Autophagy-inducing peptide increases CHO cell monoclonal antibody production in batch and fed-batch cultures.

The development of generic biopharmaceuticals is increasing the pressures for enhanced bioprocess productivity and yields. Autophagy ("self-eating") is a cellular process that allows cells to mitigate stresses such as nutrient deprivation. Reputed autophagy inhibitors have also been shown to increase autophagic flux under certain conditions, and enhance recombinant protein productivity in Chinese Hamster Ovary (CHO) cultures. Since peptides are commonly added to bioprocess culture media in hydrolysates, we evaluated the impact on productivity of an autophagy-inducing peptide (AIP), derived from the cellular autophagy protein Beclin 1. This was analyzed in CHO cell batch and fed-batch serum-free cultures producing a human Immunoglobulin G1 (IgG1). Interestingly, the addition of 1-4 µM AIP enhanced productivity in a concentration-dependent manner. Cell-specific productivity increased up to 1.8-fold in batch cultures, while in fed-batch cultures a maximum twofold increase in IgG concentration was observed. An initial drop in cell viability also occurred before cultures recovered normal growth. Overall, these findings strongly support the value of investigating the effects of autophagy pathway modulation, and in particular, the use of this AIP medium additive to increase CHO cell biotherapeutic protein production and yields.

Cytoplasmic conductivity as a marker for bioprocess monitoring: Study of Chinese hamster ovary cells under nutrient deprivation and reintroduction.

The ability to monitor the status of cells during nutrient limitation is important for optimizing bioprocess growth conditions in batch and fed-batch cultures. The activity level of Na+ /K+ ATPase pumps and cytoplasm ionic concentrations are directly influenced by the nutrient level, and thus, cytoplasm conductivity can be used as a markerless indicator of cell status. In this work, we monitored the change in cytoplasm conductivity of Chinese hamster ovary (CHO) cells during nutrient deprivation and reintroduction. Employing single cell dielectrophoresis, the change in cytoplasm conductivity was measured over a 48-hr period. The conditions under which the cytoplasm conductivity would recover to a normal level after nutrient reintroduction was determined. In addition, numerical simulations of cell ion flux, for different levels of Na+ /K+ ATPase pump inhibition, were used to predict the minimum conductivity expected for nutrient-deprived CHO cells. This predicted value is close to the minimum observed experimental cytoplasm conductivity for CHO cells that maintain the ability to restore the cytoplasm conductivity to the normal viable levels when nutrients are reintroduced. The recovery of starved cells was verified by reintroducing them to nutrient for 36 hr and measuring their proliferation using trypan blue exclusion assay. We conclude that cytoplasm conductivity can be used as a marker to indicate whether cells are in a recoverable state, such that the reintroduction of nutrients results in cells returning to a normal healthy state.

A semi-empirical glycosylation model of a camelid monoclonal antibody under hypothermia cell culture conditions.

The impact of cell culture environment on the glycan distribution of a monoclonal antibody (mAb) has been investigated through a combination of experiments and modeling. A newly developed CHO DUXB cell line was cultivated at two levels of initial Glutamine (Gln) concentrations (0, 4 mM) and incubation temperatures of (33 and 37 °C) in batch operation mode. Hypothermia was applied either through the entire culture duration or only during the post-exponential phase. Beyond reducing cell growth and increasing productivity, hypothermia significantly altered the galactosylation index profiles as compared to control conditions. A novel semi-empirical dynamic model was proposed for elucidating the connections between the extracellular cell culture conditions to galactosylation index. The developed model is based on a simplified balance of nucleotides sugars and on the correlation between sugars' levels to the galactosylation index (GI). The model predictions were found to be in a good agreement with the experimental data. The proposed empirical model is expected to be useful for controlling the glycoprofiles by manipulating culture conditions.

The changing dielectric properties of CHO cells can be used to determine early apoptotic events in a bioprocess.

