAB. Collectively, our results highlight that probiotics are not "one size fits all" health supplements and lay the foundation for personalized probiotic design.

Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion.

Therapies using mesenchymal stromal cells (MSCs) to treat immune and inflammatory conditions are now at an exciting stage of development, with many MSC-based products progressing to phase II and III clinical trials. However, a major bottleneck in the clinical translation of allogeneic MSC therapies is the variable immunomodulatory properties of MSC products due to differences in their tissue source, donor heterogeneity and processes involved in manufacturing and banking. This variable functionality of MSC products likely contributes to the substantial inconsistency observed in the clinical outcomes of phase III trials of MSC therapies; several trials have failed to reach the primary efficacy endpoint. In this review, we discuss various strategies to consistently maintain or enhance the immunomodulatory potency of MSCs during ex vivo expansion, which will enable the manufacture of allogeneic MSC banks that have high potency and low variability. Biophysical and biochemical priming strategies, the use of culture additives such as heparan sulfates, and genetic modification can substantially enhance the immunomodulatory properties of MSCs during in vitro expansion. Furthermore, robust donor screening, the use of biomarkers to select for potent MSC subpopulations, and rigorous quality testing to improve the release criteria for MSC banks have the potential to reduce batch-to-batch heterogeneity and enhance the clinical efficacy of the final MSC product. Machine learning approaches to develop predictive models of individual patient response can enable personalized therapies and potentially establish correlations between in vitro potency measurements and clinical outcomes in human trials.

Harnessing the potential of machine learning for advancing "Quality by Design" in biomanufacturing.

Ensuring consistent high yields and product quality are key challenges in biomanufacturing. Even minor deviations in critical process parameters (CPPs) such as media and feed compositions can significantly affect product critical quality attributes (CQAs). To identify CPPs and their interdependencies with product yield and CQAs, design of experiments, and multivariate statistical approaches are typically used in industry. Although these models can predict the effect of CPPs on product yield, there is room to improve CQA prediction performance by capturing the complex relationships in high-dimensional data. In this regard, machine learning (ML) approaches offer immense potential in handling non-linear datasets and thus are able to identify new CPPs that could effectively predict the CQAs. ML techniques can also be synergized with mechanistic models as a 'hybrid ML' or 'white box ML' to identify how CPPs affect the product yield and quality mechanistically, thus enabling rational design and control of the bioprocess. In this review, we describe the role of statistical modeling in Quality by Design (QbD) for biomanufacturing, and provide a generic outline on how relevant ML can be used to meaningfully analyze bioprocessing datasets. We then offer our perspectives on how relevant use of ML can accelerate the implementation of systematic QbD within the biopharma 4.0 paradigm.

HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors.

Animal cell-based expression platforms enable the production of complex biomolecules such as recombinant proteins and viral vectors. Although most biotherapeutics are produced in animal cell lines, production in human cell lines is expanding. One important advantage of using human cell lines is the increased potential that the resulting biotherapeutics would carry more "human-like" post-translational modifications. Among the human cell lines, HEK293 is widely utilized due to its high transfectivity, rapid growth rate, and ability to grow in a serum-free, suspension culture. In this review, we discuss the use of HEK293 cells and its subtypes in the production of biotherapeutics. We also compare their usage against other commonly used host cell lines in each category of biotherapeutics and summarise the factors influencing the choice of host cell lines used.

Applications and analysis of hydrolysates in animal cell culture.

Animal cells are used in the manufacturing of complex biotherapeutic products since the 1980s. From its initial uses in biological research to its current importance in the biopharmaceutical industry, many types of culture media were developed: from serum-based media to serum-free to protein-free chemically defined media. The cultivation of animal cells economically has become the ultimate goal in the field of biomanufacturing. Serum serves as a source of amino acids, lipids, proteins and most importantly growth factors and hormones, which are essential for many cell types. However, the use of serum is unfavorable due to its high price tag, increased lot-to-lot variations and potential risk of microbial contamination. Efforts are progressively being made to replace serum with recombinant proteins such as growth factors, cytokines and hormones, as well as supplementation with lipids, vitamins, trace elements and hydrolysates. While hydrolysates are more complex, they provide a diverse source of nutrients to animal cells, with potential beneficial effects beyond the nutritional value. In this review, we discuss the use of hydrolysates in animal cell culture and briefly cover the composition of hydrolysates, mode of action and potential contaminants with some perspectives on its potential role in animal cell culture media formulations in the future.

