Chinese Hamster Ovary Research Articles

Sodium-dependent glucose co-transport proteins (SGLTs) are not involved in human glucose taste detection

The sweet taste of saccharides, such as sucrose and glucose, and other sweeteners is known to result from activation of the TAS1R2/R3 receptor expressed in taste receptor cells (TRCs) of the taste bud. Recent reports have suggested the existence of an additional sweet taste signaling pathway for metabolizable saccharides that is dependent on the activity of glucose transporters, especially SGLT1, also expressed in TRCs. We have investigated the potential contribution of SGLT1 to glucose taste signaling in humans. Concentration-response analysis of glucose mediated changes in membrane potential measured in Chinese hamster ovary (CHO) cells transiently expressing the human SGLT1 (hSGLT1) yielded an EC50 value of 452 μM. The SGLT inhibitor phlorizin inhibited the membrane potential response to 10 mM glucose with an IC50 of 3.5 μM. In contrast, EC50 values of 127 and 132 mM were obtained from concentration-response analysis of glucose taste in vehicles of water or 20 mM NaCl, respectively, by rapid throughput taste discrimination with human subjects. Lactisole, an antagonist of TAS1R2/R3, at a concentration of 1 mM completely inhibited taste responses to glucose concentrations of 250 mM and below. Phlorizin (0.2 mM) and the high potency SGLT1-selective inhibitor mizagliflozin (10 μM) failed to inhibit glucose taste detection measured at peri-threshold concentrations in the rapid throughput taste discrimination assay. A Yes/No experiment using the taste discrimination assay revealed that 0.2 mM phlorizin was discriminable from water for some subjects. Taken together the results indicate that agonist activation of TAS1R2/R3 is sufficient to account for all glucose taste without contribution by an alternative SGLT-mediated signaling pathway. Furthermore, the taste of phlorizin could be a confounding variable for studies evaluating a role for SGLTs in taste.

Engineering Escherichia coli-Derived Nanoparticles for Vaccine Development

The development of effective vaccines necessitates a delicate balance between maximizing immunogenicity and minimizing safety concerns. Subunit vaccines, while generally considered safe, often fail to elicit robust and durable immune responses. Nanotechnology presents a promising approach to address this dilemma, enabling subunit antigens to mimic critical aspects of native pathogens, such as nanoscale dimensions, geometry, and highly repetitive antigen display. Various expression systems, including Escherichia coli (E. coli), yeast, baculovirus/insect cells, and Chinese hamster ovary (CHO) cells, have been explored for the production of nanoparticle vaccines. Among these, E. coli stands out due to its cost-effectiveness, scalability, rapid production cycle, and high yields. However, the E. coli manufacturing platform faces challenges related to its unfavorable redox environment for disulfide bond formation, lack of post-translational modifications, and difficulties in achieving proper protein folding. This review focuses on molecular and protein engineering strategies to enhance protein solubility in E. coli and facilitate the in vitro reassembly of virus-like particles (VLPs). We also discuss approaches for antigen display on nanocarrier surfaces and methods to stabilize these carriers. These bioengineering approaches, in combination with advanced nanocarrier design, hold significant potential for developing highly effective and affordable E. coli-derived nanovaccines, paving the way for improved protection against a wide range of infectious diseases.

Immuno-PET/CT Imaging of Trop2 with [18F]AlF-RESCA-T4 Differentiates Lung Cancer from Inflammation.

