Mapping the molecular basis for growth related phenotypes in industrial producer CHO cell lines using differential proteomic analysis.

BACKGROUND: The ability to achieve high peak viable cell density earlier in CHO cell culture and maintain an extended cell viability throughout the production process is highly desirable to increase recombinant protein yields, reduce host cell impurities for downstream processing and reduce the cost of goods. In this study we implemented label-free LC-MS/MS proteomic profiling of IgG4 producing CHO cell lines throughout the duration of the cell culture to identify differentially expressed (DE) proteins and intracellular pathways associated with the high peak viable cell density (VCD) and extended culture VCD phenotypes. RESULTS: We identified key pathways in DNA replication, mitotic cell cycle and evasion of p53 mediated apoptosis in high peak VCD clonally derived cell lines (CDCLs). ER to Golgi vesicle mediated transport was found to be highly expressed in extended culture VCD CDCLs while networks involving endocytosis and oxidative stress response were significantly downregulated. CONCLUSION: This investigation highlights key pathways for targeted engineering to generate desirable CHO cell phenotypes for biotherapeutic production.

Investigating the influence of physiologically relevant hydrostatic pressure on CHO cell batch culture.

Chinese hamster ovary (CHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins, such as monoclonal antibodies. Enhancement of productivity of these CHO cells is one of the top priorities in the biopharmaceutical industry to reduce manufacturing cost. Although there are many different methods (e.g. temperature, pH, feed) to improve protein production in CHO cells, the role of physiologically relevant hydrostatic pressure in CHO cell culture has not been reported yet. In this study, four different hydrostatic pressures (0, 30, 60, and 90 mmHg) were applied to batch CHO cells, and their cell growth/metabolism and IgG1 production were examined. Our results indicate that hydrostatic pressure can increase the maximum cell concentration by up to 50%. Moreover, overall IgG1 concentration on Day 5 showed that 30 mmHg pressure can increase IgG1 production by 26%. The percentage of non-disulphide-linked antibody aggregates had no significant change under pressure. Besides, no significant difference was observed between 30 mmHg and no pressure conditions in terms of cell clumping formation. All these findings are important for the optimization of fed-batch or perfusion culture for directing cell growth and improving antibody production.

Improving Chinese hamster ovary host cell protein ELISA using Ella®: an automated microfluidic platform.

Chinese hamster ovary (CHO) cells are a mammalian cell line used in the production of therapeutic proteins. Host cell proteins (HCPs) are process-related impurities that are derived from the host cell expression system. During biopharmaceutical drug development, removal of HCPs is required. Enzyme-linked immunosorbent assay (ELISA) is a common technique to quantitate HCPs, but is a labor-intensive process that takes up to 7 h. Ella® is an automated instrument that utilizes microfluidics and glass nanoreactors to quantitate HCPs in 75 min using similar ELISA reagents. The antibodies and antigens are captured on three distinct glass nanoreactors, resulting in sensitive reproducible data. Our results indicate that Ella quantitates CHO HCPs with precision, accuracy, sensitivity and trends comparable with our traditional CHO HCP ELISA.

A cell-based assay for the detection of neutralizing antibodies against alemtuzumab.

Aim: The humanized anti-CD52 monoclonal antibody alemtuzumab depletes lymphocytes and is currently used to treat relapsing multiple sclerosis. During treatment, anti-alemtuzumab antibodies may develop and reduce effective lymphocyte depletion in future treatment cycles. Results: Alemtuzumab-Alexa Fluor 488 conjugate binding to the CHO-CD52 cell surface was inhibited by anti-alemtuzumab antibodies. Conclusion: In this proof-of-concept study, a CHO-CD52 cell line has been developed and used to detect the presence of anti-alemtuzumab neutralizing antibodies. This platform provides the basis of an assay for routine screening of serum for neutralizing antibodies from patients treated with alemtuzumab.

Analysis of chemometric models applied to Raman spectroscopy for monitoring key metabolites of cell culture.