Multi-omics profiling of a CHO cell culture system unravels the effect of culture pH on cell growth, antibody titer, and product quality.

A robust monoclonal antibody (mAb) bioprocess requires physiological parameters such as temperature, pH, or dissolved oxygen to be well-controlled as even small variations in them could potentially impact the final product quality. For instance, pH substantially affects N-glycosylation, protein aggregation, and charge variant profiles, as well as mAb productivity. However, relatively less is known about how pH jointly influences product quality and titer. In this study, we investigated the effect of pH on culture performance, product titer, and quality profiles by applying longitudinal multi-omics profiling, including transcriptomics, proteomics, metabolomics, and glycomics, at three different culture pH set points. The subsequent systematic analysis of multi-omics data showed that pH set points differentially regulated various intracellular pathways including intracellular vesicular trafficking, cell cycle, and apoptosis, thereby resulting in differences in specific productivity, product titer, and quality profiles. In addition, a time-dependent variation in mAb N-glycosylation profiles, independent of pH, was identified to be mainly due to the accumulation of mAb proteins in the endoplasmic reticulum disrupting cellular homeostasis over culture time. Overall, this multi-omics-based study provides an in-depth understanding of the intracellular processes in mAb-producing CHO cell line under varied pH conditions, and could serve as a baseline for enabling the quality optimization and control of mAb production.

Utility of Ion-Mobility Spectrometry for Deducing Branching of Multiply Charged Glycans and Glycopeptides in a High-Throughput Positive ion LC-FLR-IMS-MS Workflow.

High-throughput glycan analysis has become an important part of biopharmaceutical production and quality control. However, it is still a significant challenge in the field of glycomics to easily deduce isomeric glycan structures, especially in a high-throughput manner. Ion mobility spectrometry (IMS) is an excellent tool for differentiating isomeric glycan structures. However, demonstrations of the utility of IMS in high-throughput workflows such as liquid chromatography-fluorescence-mass spectrometry (LC-FLR-MS) workflows have been limited with only a small amount of collision cross section (CCS) data available. In particular, IMS data of glycan fragments obtained in positive ion mode are limited in comparison to those obtained in negative ion mode despite positive ion mode being widely used for glycomics. Here, we describe IMS TWCCSN2 data obtained from a high-throughput LC-FLR-IMS-MS workflow in positive ion mode. We obtained IMS data from a selection of RapiFluor-MS (RFMS) labeled N-glycans and also glycopeptides. We describe how IMS is able to distinguish isomeric N-glycans and glycopeptides using both intact IMS and fragment-based IMS glycan sequencing experiments in positive ion mode, without significantly altering the high-throughput nature of the analysis. For the first time, we were able to successfully use IMS in positive ion mode to determine the branching of isomeric glycopeptides and RFMS labeled glycans. Further, we highlight that IMS glycan sequencing of fragments obtained from RFMS labeled glycans was similar to that of glycopeptides. Finally, we show that the IMS glycan sequencing approach can highlight shared structural features of nonisomeric glycans in a high-throughput LC-FLR-IMS-MS workflow.

Exploiting the Disialyl Galactose Activity of α2,6-Sialyltransferase from Photobacterium damselae To Generate a Highly Sialylated Recombinant α-1-Antitrypsin.

Sialic acids are sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where a lack of sialylation can reduce bioactivity. Here, we describe how α-2,6-sialyltransferase from Photobacterium damselae can be used to markedly increase the level of sialylation of CHO-produced α-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second disialyl galactose motif Neu5Ac-α2,3(Neu5Ac-α2,6)Gal was produced, which, to our knowledge, had never been detected on a mammalian glycoprotein. We exploited this disialyl galactose activity of the P. damselae in a multienzyme reaction to produce a highly sialylated α-1-antitrypsin. The influence of this unique disialylation on the in vitro activity of α-1-antitrypsin was studied, and a toolkit of mass spectrometry methods for identifying this new disialyl galactose motif in complex mixtures was developed.

