Large-scale Cell Culture Research Articles

Modeling and optimization of membrane preparation conditions of the alginate‐based microcapsules with response surface methodology

Microencapsulation has been a promising approach for drug delivery, cell implantation, cell-based gene therapy and large-scale cell culture. To make use of microcapsules more effectively, it is important to accurately construct the microcapsule membranes with desired properties including a certain thickness, strength, and so forth. To date single factor experiments have been widely used, however, they are time-consuming to obtain the desired membrane preparation conditions. Response surface methodology (RSM) is a mathematical and statistical technique for building empirical models that gained importance for optimizing reacting conditions. In this study, three signifficant effect factors that affect alginate-based microcapsule membrane properties, including membrane thickness, swelling degree, and mechanical stability, were determined with Plackett-Burman method, and then three empirical models were built to optimize the preparation conditions of the microcapsule membranes according to the responses of these three signifficant effect factors respectively with RSM. These models can be used to predict the characteristics of microcapsules under different membrane preparation conditions, which provide a guide for optimizing the microencapsulation technology.

Evaluation of an online biomass probe to monitor cell growth and cell death

Background The estimation of cell density and cell viability of mammalian cell lines in cell culture has traditionally been performed using the exclusion dye trypan blue that stains “dead” cells when their cell membrane is damaged. In large scale cell cultures using bioreactors this estimation is performed off-line. The online biomass probe is based on the principle that under the influence of an electric field between two electrodes, ions in suspension migrate toward the electrodes. The cell plasma membrane is non-conductive so that the cells with intact plasma membranes are polarized and act as tiny capacitors and it has been shown that capacitance increases as the cell concentration does. The measurement is based on the linear relationship between the permittivity difference e1e2 and the viable biomass concentration as it has been described by Ansorge et al. [1]. This study compares the data obtained using the biomass probe against the cell counts and viability determined by trypan blue exclusion with two GS-CHO cell line productions in bioreactors. Apoptosis determination measurements using rhodamine-123 and pancaspase activation by flow cytometry will be compared to permittivity values.

Microfibrous carriers for cell culture: A comparative study

Small patches of polyethylene terephthalate (PET) nonwoven microfibrous matrices have excellent properties and can be used as carriers for culturing cells in agitated bioreactors. The microfibrous carriers are highly porous and can provide large surface areas and three-dimensional space for high-density cell growth. In this work, the microfibrous carriers and several commercial microcarriers were used to study cell attachment kinetics, growth, and monoclonal antibody production with Chinese hamster ovary cells. Compared with commercial solid and macroporous microcarriers, the microfibrous carriers showed better or similar performances. In addition, the microfibrous carriers provided a wider operable range for agitation rate than commercial microcarriers, effectively protecting cells from shear stress and carrier collisions. In addition, the microfibrous carriers are available at a much lower cost than commercial microcarriers, providing an attractive alternative to microcarrier-based large-scale cell cultures.

Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.

A study of D‐lactate and extracellular methylglyoxal production in lactate Re‐Utilizing CHO cultures

In large-scale mammalian cell culture, the key toxic by-products assessed and monitored are lactate and ammonia. Often no distinction between the two isoforms of lactate is made. Here, we present profiles of both D- and L-lactate. D-Lactate is the end molecule of the methylglyoxal pathway. D-Lactate unlike L-lactate is not re-utilized, and although during normal culture time frames it represents one-tenth of total lactate, during lactate re-use it represents nearly 35%. This indicates significant carbon flow through pathways not associated with primary metabolites. We have observed that the behavior of D-lactate is radically different from that of L-lactate with the level of one isoform changing, whilst the concentration of the other remains constant. This is an example of an alternate carbon flow pathway containing metabolic intermediates that may potentially have a detrimental effect on cells. The activity of the methylglyoxal pathway when measured as a proportion of glucose consumption in this study far exceeds any previously reported. This highlights the potential importance of "non-primary" metabolisms to long lifespan mammalian fermentation practices.