The Food and Drug Administration (FDA) initiative of Process Analytical Technology (PAT) encourages the monitoring of biopharmaceutical manufacturing processes by innovative solutions. Raman spectroscopy and the chemometric modeling tool partial least squares (PLS) have been applied to this aim for monitoring cell culture process variables. This study compares the chemometric modeling methods of Support Vector Machine radial (SVMr), Random Forests (RF), and Cubist to the commonly used linear PLS model for predicting cell culture components-glucose, lactate, and ammonia. This research is performed to assess whether the use of PLS as standard practice is justified for chemometric modeling of Raman spectroscopy and cell culture data. Model development data from five small-scale bioreactors (2 × 1 L and 3 × 5 L) using two Chinese hamster ovary (CHO) cell lines were used to predict against a manufacturing scale bioreactor (2,000 L). Analysis demonstrated that Cubist predictive models were better for average performance over PLS, SVMr, and RF for glucose, lactate, and ammonia. The root mean square error of prediction (RMSEP) of Cubist modeling was acceptable for the process concentration ranges of glucose (1.437 mM), lactate (2.0 mM), and ammonia (0.819 mM). Interpretation of variable importance (VI) results theorizes the potential advantages of Cubist modeling in avoiding interference of Raman spectral peaks. Predictors/Raman wavenumbers (cm-1 ) of interest for individual variables are X1139-X1141 for glucose, X846-X849 for lactate, and X2941-X2943 for ammonia. These results demonstrate that other beneficial chemometric models are available for use in monitoring cell culture with Raman spectroscopy.

Application of Raman spectroscopy in monoclonal antibody producing continuous systems for downstream process intensification.

Monoclonal antibodies (mAbs) are biopharmaceuticals produced by mammalian cell lines in bioreactors at a variety of scales. Cell engineering, media optimization, process monitoring, and control strategies for in vitro production have become crucial subjects to meet increasing demand for these high value pharmaceuticals. Raman Spectroscopy has gained great attention in the pharmaceutical industry for process monitoring and control to maintain quality assurance. For the first time, this article demonstrated the possibility of subclass independent quantitative mAb prediction by Raman spectroscopy in real time. The developed model estimated the concentrations of different mAb isotypes with average prediction errors of 0.2 (g/L) over the course of cell culture. In situ Raman spectroscopy combined with chemometric methods showed to be a useful predictive tool for monitoring of real time mAb concentrations in a permeate stream without sample removal. Raman spectroscopy can, therefore, be considered as a reliable process analytical technology tool for process monitor, control, and intensification of downstream continuous manufacturing. The presented results provide useful information for pharmaceutical industries to choose the most appropriate spectroscopic technology for their continuous processes.

Semi-continuous scale-down models for clone and operating parameter screening in perfusion bioreactors.

Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCDmax (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPRmin (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.

Mass spectrometric evaluation of upstream and downstream process influences on host cell protein patterns in biopharmaceutical products.

For production of different monoclonal antibodies (mAbs), biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are typically characterized regarding influence of upstream and downstream process (DSP) parameters on critical quality attributes (CQAs). CQAs must be monitored strictly by an adequate control strategy. One such process-related CQA is the content of host cell protein (HCP) which is typically analyzed by immunoassay methods (e.g., HCP-ELISA). The capacity of the immunoassay to detect a broad range of HCPs, relevant for the individual mAb-production process should be proven by orthogonal proteomic methods such as 2D gel electrophoresis or mass spectrometry (MS). In particular MS has become a valuable tool to identify and quantify HCP in complex mixtures. We evaluate up- and DSP parameters of four different biopharmaceutical products, two different process variants, and one mock fermentation on the HCP pattern by shotgun MS analysis and ELISA. We obtained a similar HCP pattern in different cell culture fluid harvests compared to the starting material from the downstream process. During the downstream purification process of the mAbs, the HCP level and the number of HCP species significantly decreased, accompanied by an increase in diversity of the residual HCP pattern. Based on this knowledge, we suggest a control strategy that combines multi product ELISA for in-process control and release analytics, and MS testing for orthogonal HCP characterization, to attain knowledge on the HCP level, clusters and species. This combination supports a control strategy for HCPs addressing safety and efficacy of biopharmaceutical products.

Pyruvate Kinase Muscle-1 Expression Appears to Drive Lactogenic Behavior in CHO Cell Lines, Triggering Lower Viability and Productivity: A Case Study.