A human expression system based on HEK293 for the stable production of recombinant erythropoietin.

Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.

Multi-omics profiling of CHO parental hosts reveals cell line-specific variations in bioprocessing traits.

Chinese hamster ovary (CHO) cells are the most prevalent mammalian cell factories for producing recombinant therapeutic proteins due to their ability to synthesize human-like post-translational modifications and ease of maintenance in suspension cultures. Currently, a wide variety of CHO host cell lines has been developed; substantial differences exist in their phenotypes even when transfected with the same target vector. However, relatively less is known about the influence of their inherited genetic heterogeneity on phenotypic traits and production potential from the bioprocessing point of view. Herein, we present a global transcriptome and proteome profiling of three commonly used parental cell lines (CHO-K1, CHO-DXB11, and CHO-DG44) in suspension cultures and further report their growth-related characteristics, and N- and O-glycosylation patterns of host cell proteins (HCPs). The comparative multi-omics and subsequent genome-scale metabolic network model-based enrichment analyses indicated that some physiological variations of CHO cells grown in the same media are possibly originated from the genetic deficits, particularly in the cell-cycle progression. Moreover, the dihydrofolate reductase deficient DG44 and DXB11 possess relatively less active metabolism when compared to K1 cells. The protein processing abilities and the N- and O-glycosylation profiles also differ significantly across the host cell lines, suggesting the need to select host cells in a rational manner for the cell line development on the basis of recombinant protein being produced.

Application of maltose as energy source in protein-free CHO-K1 culture to improve the production of recombinant monoclonal antibody.

Oligosaccharides are generally considered to be un-utilized for growth of mammalian cells because their permeability across the cell membrane is low. However, in our previous study, we discovered that CHO and HEK293 cells consume maltose in culture media without serum and glucose. This is interesting because the transporter for maltose in mammalian cells has not been discovered to-date, and the only animal disaccharide transporter that is recently discovered is a sucrose transporter. The application of oligosaccharides in mammalian cell-based biopharmaceutical manufacturing can be beneficial, because it can theoretically increase carbohydrate content of the culture medium and decrease lactate production. Here, we first determined that specific maltose consumption rate in CHO cells was similar to galactose and fructose at 0.257 ng/cell/day. We then demonstrated that CHO cells can be cultivated with reasonable cell growth using higher maltose concentrations. After which, we evaluated the use of maltose supplementation in the production of a recombinant monoclonal antibody in batch and fed-batch cultures, demonstrating improvements in recombinant monoclonal antibody titer of 15% and 23% respectively. Finally, glycosylation profiles of the antibodies were analyzed.

Impact of host cell line choice on glycan profile.

Protein glycosylation is post-translational modification (PTM) which is important for pharmacokinetics and immunogenicity of recombinant glycoprotein therapeutics. As a result of variations in monosaccharide composition, glycosidic linkages and glycan branching, glycosylation introduces considerable complexity and heterogeneity to therapeutics. The host cell line used to produce the glycoprotein has a strong influence on the glycosylation because different host systems may express varying repertoire of glycosylation enzymes and transporters that contributes to specificity and heterogeneity in glycosylation profiles. In this review, we discuss the types of host cell lines currently used for recombinant therapeutic production, their glycosylation potential and the resultant impact on glycoprotein properties. In addition, we compare the reported glycosylation profiles of four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII (FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced in different mammalian cells to establish the influence of mammalian host cell lines on glycosylation.

Mammalian Systems Biotechnology Reveals Global Cellular Adaptations in a Recombinant CHO Cell Line.

