Katrin Braasch

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.

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. Biotechnol. Bioeng. 2013;110: 2902–2914.

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.

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 107 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. Biotechnol. Bioeng. 2012; 109:1228–1238.

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.

Electroporation and dielectrophoresis of single cells using a microfluidic system employing a microwave interferometric sensor

We describe a microfluidic device integrated with a microwave interferometric sensor that is able to simultaneously electroporate and measure the dielectrophoresis (DEP) response of single biological cells. The system can measure changes in dielectric properties of a cell permeabilized using a high-intensity pulsed electric field (PEF) from a few seconds after exposure to the pulses. It provides a mechanism for investigating time-dependent changes in the cell membrane and ion flux. Using this device, experiments performed on single Chinese hamster ovary (CHO) cells exposed to microsecond pulsed electric fields show significant changes in their DEP response immediately after exposure.

Multi-Frequency DEP Cytometer Employing a Microwave Sensor for Dielectric Analysis of Single Cells

We present a microfluidic device for in-flow dielectric characterization of single biological cells. The dielectric spectrum is obtained by measuring the multiple-frequency dielectrophoresis (DEP) response of individual cells as they travel over an array of sensing and actuating electrodes. The DEP induced translation of each cell is detected by measuring the differential impedance of the array using a microwave interferometer, which is capable of sub-attofarad sensitivity, and is coupled to the sensing electrodes. The DEP response of a cell at multiple frequencies in the beta-dispersion region is chosen to discern particular cell dielectric properties as it travels along the array-such as cytoplasm conductivity and membrane capacitance. The Clausius-Mossotti factor of the cell is determined from the measured response signal in conjunction with numerical simulation of its trajectory. The approach is validated through measuring polystyrene microspheres. The DEP response of Chinese hamster ovary cells using two simultaneous frequencies is demonstrated.

Multi-frequency DEP cytometer employing a microwave interferometer for the dielectric analysis of micro-particles

We present a multi-frequency dielectrophoresis (DEP) based microfluidic device for characterizing the complex dielectric properties of single micron-sized particles while in flow. The device employs a multi-electrode transmission line sensor coupled to a microwave-interferometer, capable of sub-attofarad sensitivity, for detecting the DEP-induced translation of the particle under study. DEP actuation of the particle at different frequencies - which is related to its dielectric response - is sensed as it travels along the sensor. Characterization of the dielectric response of polystyrene micro-spheres using two frequencies is demonstrated.

Dielectrophoresis study of temporal change in internal conductivity of single CHO cells after electroporation by pulsed electric fields

Applying sufficiently strong pulsed electric fields to a cell can permeabilize the membrane and subsequently affect its dielectric properties. In this study, we employ a microfluidic dielectrophoresis cytometry technique to simultaneously electroporate and measure the time-dependent dielectric response of single Chinese hamster ovary cells. Using experimental measurements along with numerical simulations, we present quantitative results for the changes in the cytoplasm conductivity of single cells within seconds after exposure to 100 μs duration pulsed electric fields with various intensities. It is shown that, for electroporation in a medium with conductivity lower than that of the cells cytoplasm, the internal conductivity of the cell decreases after the electroporation on a time scale of seconds and stronger pulses cause a larger and more rapid decrease. We also observe that, after the electroporation, the cells internal conductivity is constrained to a threshold. This implies that the cell prevents some of the ions in its cytoplasm from diffusing through the created pores to the external medium. The temporal change in the dielectric response of each individual cell is continuously monitored over minutes after exposure to pulsed electric fields. A time constant associated with the cells internal conductivity change is observed, which ranges from seconds to tens of seconds depending on the applied pulse intensity. This experimental observation supports the results of numerical models reported in the literature.