Immuno-PET/CT imaging, a branch of molecular imaging, can noninvasively and specifically visualize biomarker expression across the body. Trophoblast cell surface antigen 2 (Trop2) is a pan-cancer biomarker and plays a crucial role in tumorigenesis through multiple signaling pathways. The study aims to develop and translate novel Trop2 single-domain antibody (sdAb) tracers for clinical use. Methods: Two sdAbs (i.e., His-tagged T4 and His-tag-free RT4) are recombinantly expressed in Chinese hamster ovary cells. The purities and binding kinetics are determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, high-performance liquid chromatography, and surface plasmon resonance assays. The AlF restrained complexing agent (RESCA) method is applied to develop 18F-labeled sdAb tracers ([18F]AlF-RESCA-T4 and [18F]AlF-RESCA-RT4), followed by thorough preclinical imaging and blocking studies on tumor-bearing mice and a pilot clinical trial evaluating the clinical imaging safety and feasibility of [18F]AlF-RESCA-T4 immuno-PET/CT. Results: [18F]AlF-RESCA-T4 and [18F]AlF-RESCA-RT4 possess high radiochemical purities. Preclinical imaging in the T3M-4 tumor model revealed prominent uptake (percentage injected dose/g) of [18F]AlF-RESCA-T4 (11.13 ± 1.53, n = 4) and [18F]AlF-RESCA-RT4 (8.83 ± 1.22, n = 4), which were significantly reduced by coinjection of unlabeled T4 and RT4 in blocking studies. The His-tag removal strategy further optimized the probe's invivo pharmacokinetics and reduced renal radioactivity accumulation without significantly decreasing tumor uptake. In a pilot clinical trial, [18F]AlF-RESCA-T4 immuno-PET/CT showed promising potency in annotating Trop2 expression and differentiating tumors from inflammatory diseases such as tuberculosis. Conclusion: [18F]AlF-RESCA-T4 and [18F]AlF-RESCA-RT4 can specifically annotate Trop2 expression. Clinical [18F]AlF-RESCA-T4 immuno-PET/CT imaging can screen patients for Trop2-targeted therapies and differentiate lung inflammation from cancer.

Adaptation of Aglycosylated Monoclonal Antibodies for Improved Production in Komagataella phaffii.

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals manufactured by well-established processes using Chinese Hamster Ovary (CHO) cells. Next-generation biomanufacturing using alternative hosts like Komagataella phaffii could improve the accessibility of these medicines, address broad societal goals for sustainability, and offer financial advantages for accelerated development of new products. Antibodies produced by K. phaffii, however, may manifest unique molecular quality attributes, like host-dependent, product-related variants, that could raise potential concerns for clinical use. We demonstrate here conservative modifications to the amino acid sequence of aglycosylated antibodies based on the human IgG1 isotype that minimize product-related variations when secreted by K. phaffii. A combination of 2-3 changes of amino acids reduced variations across six different aglycosylated versions of commercial mAbs. Expression of a modified sequence of NIST mAb in both K. phaffii and CHO cells showed comparable biophysical properties and molecular variations. These results suggest a path toward the production of high-quality mAbs that could be expressed interchangeably by either yeast or mammalian cells. Improving molecular designs of proteins to enable a range of manufacturing strategies for well-characterized biopharmaceuticals could accelerate global accessibility and innovations.

Ustekinumab Biosimilars

Ustekinumab is a fully human IgG1k monoclonal antibody that binds with high affinity and specificity to the p40 subunit of interleukins (IL-) 12 and 23, inhibiting their activity by preventing binding to their receptors. The European extension of the patent (Supplementary Protection Certificate) of ustekinumab expired on 20 July 2024. Biosimilar alternatives to ustekinumab are now an additional option for treating patients. The efficacy data for this drug in moderate-to-severe psoriasis obtained both from clinical trials and indirect comparisons through meta-analyses, are superior to those of etanercept and adalimumab, and its safety profile is more favorable than that of tumor necrosis factor (TNF) inhibitors. Several ustekinumab biosimilars have already been approved by regulatory agencies: between October 2023 and October 2024, Wezlana® (Amgen ABP 654), Uzpruvo® (Alvotech AVT04) and Pyzchiva® (Samsung/Bioepis SB17) have been approved by both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). SteQeyma® (Celltrion Healthcare CT-P43) was approved by the EMA in August 2024. Otulfi® (Fresenius Kabi/Formycon) was approved by the FDA in October 2024. Several other potential biosimilar candidates are under development, including BAT2206 (Bio-Thera), DMB-3115 (Dong-A ST), QX001S (Qyuns Therapeutic), BFI-751 (BioFactura), NeuLara (Neuclone), ONS3040 (Oncobiologics), and BOW090 (Epirus Biopharmaceuticals). In most cases, these monoclonal antibodies are expressed in cell lines (e.g., Chinese Hamster Ovary, CHO) different from those used for the originator (Sp2/0 spleen cell murine myeloma); of note, the cell line of origin is not a requirement for biosimilarity in the totality-of-evidence comparison exercise and may facilitate the production and reduce the immunogenicity of biosimilars originated in CHO cultures. This narrative review summarizes the available data on characteristics of the full comparability exercises and comparative clinical trials of these drugs.