Chinese hamster ovary (CHO) cell lines are used to express a variety of therapeutic proteins. However, lactogenic behavior displayed by some CHO cell lines during manufacturing processes may result in a decline in viability, productivity, and possible alterations in product quality. In cultured cells, lactate is produced during glycolysis through irreversible conversion of phosphoenolpyruvate to pyruvate and then lactate via sequential function of pyruvate kinase and lactate dehydrogenase (LDH) enzymes. In the process of cell line development (CLD), two lactogenic cell lines expressing different antibody molecules are identified. The lactogenic behaviors of these cell lines can be differentially mitigated through optimization of either nutrient feeds or culture pH, depending on the cell line. Analysis of various proteins involved in the glycolysis pathway reveal a direct correlation between the pyruvate kinase muscle-1 (PKM-1) isoform levels and lactogenic behavior. CRISPR mediated knockout of the PKM-1 isoform abolishes lactate accumulation even under lactogenic conditions. Furthermore, a cell line lacking expression of both PKM-1 and PKM-2 enzymes capable of maintaining productivity, viability, and growth without displaying lactogenic behavior is identified. Targeted deletion of PKM in CHO cells may be tolerated due to expression of PKL (liver) and PKR (red blood cell) isoforms of pyruvate kinase. All together, these findings suggest that PKM-1 up-regulation during antibody production could trigger lactogenic behavior and that this enzyme is dispensable for CHO cell survival.

Levels of ADAM10 are reduced in Alzheimer's disease CSF.

BACKGROUND: The disintegrin metalloproteinase 10 (ADAM10) is the main α-secretase acting in the non-amyloidogenic processing of the amyloid precursor protein. This study assesses whether ADAM10 is present in cerebrospinal fluid (CSF), and whether it has potential as a biomarker for Alzheimer's disease (AD). METHODS: ADAM10 was characterized in human CSF samples by immunoprecipitation and western blotting using antibodies specific for different domains of the protein and by ultracentrifugation in sucrose density gradients. Samples from AD patients (n = 20) and age-matched non-AD controls (n = 20) were characterized for classical CSF biomarkers, Aß42, T-tau, or P-tau by ELISA, and assayed for soluble ADAM10 levels by western blotting. RESULTS: We found that ADAM10 is present in human CSF as several distinct species: an immature form retaining the prodomain (proADAM10; ~ 80 kDa), a mature unprocessed full-length form (ADAM10f; ~ 55 kDa), and a truncated large soluble form released from the membrane (sADAM10; ~ 50 kDa). Fractionation by ultracentrifugation on sucrose density gradients showed that the ADAM10f and sADAM10 species form large complexes. Immunoblotting revealed a significant decrease in ADAM10f and sADAM10 in AD CSF compared to control CSF, while proADAM10 levels remained unaltered. CONCLUSIONS: Several forms of ADAM10 are present in CSF, mainly assembled as high-molecular weight complexes. The determination of the levels of mature forms of CSF-ADAM10 may be useful as a biomarker for AD.

Inflammatory Cytokine, IL-1ß, Regulates Glial Glutamate Transporter via microRNA-181a in vitro.

Glutamate overload triggers synaptic and neuronal loss that potentially contributes to neurodegenerative diseases including Alzheimer's disease (AD). Glutamate clearance and regulation at synaptic clefts is primarily mediated by glial glutamate transporter 1 (GLT-1). We determined that inflammatory cytokines significantly upregulated GLT-1 through microRNA-181a-mediated post-transcriptional modifications. Unveiling the key underlying mechanisms modulating GLT-1 helps better understand its physiological and pathological interactions with cytokines. Primary murine astrocyte and neuron co-culture received 20 ng/mL IL-1ß, TNF-α, or IL-6 for 48 h. Soluble proteins or total RNA were extracted after treatment for further analyses. Treatment with inflammatory cytokines, IL-1ß and TNF-α, but not IL-6, significantly increased GLT-1 steady-state levels (p≤0.05) without affecting mRNA levels, suggesting the cytokine-induced GLT-1 was regulated through post-transcriptional modifications. Among the candidate microRNAs predicted to modulate GLT-1, only microRNA-181a was significantly decreased following the IL-1ß treatment (p≤0.05). Co-treatment of microRNA-181a mimic in IL-1ß-treated primary astrocytes and neurons effectively blocked the IL-1ß-induced upregulation of GLT-1. Lastly, we attempted to determine the link between GLT-1 and microRNA-181a in human AD brains. A significant reduction of GLT-1 was found in AD hippocampus tissues, and the ratio of mature microRNA-181a over primary microRNA-181a had an increasing tendency in AD. MicroRNA-181a controls rapid modifications of GLT-1 levels in astrocytes. Cytokine-induced inhibition of microRNA-181a and subsequent upregulation of GLT-1 may have physiological implications in synaptic plasticity while aberrant maturation of microRNA-181a may be involved in pathological consequences in AD.