Effective development of host cells for therapeutic protein production is hampered by the poor characterization of cellular transfection. Here, we employed a multi-omics-based systems biotechnology approach to elucidate the genotypic and phenotypic differences between a wild-type and recombinant antibody-producing Chinese hamster ovary (CHO) cell line. At the genomic level, we observed extensive rearrangements in specific targeted loci linked to transgene integration sites. Transcriptional re-wiring of DNA damage repair and cellular metabolism in the antibody producer, via changes in gene copy numbers, was also detected. Subsequent integration of transcriptomic data with a genome-scale metabolic model showed a substantial increase in energy metabolism in the antibody producer. Metabolomics, lipidomics, and glycomics analyses revealed an elevation in long-chain lipid species, potentially associated with protein transport and secretion requirements, and a surprising stability of N-glycosylation profiles between both cell lines. Overall, the proposed knowledge-based systems biotechnology framework can further accelerate mammalian cell-line engineering in a targeted manner.

Evaluation and use of disaccharides as energy source in protein-free mammalian cell cultures.

Mammalian cells are generally considered to be unable to utilize polysaccharides for cell growth because the phospholipid bilayer in the cell membrane has very low permeability to sugars. With the recent discovery of the only known animal disaccharide transporter, a sucrose transporter, we considered the potential use of polysaccharides as energy source, because that can impact biopharmaceutical manufacturing by potentially increasing carbohydrate loading in the culture medium and decreasing lactate accumulation. In this study, we found that mammalian cells can utilize maltose for growth in the absence of glucose and successfully adapted CHO-K1, CHO-DG44 and HEK293 cells to grow in glucose-free, maltose-containing serum-free protein-free media. We then cultivated a non-adapted CHO-K1 producer cell line in media containing both glucose and maltose to show that the cells can utilize maltose in a biphasic manner, that maltose enters the cells, and that maltose utilization only took place in the presence of the cells. This is the first report of a protein-free mammalian cell culture using a disaccharide as energy source.

Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy.

Amphotericin B (AMB) is a highly hydrophobic antifungal, whose use is limited by its toxicity and poor solubility. To improve its solubility, AMB was reacted with a functionalized polyethylene glycol (PEG), yielding soluble complex AmB-PEG formulations that theoretically comprise of chemically conjugated AMB-PEG and free AMB that is physically associated with the conjugate. Reverse-phase chromatography and size exclusion chromatography methods using HPLC were developed to separate conjugated AMB-PEG and free AmB, enabling the further characterization of these formulations. Using HPLC and dynamic light scattering analyses, it was observed that the AMB-PEG 2 formulation, having a higher molar ratio of 2 AMB: 1 PEG, possesses more free AMB and has relatively larger particle diameters compared to the AMB-PEG 1 formulation, that consists of 1 AMB: 1 PEG. The identity of the conjugate was also verified using mass spectrometry. AMB-PEG 2 demonstrates improved antifungal efficacy relative to AMB-PEG 1, without a concurrent increase in in vitro toxicity to mammalian cells, implying that the additional loading of free AMB in the AMB-PEG formulation can potentially increase its therapeutic index. Compared to unconjugated AMB, AMB-PEG formulations are less toxic to mammalian cells in vitro, even though their MIC50 values are comparatively higher in a variety of fungal strains tested. Our in vitro results suggest that AMB-PEG 2 formulations are two times less toxic than unconjugated AMB with antifungal efficacy on Candida albicans and Cryptococcus neoformans.

Engineering selection stringency on expression vector for the production of recombinant human alpha1-antitrypsin using Chinese Hamster ovary cells.