To ensure maximum productivity of recombinant proteins it is desirable to prolong cell viability during a mammalian cell bioprocess, and therefore important to carefully monitor cell density and viability. In this study, five different and independent methods of monitoring were applied to Chinese hamster ovary (CHO) cells grown in a batch culture in a controlled bioreactor to determine cell density and/or cell viability. They included: a particle counter, trypan blue exclusion (Cedex), an in situ bulk capacitance probe, an off-line fluorescent flow cytometer, and a prototype dielectrophoretic (DEP) cytometer. These various techniques gave similar values during the exponential growth phase. However, beyond the exponential growth phase the viability measurements diverged. Fluorescent flow cytometry with a range of fluorescent markers was used to investigate this divergence and to establish the progress of cell apoptosis: the cell density estimates by the intermediate stage apoptosis assay agreed with those obtained by the bulk capacitance probe and the early stage apoptosis assay viability measurements correlated well with the DEP cytometer. The trypan blue assay showed higher estimates of viable cell density and viability compared to the capacitance probe or the DEP cytometer. The DEP cytometer measures the dielectric properties of individual cells and identified at least two populations of cells, each with a distinct polarizability. As verified by comparison with the Nexin assay, one population was associated with viable (non-apoptotic) cells and the other with apoptotic cells. From the end of the exponential through the stationary and decline stages there was a gradual shift of cell count from the viable into the apoptotic population. However, the two populations maintained their individual dielectric properties throughout this shift. This leads to the conclusion that changes in bulk dielectric properties of cultures might be better modeled as shifts in cells between different dielectric sub-populations, rather than assuming a homogeneous dielectric population. This shows that bulk dielectric probes are sensitive to the early apoptotic changes in cells. DEP cytometry offers a novel and unique technology for analyzing and characterizing mammalian cells based on their dielectric properties, and suggests a potential application of the device as a low-cost, label-free, electronic monitor of physiological changes in cells.

Inhibition of glutamine-dependent autophagy increases t-PA production in CHO cell fed-batch processes.

Understanding the cellular responses caused by metabolic stress is crucial for the design of robust fed-batch bioprocesses that maximize the expression of recombinant proteins. Chinese hamster ovary cells were investigated in chemically defined, serum-free cultures yielding 10(7) cells/mL and up to 500 mg/L recombinant tissue-plasminogen activator (t-PA). Upon glutamine depletion increased autophagosome formation and autophagic flux were observed, along with decreased proliferation and high viability. Higher lysosomal levels correlated with decreased productivity. Chemical inhibition of autophagy with 3-methyl adenine (3-MA) increased the t-PA yield by 2.8-fold. Autophagy-related MAP1LC3 and LAMP2 mRNA levels increased continuously in all cultures. Analysis of protein quality revealed that 3-MA treatment did not alter glycan antennarity while increasing fucosylation, galactosylation, and sialylation. Taken together, these findings indicate that inhibition of autophagy can considerably increase the yield of biotechnology fed-batch processes, without compromising the glycosylation capacity of cells. Monitoring or genetic engineering of autophagy provides novel avenues to improve the performance of cell culture-based recombinant protein production.

Progression of change in membrane capacitance and cytoplasm conductivity of cells during controlled starvation using dual-frequency DEP cytometry.

The dielectric properties of cells are directly related to their morphological and physiological properties and can be used to monitor their status when exposed to stress conditions. In this work, dual-frequency dielectrophoresis (DEP) cytometry was employed to measure changes in the membrane capacitance and cytoplasm conductivity of single Chinese hamster ovary (CHO) cells during the progression of starvation-induced apoptosis. Our dual-frequency DEP cytometer enables simultaneous measurement of multiple dielectric properties of single cells and identification of their state (viable or apoptotic) within a heterogeneous sample. We employed one frequency to determine each cell's viability state and the other frequency to characterize the change in membrane capacitance or cytoplasm conductivity. Cells were starved by incubation in a medium lacking glucose and glutamine and monitored every 12 h over a 64 h period. Our results showed a subpopulation of early apoptotic cells emerged after 40 h in the starvation medium, which rapidly increased during the next 12 h. After 52 h, a complete transition from viable to apoptotic state was observed. Analyzing the subpopulation of viable cells over the first 52 h showed that the membrane capacitance gradually declined from an initial value of 2.0 to 1.2 µF/cm2, and was 0.9 µF/cm2 for apoptotic cells. The cytoplasm conductivity of viable cells initially remained constant and then declined from 0.40 to 0.27 S/m after 40 h, coinciding with onset of apoptotic processes. A dramatic decrease in cytoplasm conductivity from 0.27 to 0.07 S/m was observed after 52 h, corresponding to apoptotic cells. As membrane capacitance is related to membrane morphology and cytoplasm conductivity is related to intracellular ion concentrations, the results indicate that during controlled starvation the cell membrane smooths gradually whereas intracellular ion concentrations are initially maintained near homeostatic levels until a later dramatic decline occurs.

Quantitative Model for Ion Transport and Cytoplasm Conductivity of Chinese Hamster Ovary Cells.