Proteomics in biomanufacturing control: Protein dynamics of CHO-K1 cells and conditioned media during apoptosis and necrosis.

Cell viability has a critical impact on product quantity and quality during the biomanufacturing of therapeutic proteins. An advanced understanding of changes in the cellular and conditioned media proteomes upon cell stress and death is therefore needed for improved bioprocess control. Here, a high pH/low pH reversed phase data independent 2D-LC-MSE discovery proteomics platform was applied to study the cellular and conditioned media proteomes of CHO-K1 apoptosis and necrosis models where cell death was induced by staurosporine exposure or aeration shear in a benchtop bioreactor, respectively. Functional classification of gene ontology terms related to molecular functions, biological processes, and cellular components revealed both cell death independent and specific features. In addition, label free quantitation using the Hi3 approach resulted in a comprehensive shortlist of 23 potential cell viability marker proteins with highest abundance and a significant increase in the conditioned media upon induction of cell death, including proteins related to cellular stress response, signal mediation, cytoskeletal organization, cell differentiation, cell interaction as well as metabolic and proteolytic enzymes which are interesting candidates for translating into targeted analysis platforms for monitoring bioprocessing response and increasing process control.

Effect of propeptide amino acid substitution in γ-carboxylation, activity and expression of recombinant human coagulation factor IX.

The production of recombinant vitamin K dependent (VKD) proteins for therapeutic purposes is an important challenge in the pharmaceutical industry. These proteins are primarily synthesized as precursor molecules and contain pre-propeptide sequences. The propeptide is connected to γ-carboxylase enzyme through the γ-carboxylase recognition site for the direct γ-carboxylation of VKD proteins that has a significant impact on their biological activity. Propeptides have different attitudes toward γ-carboxylase and certain amino acids in propeptide sequences are responsible for the differences in γ-carboxylase affinity. By aiming to replace amino acids in hFIX propeptide domain based on the prothrombin propeptide, pMT-hFIX-M14 expression cassette, containing cDNA of hFIX with substituted -14 residues (Asp to Ala) was made. After transfection of Drosophila S2 cells, expression of the active hFIX was analyzed by performing ELISA and coagulation test. A 1.4-fold increase in the mutant recombinant hFIX expression level was observed in comparison with that of a native recombinant hFIX. The enhanced hFIX activity and specific activity of the hFIXD-14A (2.2 and 1.6 times, respectively) were further confirmed by comparing coagulation activity levels of substituted and native hFIX. Enrichment for functional, fully γ-carboxylated hFIX species via barium citrate adsorption demonstrated 2-fold enhanced recovery in the S2-expressing hFIXD-14A relative to that expressed native hFIX. These results show that changing -14 residues leads to a decrease in the binding affinity to substrate, increase in γ-carboxylation and activity of recombinant hFIX. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:515-520, 2018.

Produção da proteína recombinante humana TGF-ß1 (fator do crescimento transformante beta 1) em células de mamífero / Production of recombinant human protein TGF-ß1 (Transforming Growth Factor Beta 1) in mammalian cells