Experimental determination of maximum shear stress in Mobius® Breez perfusion microbioreactors and comparative analysis with stirred tank bioreactors

Perfusion processes have experienced increased popularity in recent years due to their ability to sustain high cell densities and productivities in biopharmaceutical production, offering advantages over traditional batch and fed-batch cultivation methods. The Mobius® Breez microbioreactor significantly reduces experimental effort by downsizing the classical volume of perfusion bioreactors to the mL range and thus represents a valuable tool for process development. However, miniaturization has raised questions regarding comparability with traditional bioreactors in terms of the physical environment, such as hydrodynamic shear stress. Therefore, the maximum hydrodynamic shear stress, cultivation performance, and membrane-wall contact were evaluated to elucidate the system's behavior. Findings reveal two distinct operational conditions, distinguished by the presence or absence of membrane-wall contact, resulting in varying levels of hydrodynamic stress. Conditions lacking membrane contact demonstrate stress levels within safe operating thresholds for CHO cells, while those involving membrane contact exceed these thresholds, potentially leading to cell damage. Through the identification of critical frequencies of membrane motion, this study offers insights for optimizing microbioreactor operation and enhancing overall bioprocess efficiency.

Investigating the effect of linker peptides on the fragmentation of Fc-fusion proteins in transient gene expression of mammalian cells

Protein fragmentation is a critical quality attribute for Fc-fusion protein production in mammalian cells. In the production of viral non-structural proteins as the form of Fc-fusion protein, fragmentation of Fc-fusion proteins occurred in two transient gene expression (TGE) systems with human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. The introduction of a flexible empirical linker reduced fragmentation in HEK293 cells, but not in CHO cells. Additionally, two rigid empirical linkers failed to restore impaired Fc-fusion proteins in CHO cells. In vitro incubation assay using conditioned culture medium and cultures supplemented with protease inhibitor cocktail suggest that fragmentation of Fc-fusion proteins in CHO cells may be due to various host cell proteins released into the culture medium. These findings suggest that the introduction of linker peptides can improve the fragmentation of Fc-fusion proteins in mammalian cells, but exhibit different fragment patterns depending on the cell type.

Genome-Scale Modeling of CHO Cells Unravel the Critical Role of Asparagine in Cell Culture Feed Media.

Amino acids, including asparagine, aspartate, glutamine, and glutamate, play important roles in purine and pyrimidine biosynthesis as well as serve as anaplerotic sources fueling the tricarboxylic acid (TCA) cycle for mitochondrial energy generation. Despite extensive studies on glutamine and glutamate in CHO cell cultures, the roles of asparagine and aspartate, especially in feed media, remain underexplored. In this study, we utilized a CHO genome scale model to first deeply characterize the intracellular metabolic states of CHO cells cultured in different combinations of basal and feed media to understand the traits of asparagine/aspartate-dependent and glutamate-dependent feeds. Subsequently, we identified the critical role of asparagine and aspartate in the feed media as anaplerotic sources and conducted in silico simulations to ascertain their optimal ratios to improve cell culture performance. Finally, based on the model simulations, we reformulated the feed media by tailoring the concentrations of asparagine and aspartate. Our experimental data reveal a CHO cell preference for asparagine compared with aspartate, and thus maintaining an optimal ratio of these amino acids is a key factor for achieving optimal CHO cell culture performance in biopharmaceutical production.