Monitoring of Cell Viability and Proliferation in Hydrogel-Encapsulated System by Resazurin Assay

Cell microencapsulation is a promising approach for cell implantation, cell-based gene therapy and large-scale cell culture. For better quality control, it is important to accurately measure the microencapsulated cell viability and proliferation in the culture. A number of assays have been used for this purpose, but limitations arise. In this study, we investigated the feasibility and reliability of resazurin as a cell growth indicator in microencapsulated culture system. According to the experiment data, there was a reversible, time- and dose-dependent growth inhibition as observed for resazurin application in encapsulated cells. A positive relationship was observed between reduction of resazurin and CHO cell number in microcapsule. Moreover, the resazurin assay provided an equivalent result to the commonly used MTT method in determining CHO cell proliferation in APA microcapsule with no notable influence on cell distribution and organization pattern. In conclusion, resazurin assay is offered as a simple, rapid and non-invasive method for in vitro microencapsulated cell viability and proliferation measurement.

Human IgG1 Hinge Fragmentation as the Result of H2O2-mediated Radical Cleavage

Hinge cleavage of a recombinant human IgG1 antibody, generated during production in a Chinese hamster ovary cell culture, was observed in the purified material. The cleavage products could be reproduced by incubation of the antibody with H(2)O(2) and featured complementary ladders of the C- and N-terminal residues (Asp(226)-Lys(227)-Thr(228)-His(229)-Thr(230)) in the heavy chain of the Fab domain and the upper hinge of one of the Fc domains, respectively. Two adducts of +45 and +71 Da were also observed at the N-terminal residues of some Fc fragments and were identified as isocyanate and alpha-ketoacyl derivatives generated by radical cleavage at the alpha-carbon position through the diamide and alpha-amidation pathways. We determined that the hinge cleavage was initiated by radical-induced breakage of the disulfide bond between the two hinge cysteines at position 231 (Cys(231)-Pro-Pro-Cys-Pro), followed by the formation of a thiyl radical (Cys(231)-S(*)) on one cysteine and sulfenic acid (Cys(231)-SOH) on the other. The location of the initial radical attack and the critical role of Cys(231) were demonstrated by the observation that 5,5-dimethyl-1-pyrroline N-oxide only reacted with the Cys(231) radical and completely blocked hinge cleavage, suggesting the necessity of an electron/radical transfer from the Cys(231) radical to the hinge residues where cleavage was observed. As a precursor of hydroxyl radicals, H(2)O(2) is widely produced in healthy cells and tissues and therefore could be the source for the radical-induced fragmentation of human IgG1 antibodies in vivo.

BAK and BAX deletion using zinc‐finger nucleases yields apoptosis‐resistant CHO cells

Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX- and BAK-deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production.

Identification by Automated Screening of a Small Molecule that Selectively Eliminates Neural Stem Cells Derived from hESCs but Not Dopamine Neurons

BackgroundWe have previously described fundamental differences in the biology of stem cells as compared to other dividing cell populations. We reasoned therefore that a differential screen using US Food and Drug Administration (FDA)-approved compounds may identify either selective survival factors or specific toxins and may be useful for the therapeutically-driven manufacturing of cells in vitro and possibly in vivo.Methodology/Principal FindingsIn this study we report on optimized methods for feeder-free culture of hESCs and hESC-derived neural stem cells (NSCs) to facilitate automated screening. We show that we are able to measure ATP as an indicator of metabolic activity in an automated screening assay. With this optimized platform we screened a collection of FDA-approved drugs to identify compounds that have differential toxicity to hESCs and their neural derivatives. Nine compounds were identified to be specifically toxic for NSCs to a greater extent than for hESCs. Six of these initial hits were retested and verified by large-scale cell culture to determine dose-responsive NSC toxicity. One of the compounds retested, amiodarone HCL, was further tested for possible effects on postmitotic neurons, a likely target for transplant therapy. Amiodarone HCL was found to be selectively toxic to NSCs but not to differentiated neurons or glial cells. Treated and untreated NSCs and neurons were then interrogated with global gene expression analysis to explore the mechanisms of action of amiodarone HCl. The gene expression analysis suggests that activation of cell-type specific cationic channels may underlie the toxicity of the drug.Conclusions/SignificanceIn conclusion, we have developed a screening strategy that allows us to rapidly identify clinically approved drugs for use in a Chemistry, Manufacture and Control protocol that can be safely used to deplete unwanted contaminating precursor cells from a differentiated cell product. Our results also suggest that such a strategy is rich in the potential of identifying lineage specific reagents and provides additional evidence for the utility of stem cells in screening and discovery paradigms.

Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells

This work investigates an application of the alginate encapsulation technology to the differentiation of embryonic stem (ES) cells into insulin-producing cells. It shows that the ES cells can efficiently be encapsulated within the alginate beads, retaining a high level of cell viability. The alginate encapsulation achieves approximately 10-fold increase in the cell density in the culture, in comparison to the two-dimensional conditions, opening a potential benefit of the technology in large-scale cell culture applications. Manipulations of encapsulation conditions, particularly of the initial alginate concentration, allow the control over both the diffusion of molecules into the alginate matrix (e.g. differentiation factors) as well as control over the matrix porosity/flexibility to permit the proliferation and growth of encapsulated ES aggregates within the bead. Post-differentiation analysis confirms the presence of insulin-positive cells, as judged from immunostaining, insulin ELISA and RT-PCR analysis. The functionality of the encapsulated and differentiated cells was confirmed by their insulin production capability, whereby on glucose challenge the insulin production by the cells differentiated within alginate beads was found to be statistically significantly higher than for the cells from conventional two-dimensional differentiation system.

S22: Assessment of chronic toxicity for biopharmaceuticals – Just another NCE?

The development of recombinant DNA, large-scale cell culture technologies, and advances in synthetic DNA and protein chemistry introduced a wide range of pharmaceutical products including cytokines, receptor agonists/antagonists, hormones, growth factors, monoclonal antibodies, and gene therapy products for the diagnosis, prevention, and treatment of various diseases. Because of their complex structural and biological characteristics, as well as the complexity of the manufacturing process a new approach to their preclinical safety evaluation has developed over the last 20 years. Although most of these products require a case-by-case assessment using knowledge gained from both non-clinical and clinical evaluation of these products, some general principles have been highlighted with regards to their safety evaluation. The critical review of this experience formed the basis for the ICHS6 guidance (Preclinical Safety Evaluation of Biotechnology-derived Pharmaceuticals) which was published in 1997. As the ICHS6 guidance has been in practical use for 10 years, the additional marketed products allow for the interrogation of a larger pre-clinical as well clinical database. The primary purpose of this presentation is to discuss the special considerations for the pre-clinical safety assessment for biopharmaceuticals, in particular those intended for chronic indication. What have we learned over the past 20 years? Considerations such as relevancy of animal species, impact of potential immunogenicity and type of toxicity of biopharmaceuticals will impact the design of the chronic study. For example, the statement in the ICHS6, “for those biopharmaceuticals intended for chronic use indications, studies of 6 months duration have generally been appropriate…”; is this still relevant? Have pre-clinical studies been predictive for the clinical situation? Also, the need for assessment of potential carcinogenicity will be discussed briefly, and again, the question, how the unique characteristics of biopharmaceuticals influence the requirement for and/or the type of pre-clinical studies needed to support chronic administration will be discussed.

Crystal Structure of a Two-domain Multicopper Oxidase

The two-domain multicopper oxidases are proposed to be key intermediates in the evolution of three-domain multicopper oxidases. A number of two-domain multicopper oxidases have been identified from genome sequences and are classified as type A, type B, or type C on the basis of the predicted location of the type 1 copper center. The crystal structure of blue copper oxidase, a type C two-domain multicopper oxidase from Nitrosomonas europaea, has been determined to 1.9 A resolution. Blue copper oxidase is a trimer, of which each subunit comprises two cupredoxin domains. Each subunit houses a type 1 copper site in domain 1 and a type 2/type 3 trinuclear copper cluster at the subunit-subunit interface. The coordination geometry at the trinuclear copper site is consistent with reduction of the copper ions. Although the overall architecture of blue copper oxidase is similar to nitrite reductases, detailed structural alignments show that the fold and domain orientation more closely resemble the three-domain multicopper oxidases. These observations have important implications for the evolution of nitrite reductases and multicopper oxidases.

Scale-Up Considerations Based on Studies at the Bench Scale in Stirred Bioreactors