O fator de crescimento transformante beta tipo 1, TGF-ß1, é uma proteína extracelular homodimérica secretada por vários tipos celulares, que pode ter ação parácrina ou endócrina. Essa proteína está envolvida em processos celulares de diferenciação, proliferação, mobilidade e formação de matriz extracelular. Além disso, é parte importante dos processos de regeneração tecidual, atuando, de maneira decisiva, no reparo, atraindo macrófagos e fibroblastos para o local da injúria e estimulando a angiogênese. Assim, considerando o papel desse peptídeo no processo regenerativo, o uso de TGF-ß1 como proteína terapêutica na área de Bioengenharia Tecidual é bastante promissor. Apesar disso, a venda dessa proteína, para fins terapêuticos, é inexistente no mercado e a proteína recombinante vendida, que só pode ser utilizada em pesquisas científicas, não é produzida nacionalmente e chega a custar R$200.000,00/mg. Nesse contexto, o objetivo do presente trabalho é desenvolver uma metodologia de produção do fator recombinante TGF-ß1 em células de ovário de hamster chinês (CHO), visando à obtenção de níveis altos de rendimento, e, futuramente, a transferência da tecnologia de produção para a iniciativa privada, tornando possível seu uso na Medicina Regenerativa, sozinho ou em combinação com outros fatores de crescimento. O cDNA de TGF-ß1 foi amplificado a partir de um banco de cDNA humano e clonado no vetor proprietário pNU1 de expressão de mamífero. A construção pNU1/TGF-ß1 foi utilizada para transfectar estavelmente células CHO DG44 e uma estratégia de co-amplificação foi utilizada para selecionar células transfectantes com maior número de cópias da sequência correspondente a TGF-ß1. Estas culturas foram submetidas ao processo de amplificação gênica com concentrações crescentes de metotrexato. Ensaios de Western Blot e ELISA foram realizados utilizando-se o meio condicionado pelas populações selecionadas e por clones superprodutores. Entre os 41clones obtidos, cinco apresentaram maiores níveis de produção de TGF-ß1, entre 1.000 e 2.000 ng/mL. Estes clones foram selecionados para a realização de testes de atividade in vitro utilizando-se células A549, que permitem avaliar a transição epitélio-mesênquima. Um ensaio de cicatrização de feridas em peles do dorso de camundongos foi padronizado e utilizado para avaliar a atividade in vivo do clone que apresentou melhor resultado in vitro. A proteína TGF-ß1 foi parcialmente purificada por HPLC em uma coluna de afinidade. Portanto, a proteína TGF-ß1 humana recombinante foi produzida, apresentando atividade biológica in vitro e in vivo, sendo capaz de reparar eficientemente feridas cutâneas. Essa iniciativa pode oferecer aos pacientes uma alternativa para o tratamento de lesões teciduais, acelerando a cicatrização de feridas e o reparo de tecidos

The transforming growth factor beta 1, TGF-ß1, is a homodimeric extracellular protein secreted by several cell types, which may have paracrine or endocrine action. This protein is involved in cellular processes of differentiation, proliferation, mobility and formation of extracellular matrix. In addition, it is an important part of the tissue regeneration processes, acting decisively on repair, attracting macrophages and fibroblasts to the site of injury and stimulating angiogenesis. Therefore, considering the role of this peptide in the regenerative process and the use of TGF-ß1 as a therapeutic protein in the field of Tissue Bioengineering is very promising. Despite this, the sale of this protein for therapeutic purposes is nonexistent in the market and the recombinant protein available in the market, which can only be used in scientific research, is not produced nationally and the costs are in the order of R$ 200,000.00/mg. In this context, the objective of the present work is to develop a methodology for the production of the TGF-ß1 recombinant factor in Chinese hamster ovary (CHO) cells, aiming at obtaining high yields, and, in the future, transfering the production technology to the private initiative, allowing its use in Regenerative Medicine, alone or in combination with other growth factors. The TGF-ß1 cDNA was amplified from a human cDNA library and cloned into the proprietary pNU1 mammalian expression vector. The pNU1/TGF-ß1 construct was used to stably transfect CHO DG44 cells, and a co-amplification strategy was used to select transfectant cells with the largest number of gene copies. These cultures were subjected to the process of gene amplification with methotrexate. Western Blot and ELISA were used to assay the conditioned medium obtained from the selected cell populations and from overproducing cell clones. Among the 41 clones obtained, five presented higher levels of TGF-ß1 production, between 1,000 and 2,000 ng/mL. These clones were selected for in vitro activity testing using A549 cells to evaluate the epithelial-mesenchymal transition. Awound healing assay on mouse dorsal skin was standardized and used to evaluate the in vivo activity of the cell clone which displayed the highest result in vitro. The TGF-ß1 protein was partially purified by HPLC on an affinity column. Therefore, the recombinant human TGF-ß1 protein was produced and shown to display biological activity both in vitro and in vivo, being able to eficiently repair cutaneous wounds. This initiative may provide patients with an alternative treatment for tissue damage, accelerating wound healing and tissue repair

Evaluation of solvent systems for optimized extractables studies of single use bioprocessing solutions.