Application of bacterial-derived long cellulose nanofiber to suspension culture of mammalian cells as a shear protectant

Nanofibrillated bacterial cellulose (NFBC) is a bio-compatible long-fiber nanocellulose produced by cellulose-synthesizing bacteria. It forms an entangled network structure in the suspension state, thereby imparting greater viscosity than conventional media additives. In this study, we examined its application as a shear protectant in the suspension culture of mammalian cells to mitigate hydrodynamic stress imposed on the cells. The media supplemented with hydroxypropyl cellulose-adsorbed NFBC (HP-NFBC) exhibited an increase in shear viscosity according to rheometric analysis, similar to FP003, a commercially available medium additive. Suspension culture of Chinese hamster ovary cells in HP-NFBC-containing media under a high stirring rate (120 rpm) demonstrated higher cell growth and lower cell death compared to those in the medium without additives and in FP003. A 0.10 (w/v)% concentration of HP-NFBC showed the highest viable cell number among the tested concentrations. Computational fluid dynamics simulation revealed a decrease in shear rate and flow velocity within the spinner flask owing to the addition of HP-NFBC or FP003. It is suggested that the decline of these parameters in high-viscosity media suppresses the hydrodynamic stress on cells. This study highlights the potential of HP-NFBC as a shear protectant in mammalian cell suspension culture.

ARMC5 selectively degrades SCAP-free SREBF1 and is essential for fatty acid desaturation in adipocytes

SREBF1 plays the central role in lipid metabolism. It has been known that full-length SREBF1 that did not associate with SCAP (SCAP-free SREBF1) is actively degraded, but its molecular mechanism and its biological meaning remain unclear. ARMC5-CUL3 complex was recently identified as E3 ubiquitin ligase of full-length SREBF. Although ARMC5 was involved in SREBF pathway in adrenocortical cells, the role of ARMC5 in adipocytes has not been investigated. In this study, adipocyte-specific Armc5 knockout mice were generated. In the white adipose tissue (WAT) of these mice, all the stearoyl-CoA desaturase (Scd) were drastically downregulated. Consistently, unsaturated fatty acids were decreased and saturated fatty acids were increased. The protein amount of full-length SREBF1 were increased, but ATAC-Seq peaks at the SREBF1-binding sites were markedly diminished around the Scd1 locus in the WAT of Armc5 knockout mice. Armc5-deficient 3T3-L1 adipocytes also exhibited downregulation of Scd. Mechanistically, disruption of Armc5 restored decreased full-length SREBF1 in CHO cells deficient for Scap. Overexpression of Scap inhibited ARMC5-mediated degradation of full-length SREBF1, and overexpression of Armc5 increased nuclear SREBF1/full-length SREBF1 ratio and SREBF1 transcriptional activity in the presence of exogenous SCAP. These results demonstrated that ARMC5 selectively removes SCAP-free SREBF1 and stimulates SCAP-mediated SREBF1 processing, hence is essential for fatty acid desaturation in vivo.

New prospects for 89Zr-immuno-PET in brain applications – Alpha-synucleinopathies