BACKGROUND: Expression vector engineering technology is one of the most convenient and timely method for cell line development to meet the rising demand of novel production cell line with high productivity. Destabilization of dihydrofolate reductase (dhfr) selection marker by addition of AU-rich elements and murine ornithine decarboxylase PEST region was previously shown to improve the specific productivities of recombinant human interferon gamma in CHO-DG44 cells. In this study, we evaluated novel combinations of engineered motifs for further selection marker attenuation to improve recombinant human alpha-1-antitrypsin (rhA1AT) production. Motifs tested include tandem PEST elements to promote protein degradation, internal ribosome entry site (IRES) mutations to impede translation initiation, and codon-deoptimized dhfr selection marker to reduce translation efficiency. RESULTS: After a 2-step methotrexate (MTX) amplification to 50 nM that took less than 3 months, the expression vector with IRES point mutation and dhfr-PEST gave a maximum titer of 1.05 g/l with the top producer cell pool. Further MTX amplification to 300 nM MTX gave a maximum titer of 1.15 g/l. Relative transcript copy numbers and dhfr protein expression in the cell pools were also analysed to demonstrate that the transcription of rhA1AT and dhfr genes were correlated due to the IRES linkage, and that the strategies of further attenuating dhfr protein expression with the use of a mutated IRES and tandem PEST, but not codon deoptimization, were effective in reducing dhfr protein levels in suspension serum free culture. CONCLUSIONS: Novel combinations of engineered motifs for further selection marker attenuation were studied to result in the highest reported recombinant protein titer to our knowledge in shake flask batch culture of stable mammalian cell pools at 1.15 g/l, highlighting applicability of expression vector optimization in generating high producing stable cells essential for recombinant protein therapeutics production. Our results also suggest that codon usage of the selection marker should be considered for applications that may involve gene amplification and serum free suspension culture, since the overall codon usage and thus the general expression and regulation of host cell proteins may be affected in the surviving cells.

Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed-batch bioreactors.

CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures.

DOK3 is required for IFN-ß production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

The downstream of kinase (DOK) family of adaptors is generally involved in the negative regulation of signaling pathways. DOK1, 2, and 3 were shown to attenuate TLR4 signaling by inhibiting Ras-ERK activation. In this study, we elucidated a novel role for DOK3 in IFN-ß production. Macrophages lacking DOK3 were impaired in IFN-ß synthesis upon influenza virus infection or polyinosinic-polyribocytidylic acid stimulation. In the absence of DOK3, the transcription factor IFN regulatory factor 3 was not phosphorylated and could not translocate to the nucleus to activate ifn-ß gene expression. Interestingly, polyinosinic-polyribocytidylic acid-induced formation of the upstream TNFR-associated factor (TRAF) 3/TANK-binding kinase (TBK) 1 complex was compromised in dok3(-/-) macrophages. DOK3 was shown to bind TBK1 and was required for its activation. Furthermore, we demonstrated that overexpression of DOK3 and TBK1 could significantly enhance ifn-ß promoter activity. DOK3 was also shown to bind TRAF3, and the binding of TRAF3 and TBK1 to DOK3 required the tyrosine-rich C-terminal domain of DOK3. We further revealed that DOK3 was phosphorylated by Bruton's tyrosine kinase. Hence, DOK3 plays a critical and positive role in TLR3 signaling by enabling TRAF3/TBK1 complex formation and facilitating TBK1 and IFN regulatory factor 3 activation and the induction of IFN-ß production.

Advances in Mammalian cell line development technologies for recombinant protein production.

From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA) annually. In addition, the expiration of blockbuster biologics has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX) amplification technology or the glutamine synthetase (GS) system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics.

Identification of autocrine growth factors secreted by CHO cells for applications in single-cell cloning media.

Chinese hamster ovary (CHO) cell lines are widely used for the expression of therapeutic recombinant proteins, including monoclonal antibodies and other biologics. For manufacturing, cells derived from a single-cell clone are typically used to ensure product consistency. Presently, fetal bovine serum (FBS) is commonly used to support low cell density cultures to obtain clonal cell populations because cells grow slowly, or even do not survive at low cell densities in protein-free media. However, regulatory authorities have discouraged the use of FBS to reduce the risk of contamination by adventitious agents from animal-derived components. In this study, we demonstrated how a complementary mass spectrometry-based shotgun proteomics strategy enabled the identification of autocrine growth factors in CHO cell-conditioned media, which has led to the development of a fully defined single-cell cloning media that is serum and animal component-free. Out of 290 secreted proteins that were identified, eight secreted growth factors were reported for the first time from CHO cell cultures. By supplementing a combination of these growth factors to protein-free basal media, single cell growth of CHO cells was improved with cloning efficiencies of up to 30%, a 2-fold improvement compared to unsupplemented basal media. Complementary effects of these autocrine growth factors with other paracrine growth factors were also demonstrated when the mixture improved cloning efficiency to 42%, similar to that for the conditioned medium.