Despite their advantages, there is concern that single-use systems used in biopharmaceutical manufacture might release potentially toxic substances during standard unit operations that negatively impact cell growth. Characterization of the extractables profile for single-use systems is necessary to know which compounds potentially become leachables under operational cell culture conditions. A key issue in the design of extractables studies is the composition of the model solvent, in particular its pH and polarity. In this study, a new approach, based on design of experiments (DoE), has been applied to determine the composition of the model solvent for extractable profiling of single-use bags (SUBs). Particular focus was placed on the determination of the degradation products of the antioxidant Irgafos 168®, due to evidence that some of these degradation products have cytotoxic effects on CHO cells. Results indicated that 2-propanol:water is the most appropriate solvent for the extraction of highly hydrophobic compounds with polar groups and/or acid-base properties from SUBs. The described DoE approach simplifies the number of experiments, evaluates all possible solvent water mixtures to select the best extraction solvent based on polarity, establishes the influence of each variable and provides information about variable interaction, which represents an important improvement over current best practice. The developed approach was applied to seven SUBs from different vendors and production dates facilitating the identification of potentially non-satisfactory films for cultivation of CHO cell lines under process conditions.

Optimizing cell-free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations.

Cell-free protein synthesis (CFPS) is an ideal platform for rapid and convenient protein production. However, bioreactor design remains a critical consideration in optimizing protein expression. Using turbo green fluorescent protein (tGFP) as a model, we tracked small molecule components in a Chinese Hamster Ovary (CHO) CFPS system to optimize protein production. Here, three bioreactors in continuous-exchange cell-free (CECF) format were characterized. A GFP optical sensor was built to monitor the product in real-time. Mass transfer of important substrate and by-product components such as nucleoside triphosphates (NTPs), creatine, and inorganic phosphate (Pi) across a 10-kDa MWCO cellulose membrane was calculated. The highest efficiency measured by tGFP yields were found in a microdialysis device configuration; while a negative effect on yield was observed due to limited mass transfer of NTPs in a dialysis cup configuration. In 24-well plate high-throughput CECF format, addition of up to 40 mM creatine phosphate in the system increased yields by up to ∼60% relative to controls. Direct ATP addition, as opposed to creatine phosphate addition, negatively affected the expression. Pi addition of up to 30 mM to the expression significantly reduced yields by over ∼40% relative to controls. Overall, data presented in this report serves as a valuable reference to optimize the CHO CFPS system for next-generation bioprocessing. Biotechnol. Bioeng. 2017;114: 1478-1486. © 2017 Wiley Periodicals, Inc.

Spatial Chemical Stimulation Control in Microenvironment by Microfluidic Probe Integrated Device for Cell-Based Assay.

Cell-cell interactions play an important role in the development and function of multicellular organisms. To investigate these interactions in detail, it is necessary to evaluate the behavior of a cell population when the minimum number of cells in the population is stimulated by some chemical factors. We propose a microfluidic device integrated with microfluidic probe (MFP) functionality; this device is capable of imparting a chemical stimulus to cells within a microenvironment, for cell-based assays. The device contains MFP channels at the walls of the cell culture microchannels, and it can control a localized chemical stimulation area at the scale of a single cell to a few cells using MFP fluid control in a microspace. The results of a finite element method-based simulation indicated that it is possible to control the chemical stimulation area at the scale of a single cell to a few cells by optimizing the MFP channel apex width and the flow ratio. In addition, localized cell staining was demonstrated successfully using a spatial chemical stimulus. We confirmed the device functionality as a novel cell-based assay tool. We succeeded in performing localized cell collection using this method, which suggested that the single cell analysis of a cell monolayer that is subjected to a specific chemical stimulus is possible. The method proposed in this paper can contribute significantly to the fields of cell biology and drug development.