In mammalian cells cytoplasm ion concentrations and hence cytoplasm conductivity is an important indicator of their physiological state. Changes in the cytoplasm conductivity has been associated with physiological changes such as progression of cancer and apoptosis. In this work, a model that predicts the effects of physiological changes in ion transport on the cytoplasm conductivity of Chinese hamster ovary (CHO) cells is demonstrated. We determined CHO-specific model parameters, Na+/K+ ATPase pumps and ion channels densities, using a flux assay approach. The obtained sodium (PNa), potassium (PK) and chloride (PCl) permeability and Na+/K+ ATPase pump density were estimated to be 5.6 × 10-8 cm/s, 5.6 × 10-8 cm/s, 3.2 × 10-7 cm/s and 2.56 × 10-11 mol/cm2, respectively. The model was tested by comparing the model predictions with the experimentally determined temporal changes in the cytoplasm conductivity of Na+/K+ ATPase pump inhibited CHO cells. Cells' Na+/K+ ATPase pumps were inhibited using 5 mM Ouabain and the temporal behavior of their cytoplasm conductivity was measured using dielectrophoresis cytometry. The measured results are in close agreement with the model-calculated values. This model will provide insight on the effects of processes such as apoptosis or external media ion concentration on the cytoplasm conductivity of mammalian cells.

Single cell dielectrophoresis study of apoptosis progression induced by controlled starvation.

Nutrient depletion in fed-batch cultures and at the end of batch cultures is among the main causes of stress on cells and a trigger of apoptosis. In this study, we investigated changes in the cytoplasm conductivity of Chinese hamster ovary (CHO) cells under controlled starvation. Employing a single-cell dielectrophoresis (DEP) cytometer, we measured the DEP response of CHO cells incubated in a medium without glucose and glutamine over a 48-h period. Using the measured data in conjunction with numerical simulations, we determined the cytoplasm conductivity of viable and apoptotic cell subpopulations. The results show that a small subpopulation of apoptotic cells emerges after 24 to 36 h of starvation and increases rapidly over a short period of time, <12 h. The apoptotic cells have a dramatically lower cytoplasm conductivity, ∼0.05 S/m, than viable cells, ∼0.45 S/m. Viability of starvation cultures was measured by fluorescent cytometry, DEP cytometry, and trypan blue exclusion assays. DEP, Annexin V, caspase-8, and 7-AAD assays show a similar decline in viability after 36 h of starvation and indicate a very low viability after 48 h. Trypan blue exclusion assay fails to detect early-stage viability decline and estimates a much higher viability after 48 h.

Evaluation of Quenching and Extraction Methods for Nucleotide/Nucleotide Sugar Analysis.

Nucleotide sugars are the donor substrates of glycosyltransferases and their availability is known to have an impact on the glycosylation of recombinant proteins including monoclonal antibodies. In addition, the intracellular concentration levels of these metabolites can provide information about the physiological/energetic state of the cell. Therefore, the ability to qualitatively and quantitatively determine the intracellular nucleotides and nucleotide sugars can give valuable insight into the metabolism associated with the glycosylation processes in cells. However, in order to be able to perform a consistent and reliable time specific analysis of these metabolites during a cell culture the metabolism of the cell needs to be stopped immediately at the point of sampling and an efficient extraction needs to be performed. Once the nucleotides and nucleotide sugars are extracted from the cell sample an efficient HPLC method is needed to separate all or most of the metabolites of interest to allow for their identification and quantification. Here, we describe an optimized method for the analysis of the intracellular nucleotide/nucleotide sugar pool in CHO suspension cells which includes protocols for quenching, extraction and HPLC analysis.

Tuning a MAb glycan profile in cell culture: Supplementing N-acetylglucosamine to favour G0 glycans without compromising productivity and cell growth.

Glycosylation is a critical quality attribute of many therapeutic proteins, particularly monoclonal antibodies (MAbs). Nucleotide-sugar precursors supplemented to growth medium to affect the substrate supply chain of glycosylation has yielded promising but varied results for affecting glycosylation. Glucosamine (GlcN), a precursor for N-acetylglucosamine (GlcNAc), is a major component of mammalian glycans. The supplementation of GlcN to CHO cells stably-expressing a chimeric heavy-chain monoclonal antibody, EG2-hFc, reduces the complexity of glycans to favour G0 glycoforms, while also negatively impacting cell growth. Although several researchers have examined the supplementation of glucosamine, no clear explanation of its impact on cell growth has been forthcoming. In this work, the glucosamine metabolism is examined. We identified the acetylation of GlcN to produce GlcNAc to be the most likely cause for the negative impact on growth due to the depletion of intracellular acetyl-CoA pools in the cytosol. By supplementing GlcNAc in lieu of GlcN to CHO cells producing EG2-hFc, we achieve the same shift in glycan complexity with marginal impacts on the cell growth and protein production.