This paper considers recent work, mainly from the University of Birmingham, on stirred bench scale bioreactors. It focuses on mixing as it affects biological performance on scale-up, particularly the impact of stress on organisms as a result of fluid dynamic phenomena (mixing), namely mechanical stresses or spatial and temporal inhomogeneities. The work uses new experimental techniques (flow cytometry and advanced image analysis) and covers mycelial, bacterial (both including GMO’s) and yeast (including beer) fermentations and animal cell culture in batch, fed-batch and chemostat conditions. By using gas blending to control dissolved oxygen (dO2), the impact of mechanical stresses due to agitation and aeration can be decoupled from dO2 effects. With mycelia, image analysis of all biomass shows damage from mechanical stresses generated by agitation but not by aeration; and that productivity may be affected. Also, on scale-up, such damage is reduced. With yeast and bacteria, flow cytometry does not indicate damage. On the other hand, plant scale studies with E. coli give lower biomass but higher cell viability compared to the well-mixed bench-scale. When the locally high nutrient, low dO2 and high pH values found on the plant scale near feed points are simulated at bench-scale, similar results to plant scale are obtained. Similar scale-down results are obtained with a recombinant E. coli. Simulated large-scale beer production, where the inhomogeneities are associated with yeast distribution and temperature, also indicates a poorer performance. Though perceived to be more sensitive to mechanical stresses, somewhat similar conclusions can be drawn for animal cell culture, with stresses associated with bursting bubbles being potentially the most significant. These perceptions lead to problems associated with high dissolved carbon dioxide (pCO2) and osmolality in large-scale cell culture. A related topic is the search for ‘low shear’ impellers, a concept useful to mixer manufacturers but misleading to users.

Characterization of Fluorescent Sterol Binding to Purified Human NPC1

Mutations in the NPC1 gene cause Niemann-Pick type C disease, which appears to result from a defect in intracellular cholesterol trafficking. NPC1 is a member of the resistance-nodulation-cell division (RND) permease superfamily and contains a sterol-sensing domain, yet its cellular function and the identity of its substrates remain unknown. FLAG-tagged human NPC1 was purified from NPC1-expressing Chinese hamster ovary cells by solubilization in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), followed by affinity chromatography. Purified NPC1 in detergent solution appeared to be oligomeric as determined by gel filtration fast protein liquid chromatography and was photolabeled by an azido-cholesterol derivative. Fluorescent cholesterol analogs, including dehydroergosterol, cholestatrienol, and 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), displayed enhanced fluorescence upon binding to NPC1 and also resulted in saturable, concentration-dependent quenching of NPC1 intrinsic Trp fluorescence. The apparent binding affinity for these three sterols was in the 0.5-6 microm range. Binding of NBD-cholesterol to NPC1 at low detergent concentration (2 mm CHAPS) was of high apparent affinity (0.5-0.6 microm) and occurred rapidly (<1 min). However, binding of a BODIPY-labeled cholesterol derivative was very slow, requiring approximately 3 h to reach equilibrium. The apparent NBD-cholesterol binding affinity was greatly reduced at higher detergent concentration. The stoichiometry of NBD-cholesterol binding to NPC1 was approximately 1. Various sterols, including native cholesterol and 25-hydroxycholesterol, inhibited NBD-cholesterol binding, suggesting that they compete for binding to the protein. Dynamic quenching studies showed that bound NBD-cholesterol was almost completely shielded from the aqueous medium, suggesting that it is buried in a deep hydrophobic pocket in NPC1. The use of fluorescent cholesterol analogs provides novel information on the molecular properties of the sterol-binding site in the full-length NPC1 protein.

Scalable culture and cryopreservation of human embryonic stem cells on microcarriers

As a result of their pluripotency and potential for unlimited self-renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large-scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor-intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel-coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF-microcarriers was less than that on MEF-plates, the doubling time of hESCs on Matrigel-microcarriers was indistinguishable from that of hESCs expanded on Matrigel-coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier-based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications.

Effects of Elevated pCO2 and Osmolality on Growth of CHO Cells and Production of Antibody-Fusion Protein B1: A Case Study

Partial pressure of CO2 (pCO2) and osmolality as high as 150 mmHg and 440 mOsm/kg, respectively, were observed in large-scale CHO cell culture producing an antibody-fusion protein, B1. pCO2 and osmolality, when elevated to high levels in bioreactors, can adversely affect cell culture and recombinant protein production. To understand the sole impact of pCO2 or osmolality on CHO cell growth, experiments were performed in bench-scale bioreactors allowing one variable to change while controlling the other. Elevating pCO2 from 50 to 150 mmHg under controlled osmolality (about 350 mOsm/kg) resulted in a 9% reduction in specific cell growth rate. In contrast, increasing osmolality resulted in a linear reduction in specific cell growth rate (0.008 h(-1)/100 mOsm/kg) and led to a 60% decrease at 450 mOsm/kg as compared to the control at 316 mOsm/kg. This osmolality shift from 316 to 445 mOsm/kg resulted in an increase in specific production rates of lactate and ammonia by 43% and 48%, respectively. To elucidate the effect of high osmolality and/or pCO2 on the production phase, experiments were conducted in bench-scale bioreactors to more closely reflect the pCO2 and osmolality levels observed at large scale. Increasing osmolality to 400-450 mOsm/kg did not result in an obvious change in viable cell density and product titer. However, a further increase in osmolality to 460-500 mOsm/kg led to a 5% reduction in viable cell density and a 8% decrease in cell viability as compared to the control. Final titer was not affected as a result of an apparent increase in specific production rate under this increased osmolality. Furthermore, the combined effects from high pCO2 (140-160 mmHg) and osmolality (400-450 mOsm/kg) caused a 20% drop in viable cell density, a more prominent decrease as compared to elevated osmolality alone. Results obtained here illustrate the sole effect of high pCO2 (or osmolality) on CHO cell growth and demonstrate a distinct impact of high osmolality and/or pCO2 on production phase as compared to that on growth phase. These results are useful to understand the response of the CHO cells to elevated pCO2 (and/or osmolality) at a different stage of cultivation in bioreactors and thus are valuable in guiding bioreactor optimization toward improving protein production.