Host cell protein impurities in chromatographic polishing steps for monoclonal antibody purification.

Downstream purification of monoclonal antibodies (mAbs) is normally performed using a platform process that is empirically tuned to optimize impurity removal for each new product. A more fundamental understanding of impurities and the product itself would provide insights into the rational design of efficient downstream processes. This work examines the chromatographic properties of Chinese hamster ovary host cell protein (HCP) impurities in non-affinity chromatographic resins commonly used in polishing steps for monoclonal antibody purification: ion-exchange, hydrophobic interaction, and multimodal. Using proteomic analysis, the specific HCP impurities that elute close to mAb products are identified for these resins at typical downstream processing conditions. Additionally, the interactions of HCP impurities with mAb products are profiled to determine the total extent of product association and the specific HCP species that form associative complexes under conditions encountered in polishing columns. Product association and co-elution were both identified as viable mechanisms of HCP retention for the non-affinity resins tested here. A relatively large sub-population of HCP impurities was found to co-elute or associate with mAbs in each polishing column, but only a small population of HCPs-including lipoprotein lipase, chrondroitin sulfate proteoglycan 4, nidogen-1, and SPARC-were identified as difficult to remove across an entire downstream mAb process. Biotechnol. Bioeng. 2016;113: 1260-1272. © 2015 Wiley Periodicals, Inc.

High throughput chromatography strategies for potential use in the formal process characterization of a monoclonal antibody.

High throughput experimental strategies are central to the rapid optimization of biologics purification processes. In this work, we extend common high throughput technologies towards the characterization of a multi-column chromatography process for a monoclonal antibody (mAb). Scale-down strategies were first evaluated by comparing breakthrough, retention, and performance (yields and clearance of aggregates and host cell protein) across miniature and lab scale columns. The process operating space was then evaluated using several integrated formats, with batch experimentation to define process testing ranges, miniature columns to evaluate the operating space, and comparison to traditional scale columns to establish scale-up correlations and verify the determined operating space. When compared to an independent characterization study at traditional lab column scale, the high throughput approach identified the same control parameters and similar process sensitivity. Importantly, the high throughput approach significantly decreased time and material needs while improving prediction robustness. Miniature columns and manufacturing scale centerpoint data comparisons support the validity of this approach, making the high throughput strategy an attractive and appropriate scale-down tool for the formal characterization of biotherapeutic processes in the future if regulatory acceptance of the miniature column data can be achieved. Biotechnol. Bioeng. 2016;113: 1273-1283. © 2015 Wiley Periodicals, Inc.

The distribution pattern of α2,3- and α2,6-linked sialic acids affects host cell preference in Toxoplasma gondii.

Tachyzoites of Toxoplasma gondii, an obligate intracellular parasite, actively invade almost all types of nucleated cells. However, T. gondii tachyzoites preferentially infect particular types of animal tissue cells. The mechanism underlying the host cell preference of T. gondii is not yet known. In this study, we found that enzymatic removal of α2,3- but not α2,6-linked sialic acids on the surface of Vero cells decreased T. gondii tachyzoite adhesion or invasion to the treated cells. Although Chinese hamster ovary (CHO) cells express only α2,3-linked sialic acid, a genetically modified CHO cell line constructed by transfection with the α2,6-sialiltransferase gene contains subpopulations with a variety of expression patterns of α2,3- and α2,6-linked sialic acids. When T. gondii tachyzoites were added to the modified CHO cells, the tachyzoites preferentially attached to cells belonging to a subpopulation of cells that highly expressed α2,3-linked sialic acids. Additionally, multiple regression analysis performed to analyse the relationship between the amount of α2,3-linked/α2,6-linked sialic acids and parasite-expressed fluorescence intensity suggested that more tachyzoites adhered to individual α2,3-linked sialic acid rich-cells than to α2,3-linked sialic acid-poor/null cells. The results of confocal laser microscopy confirmed this finding. These results indicate that the host cell preference of T. gondii was, at least partially, affected by the distribution pattern of α2,3-, but almost never α2,6-linked sialic acids on host cells.