Monitoring cell growth, viability, and apoptosis.

The accurate determination of cell growth and viability is pivotal to monitoring a bioprocess. Direct methods to determine the cell growth and/or viability in a bioprocess include microscopic counting, electronic particle counting, image analysis, in situ biomass monitoring, and dieletrophoretic cytometry. These methods work most simply when a fixed volume sample can be taken from a suspension culture. Manual microscopic counting is laborious but affords the advantage of allowing cell viability to be determined if a suitable dye is included. Electronic particle counting is a rapid total cell count method for replicate samples, but some data distortion may occur if the sample has significant cell debris or cell aggregates. Image analysis based on the use of digital camera images acquired through a microscope has advanced rapidly with the availability of several commercially available software packages replacing manual microscopic counting and viability determination. Biomass probes detect cells by their dielectric properties or their internal concentration of NADH and can be used as a continuous monitor of the progress of a culture. While the monitoring of cell growth and viability is an integral part of a bioprocess, the monitoring of apoptosis induction is also becoming more and more important in bioprocess control to increase volumetric productivity by extending bioprocess duration. Different fluorescent assays allow for the detection of apoptotic characteristics in a cell sample.Indirect methods of cell determination involve the chemical analysis of a culture component or a measure of metabolic activity. These methods are most useful when it is difficult to obtain intact cell samples. However, the relationship between these parameters and the cell number may not be linear through the phases of a cell culture. The determination of nucleic acid (DNA) or total protein can be used as an estimate of biomass, while the depletion of glucose from the media can be used as an estimate of cellular activity. The state of cellular viability may be measured by the release of an enzyme such as lactate dehydrogenase or more directly from the intracellular adenylate energy charge from cell lysates. Alternatively, radioactive techniques may be used for an accurate determination of cellular protein synthesis.

Fed-batch CHO cell t-PA production and feed glutamine replacement to reduce ammonia production.

Industrial therapeutic protein production has been greatly improved through fed-batch development. In this study, improvement to the productivity of a tissue-plasminogen activator (t-PA) expressing Chinese hamster ovary (CHO) cell line was investigated in shake flask culture through the optimization of the fed-batch feed and the reduction of ammonia generation by glutamine replacement. The t-PA titer was increased from 33 mg/L under batch conditions to 250 mg/L with daily feeding starting after three days of culture. A commercially available fed-batch feed was supplemented with cotton seed hydrolysate and the four depleted amino acids, aspartic acid, asparagine, cysteine, and tyrosine. The fed-batch operation increased the generation of by-products such as lactate and ammonia that can adversely affect the fed-batch performance. To reduce the ammonia production, a glutamine-containing dipeptide, pyruvate, glutamate, and wheat gluten hydrolysate, were investigated as glutamine substitutes. To minimize the lag phase as the cells adjusted to the new energy source, a feed glutamine replacement process was developed where the cells were initially cultured with a glutamine containing basal medium to establish cell growth followed by feeding with a feed containing the glutamine substitutes. This two-step feed glutamine replacement process not only reduced the ammonia levels by over 45% but, in the case of using wheat gluten hydrolysate, almost doubled the t-PA titer to over 420 mg/L without compromising the t-PA product quality or glycosylation pattern. The feed glutamine replacement process combined with optimizing other feed medium components provided a simple, practical, and effective fed-batch strategy that could be applied to the production of other recombinant therapeutic proteins.

Differential electronic detector to monitor apoptosis using dielectrophoresis-induced translation of flowing cells (dielectrophoresis cytometry).

The instrument described here is an all-electronic dielectrophoresis (DEP) cytometer sensitive to changes in polarizability of single cells. The important novel feature of this work is the differential electrode array that allows independent detection and actuation of single cells within a short section ([Formula: see text]) of the microfluidic channel. DEP actuation modifies the altitude of the cells flowing between two altitude detection sites in proportion to cell polarizability; changes in altitude smaller than 0.25 µm can be detected electronically. Analysis of individual experimental signatures allows us to make a simple connection between the Clausius-Mossotti factor (CMF) and the amount of vertical cell deflection during actuation. This results in an all-electronic, label-free differential detector that monitors changes in physiological properties of the living cells and can be fully automated and miniaturized in order to be used in various online and offline probes and point-of-care medical applications. High sensitivity of the DEP cytometer facilitates observations of delicate changes in cell polarization that occur at the onset of apoptosis. We illustrate the application of this concept on a population of Chinese hamster ovary (CHO) cells that were followed in their rapid transition from a healthy viable to an early apoptotic state. DEP cytometer viability estimates closely match an Annexin V assay (an early apoptosis marker) on the same population of cells.