BackgroundRecently, 89Zr-immuno-PET imaging of therapeutic antibodies, actively transported over the blood-brain-barrier via transferrin-mediated transcytosis, was demonstrated using the chelator DFO*. In these studies, aducanumab targeting amyloid-beta was fused with a transferrin binding unit: a single-chain Fab fragment derived from 8D3 (scFab8D3). Alpha-synuclein is a hallmark protein of several neurodegenerative diseases such as Parkinson's Disease, Lewy-Body-Dementia, and Multiple System Atrophy. 89Zr-immuno-PET imaging of alpha-synuclein can be a valuable tool for image-guided drug development and assessment of target engagement. The goal of this study was to compare two currently used constructs of 8D3 for targeting potential, namely a single moiety of scFab8D3 fused to the alpha-synuclein antibody HLu-3 (HLu-3-scFab8D3) versus HLu-3 fused with two 8D3 single-chain variable fragments (HLu-3-(scFv8D3)2), by 89Zr-immuno-PET in an alpha-synuclein pre-formed fibril (PFF) deposition model. HLu-3 and the HIV-targeting B12-scFab8D3 were used as controls. The best-performing compound was further investigated in an animal model with predominantly intraneural target aggregation. MethodsAntibodies were conjugated with DFO* using DFO*-NCS and subsequently radiolabeled with 89Zr. Assessment of binding affinity was done by alpha-synuclein ELISA and with FACS analysis using mTfR1 expressing CHO-S cells. Radioimmunoconjugates were first evaluated in an extracellular alpha-synuclein deposition model established by intracranial injection of non-sonicated PFFs into the left striatum of C57Bl/6 WT mice, whereas saline was injected into the contralateral side as control. PET imaging was performed 1, 3, and 7 days post-injection, followed by ex vivo biodistribution, autoradiography and immunofluorescence analysis. Based on the results from these studies, the better-performing antibody candidate was tested similarly in an alpha-synuclein seeding model. The seeding model has intraneural alpha-synuclein aggregation and was established by intracranial injection of sonicated PFFs into both striata of F28tg mice, which overexpress human wild-type alpha-synuclein. Untreated F28tg and C57Bl/6 WT mice served as controls. ResultsThe radioconjugate was produced in sufficient radiochemical yields and purity. There was no impairment of binding affinity towards alpha-synuclein, and acceptable binding with negligible losses to mTfR1. PET imaging with [89Zr]Zr-HLu-3-scFab8D3 and [89Zr]Zr-HLu-3-(scFv8D3)2 in the deposition model showed uptake at the site of alpha-synuclein deposits. However, uptake was variable between mice. Reliable PET quantification was hampered due to the small deposition volume (~2 μL). Immunofluorescence revealed specific alpha-synuclein target engagement of both constructs with PFF deposits in the striatum, in contrast to the [89Zr]Zr-B12-scFab8D3 control. Unexpectedly, ex vivo autoradiography showed uptake in some controls ([89Zr]Zr-B12-scFab8D3 in the contralateral striatum without PFFs), potentially related to astrocyte activation at the injection sides. Ex vivo and PET brain uptake was higher for [89Zr]Zr-HLu-3-scFab8D3 when compared to [89Zr]Zr-HLu-3-(scFv8D3)2 and was therefore selected for further testing in the alpha-synuclein seeding model. No significant difference in in vivo and ex vivo brain uptake of [89Zr]Zr-HLu-3-scFab8D3 between PFF-injected F28tg, F28tg and C57Bl/6 mice was observed. Furthermore, ex vivo immunofluorescence and autoradiography showed no specific alpha-synuclein target engagement. ConclusionsSuccessful target engagement of [89Zr]Zr-HLu-3-scFab8D3 and [89Zr]Zr-HLu-3-(scFv8D3)2 with alpha-synuclein was shown in a PFF deposition model. PET imaging showed variable results, and in vivo detection of the depositions was possible in some cases. Due to the better performance in the deposition model, [89Zr]Zr-HLu-3-scFab8D3 was further investigated in an alpha-synuclein seeding model with intraneural Lewy-body pathology, showing no difference between the control groups and PFF-seeded mice. Furthermore, immunostaining of seeded F28tg mice manifested sufficient intraneural alpha-synuclein pathology but no corresponding antibody accumulation. These results underscore the ongoing challenge of imaging intraneural inclusions via immuno-PET.

Analysis of population heterogeneity in CHO cells by genome-wide DNA methylation analysis and by multi-modal single-cell sequencing

CHO cells are major hosts for the industrial production of therapeutic proteins and their production stability is of considerable economic significance. It is widely known that CHO cells can rapidly acquire genetic alterations, which affects their genetic homogeneity over time. However, the role of non-genetic mechanisms, including epigenetic mechanisms such as DNA methylation, remains poorly understood. We have now used whole-genome bisulfite sequencing to establish single-base methylation maps of eight independent CHO cell lines. Our results identify CpG islands and low-methylated regions as conserved elements with dynamic DNA methylation. Interestingly, methylation patterns were found to cluster clearly along the three main branches of CHO evolution, with no directional changes over short culture periods. Furthermore, multi-ome single-cell sequencing of 9833 nuclei from three independent cultures revealed dynamic subpopulation structures characterized by robust expression differences in pathways related to protein production. Our findings thus provide novel insights into the epigenetic landscape and heterogeneity of CHO cells and support the development of epigenetic biomarkers that trace the emergence of subpopulations in CHO cultures.

Glycocalyx Interactions Modulate the Cellular Uptake of Albumin-Coated Nanoparticles.