The fate of Pluronic F‐68 in chondrocytes and CHO cells

The surfactant Pluronic F-68 (PF-68) is widely used in large-scale mammalian cell culture to protect cells from shear stress that arises from agitation and gas sparging. Several studies suggested that PF-68 is incorporated into the cell plasma membrane and could enter the cells, but without providing any direct evidence. The current study has examined this question for two cell types, one of pharmaceutical interest (CHO cells) and the other of biomedical interest (chondrocytes or cartilage cells). A fluorescent derivative of PF-68 was synthesized to detect and localize internalized Pluronic with culture time. PF-68 uptake by the cells was quantified and characterized. We clearly demonstrate that PF-68 enters the cells, and possibly accumulates in the endocytic pathway. CHO cells showed an average uptake of 11.7 +/- 6.7 (SEM) microg PF-68/10(6) cells while the uptake of chondrocytes was 56.0 +/- 10.9 (SEM) microg PF-68/10(6) cells, independently of the initial PF-68 concentration (between 0.01 and 0.2%, w/v) and of cell concentration (from 1 x 10(6) to 4 x 10(6) cells/mL). These uptake values were identical for both static and agitated culture conditions. Finally, we found that CHO cells are able to eliminate intracellular fluorescent PF-68 but chondrocytes are not. These results show that the uptake of PF-68 by the cells can severely affect PF-68 concentration in the culture medium and thus shear protection effect.

Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a non-animal, non-human and non-plant-derived medium

For the production of a chemically inactivated Parapoxvirus ovis (PPVO), an adherent bovine kidney cell line was cultivated on Cytodex-3 microcarriers in suspension culture. The inactivated and purified virus particles have shown immune modulatory activity in several animal models. PPVO was produced by a biphasic batch process at the 3.5 and 10 L scale. Aeration was realised by bubble-free membrane oxygenation via a tube stator with a central two-blade anchor impeller. In order to increase efficiency, process robustness and safety, the established process was optimised. The cell line was adapted to a protein-free medium (except recombinant insulin) in order to increase biosafety. A scale up to a 50 L pilot plant with direct cell expansion was performed successfully. In parallel, the biphasic batch process was optimised with special emphasis on different operating conditions (cell number, Multiplicity of Infection (MOI), etc.) and process management (fed-batch, dialysis, etc.). The quality and concentration of the purified virus particles was assessed by quantitative electron microscopy, residual host cell protein and DNA-content and, finally, biologic activity in a transgenic mouse model. This integrated approach led to a new, safe, robust and highly productive large-scale production process, called "Volume-Expanded-Fed" Batch with cell densities up to 6-7e06 cells/mL. By subsequent dilution of infected cells into the next process scale, an increase in total productivity by a factor of 40 (related to an established biphasic batch process) was achieved.

Stem cells: moving the biology towards the clinic

Stem cells: moving the biology towards the clinic

Proteomic profiling of recombinant cells from large-scale mammalian cell culture processes

Global expression profiling of mammalian cells used for the production of biopharmaceuticals will allow greater insights into the molecular mechanisms that result in a high producing cellular phenotype. These studies may give insights for genetic intervention to possibly create better host cell lines or even to provide clues to more rational strategies for cell line and process development. In this review I will focus on the contribution of proteomic technologies to a greater understanding of the biology of Chinese hamster ovary cells and other producing cell lines such as NS0 mouse cells.