Albumin-based nanoparticles (ABNPs) represent promising drug carriers in nanomedicine due to their versatility and biocompatibility, but optimizing their effectiveness in drug delivery requires understanding their interactions with and uptake by cells. Notably, albumin interacts with the cellular glycocalyx, a phenomenon particularly studied in endothelial cells. This observation suggests that the glycocalyx could modulate ABNP uptake and therapeutic efficacy, although this possibility remains unrecognized. In this study, we elucidate the critical role of the glycocalyx in the cellular uptake of a model ABNP system consisting of silica nanoparticles (NPs) coated with native, cationic, and anionic albumin variants (BSA, BSA+, and BSA-). Using various methodologies-including fluorescence anisotropy, dynamic light scattering, microscale thermophoresis, surface plasmon resonance spectroscopy, and computer simulations─we found that both BSA and BSA+, but not BSA-, interact with heparin, a model glycosaminoglycan (GAG). To explore the influence of albumin-GAG interactions on NP uptake, we performed comparative uptake studies in wild-type and GAG-mutated Chinese hamster ovary cells (CHO), along with complementary approaches such as enzymatic GAG cleavage in wild-type cells, chemical inhibition, and competition assays with exogenous heparin. We found that the glycocalyx enhances the cell uptake of NPs coated with BSA and BSA+, while serving as a barrier to the uptake of NPs coated with BSA-. Furthermore, we showed that harnessing albumin-GAG interactions increases cancer cell death induced by paclitaxel-loaded albumin-coated NPs. These findings underscore the importance of albumin-glycocalyx interactions in the rational design and optimization of albumin-based drug delivery systems.

Procollagen-lysine 2-oxoglutarate 5-dioxygenases are responsible for 5R-hydroxylysine modification of therapeutic T-cell bispecific monoclonal antibodies produced by Chinese hamster ovary cells.

We present a detailed mass spectrometric analysis of three 2 + 1 T-cell bispecific monoclonal antibodies (TCB mAbs), where an unexpected +15.9950Da mass shift in tryptic peptides was observed. This modification was attributed to the occurrence of 5R-hydroxylysine (Hyl) using a hybrid LC-MS/MS molecular characterization and CRISPR/Cas9 gene deletion approach. The modification was found at various sites within TCB mAbs, with a conspicuous hot spot motif mirroring a prior observation where Hyl was mapped to the CH1-VH Fab domain interface of IgGs. In contrast to the preceding report, our structural modeling analysis on TCB mAbs unveiled substantial differences in the orientation and flexibility of motifs in immediate proximity and across the artificial CH1-VL cross Fab interface and upstream elbow segment. Utilizing a hybrid database search, RNAseq, and a CRISPR/Cas9 knockout methodology in Chinese hamster ovary (CHO) production cell lines, procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) were conclusively identified as the catalyzing enzymes accountable for the 5R-Hyl modification in TCB mAbs. To quantitatively inhibit Hyl formation in TCB mAbs, the activity of all three Chinese hamster PLOD isoenzymes needs to be depleted via CRISPR/Cas9 gene knockout. Moreover, our investigation identified cell culture iron availability, process duration, and clonal variability in CHO cells as elements influencing the levels of Hyl formation in TCB mAbs. This research offers a solution for circumventing Hyl formation in therapeutic complex mAb formats, such as TCB mAbs, produced in CHO cell culture processes, thereby addressing potential technical and biological challenges associated with unintended Hyl modification.

MiRNA Chaining for Efficient Stable Overexpression to Improve Protein Quantity and Quality in CHO Cells.

MicroRNAs (miRNAs), small noncoding RNAs with a length of about 22 nucleotides, harbor the potential to be powerful tools for the genetic engineering of production cell lines like Chinese hamster ovary (CHO) cells. Their ability to regulate multiple targets at once and their potential to fine-tune effect strengths contrast withclassical engineering approaches. However, most studies of miRNAs rely on transiently flooding the cells with miRNA mimics. Since this approach is not suitable for long-term cultivation in a bioprocess, stable overexpression of miRNAs becomes more and more important for the biotech industry. Here, the user might be confronted with insufficient overexpression of the miRNA of interest. In this chapter, we present a method for the generation of stable CHO cell lines expressing a miRNA from a plasmid-based system containing multiple copies of the miRNA, allowing tuning of overexpression and regulation.

The 'Omics Revolution in CHO Biology: Roadmap to Improved CHO Productivity.

Chinese hamster ovary (CHO) cell physiology understanding has advanced very rapidly in the past few years with incredible improvements in long-read sequencing, improved resolution, and increased computational power. Multiple parental lines have been sequenced and the resultant pan-genome can be leveraged to increase our understanding of the diverse pathways CHO cells can take to get high-productivity phenotypes. The same improvements in workflows have complemented transcriptomic studies. Microfluidics and label-free innovations have further increased the sensitivity and accuracy of proteomic methods, while also making proteomics more accessible. In this 'omics era, high-throughput screening methods, sophisticated informatic tools, and models continually drive major innovations in cell line development and process engineering. This review describes the various recent achievements in 'omics techniques and their application to improve recombinant protein expression from CHO cell lines.

Recent Advancements in Proteomic Sample Preparation from Recombinant Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell line for biopharmaceutical production because of their ability to correctly fold and post-translationally modify recombinant proteins that are compatible with human use. Proteomics, along with other "omic platforms," are being used to understand the biology of CHO cells with the ultimate aim to enhance CHO cell factories for more efficient production of biopharmaceuticals. Liquid chromatography-mass spectrometry (LC-MS) for proteomic analysis has become the standard technique for profiling proteomes. There are three stages to a typical bottom-up approach, namely, sample preparation, LC-MS/MS, and data analysis. Although there have been major advances in LC-MS/MS instrumentation and data analysis tools, sample preparation is still the crucial stage that affects the overall efficiency of any proteomic study. In this chapter, we present a comparison of a number of widely used sample preparation methods for proteomic applications, including in-solution digestion and commercially available device-based kits. All methods performed well; however, each method has inherent advantages and disadvantages.

Separation and Purification of CHO Secretome and Extracellular Vesicles for Proteome Analysis.

For decades, host cell proteins (HCPs) have been investigated as putative contaminants in downstream processing of biopharmaceutical products of Chinese hamster ovary (CHO) cells. However, little is still known about the composition of the entire protein and vesicle environment in CHO cultivations. Ever evolving mass spectrometry techniques allow more and more insights into cell-cell communication processes and the composition of extracellular matrix, proteases, and further actively segregated compounds such as extracellular vesicles (EVs). EVs themselves are a heterologous group consisting of exosomes, ectosomes, and apoptotic vesicles. To specifically analyze these subsets of the secretome and determine beneficial and detrimental factors for a production process, targeted separation and purification techniques are necessary.In this chapter, we present our optimized workflows for a clear differentiation between directly secreted proteins and the vesicular protein content of different fractions (especially exosomal small EVs) from CHO cell supernatant for proteomic analysis by NanoLC ESI-MS.

Serum-Free and Protein-Free Media for the Cultivation of Recombinant Chinese Hamster Ovary (CHO) Cell Lines.

We describe the preparation of two cell culture media formulations for the culture in suspension of Chinese hamster ovary (CHO) cell lines. The first medium, Cell growth SFM Medium, is a serum-free medium designed to maintain cell growth with high-viability profiles. The second corresponds to a protein-free version optimized to increase CHO recombinant protein production (Production PFM Medium). Considerations on the use of these formulations for other cell culture assays (such as transfections) are also described.

Phosphopeptide Enrichment and LC-MS/MS Analysis to Study the Phosphoproteome of Recombinant Chinese Hamster Ovary Cells.

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is one of the most important post-translational modifications that regulates many biological processes. There have been relatively few studies on the phosphoproteome of recombinant Chinese hamster ovary (CHO) cells to date despite phosphorylation playing a crucial role in regulating many molecular and cellular processes relevant to bioprocess phenotypes including, for example, transcription, translation, growth, apoptosis, and signal transduction. In this chapter, we provide a protocol for phosphoproteomic analysis of CHO cells using phosphopeptide enrichment with metal oxide affinity chromatography (MOAC) and immobilized metal affinity chromatography (IMAC) techniques, followed by site-specific identification of phosphorylated residues using liquid chromatography mass spectrometry (LC-MS), multistage activation (MSA), and MS3 strategies. This protocol can also be used for quantitative phosphoproteomic analysis using both labeled and label-free approaches.