Michael Butler

National Institute for Bioprocessing Research and Training (NIBRT) | NIBRT · Cell Technology Group

With the global spread of the corona virus disease-2019 pandemic, new spike variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continuously emerge due to increased possibility of virus adaptive amino acid mutations. However, the N-glycosylation profiles of different spike variants are yet to be explored extensively, although t...

Disease development and progression are often associated with aberrant glycosylation, indicating that changes in biological fluid glycome may potentially serve as disease signatures. The corona virus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a significant threat to global huma...

Glycosylation is well-recognized as a critical quality attribute of biotherapeutics being routinely monitored to ensure desired product quality, safety, and efficacy. Additionally, as one of the most prominent and complex post-translational modifications, glycosylation plays a key role in disease manifestation. Changes in glycosylation may serve as...

Previous reports present different models for the stabilization of the Fc—FcγRI immune complex. Although accord exists on the importance of L235 in IgG1 and some hydrophobic contacts for complex stabilization, discord exists regarding the existence of stabilizing glycoprotein contacts between glycans of IgG1 and a conserved FG-loop (¹⁷¹MGKHRY¹⁷⁶) o...

The N-glycosylation profile of a monoclonal antibody (mAb) is a critical quality attribute in relation to its therapeutic application. The control of this profile during biomanufacture is difficult because of the multiple parameters that affect the glycosylation metabolism within the cell and the environment in which the cell is grown. One of the a...

This document presents hydrogen-deuterium exchange mass spectrometry (HDX-MS) data from measurements of three purified IgG1 glycoform samples, predominantly G0F, G2F, and SAF, in isolation and in complexation with the high-affinity receptor, FcγRIa (CD64). The IgG1 antibody used in this study, aIL8hFc, is a murine-human chimeric IgG1, which inhibit...

The control of glycosylation profiles is essential to the consistent manufacture of therapeutic monoclonal antibodies that may be produced from a variety of cell lines including CHO and NS0. Of particular concern is the potential for generating non-human epitopes such as N-glycolylneuraminic acid (Neu5Gc) and Galα1−3 Gal that may be immunogenic. We...

Four independent mAb-producing CHO cell lines were grown in media supplemented with one of seven protein hydrolysates of animal and plant origin. This generated a 7x4 matrix of replicate cultures which was analysed for viable cell density and mAb productivity. In all cultures, a consistent growth rate was shown in batch culture up to 4 to 5 days. D...

Protein glycosylation contributes to critical biological function of glycoproteins. Glycan analysis is essential for the production of biopharmaceuticals as well as for the identification of biomarkers of disease. However, glycans are highly heterogeneous, which has considerably hampered the progress of glycomics. Here, we present an improved 96-we...

The dielectric properties of biological cells can be used to gain information on their physiology and morphology. This paper reports the first measurements of the dielectric spectra of viable and non-viable cells over the full beta-dispersion (interfacial) frequency range. Dielectrophoresis (DEP) single cell in-flow techniques were employed to quan...

Electrospray mass spectrometry (ESI-MS) was used to measure the masses of an intact dimeric monoclonal antibody (Mab) and assess the fucosylation level. The Mab under study was EG2-hFc, a chimeric human-camelid antibody of about 80 kDa (A. Bell et al., Cancer letters 2010, 289 (1), 81-90). It was obtained from cell culture with and without a fucosy...

In this work, we present an optical transit dielectrophoresis (DEP) flow cytometer for parallel single‐cell analysis. Each cell's dielectric property is inferred from velocity perturbations due to DEP actuation in a microfluidic channel. Dual LED sources facilitate velocity measurement by producing two transit shadows for each cell passing through...

Core fucosylation of an Fc N-linked glycan affects antibody effector functions, as the absence of fucose increases the antibody dependent cell cytotoxicity (ADCC) response with increased binding to the Fcγ receptors. The work presented here compares two different approaches to incrementally reduce core fucosylation of a camelid heavy chain antibody...

Amino acid availability is a key factor that can be controlled to optimize the productivity of fed‐batch cultures. To study amino acid limitation effects, a serum‐free chemically defined basal medium was formulated to exclude the amino acids that became depleted in batch culture. The effect of limiting glutamine, asparagine and cysteine on the cell...

Trypan blue dye exclusion first was proposed as a means of measuring mammalian cell damages over a century ago in 1917. Despite extensive documentation of its limitations, it remains the “gold standard” method of measuring cell viability in common use today. But can this method truly measure viability? And how do we define cell viability, for that...

Glucose depletion

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 marker‐less indi...

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...

Glycosylation is the most prevalent form of posttranslational modification of recombinant glycoproteins. N‐linked glycans are added to protein as a co‐translational modification in the endoplasmic reticulum (ER). Recombinant proteins may also contain O‐linked glycans attached through an N‐acetylgalactosamine residue that are structurally different...

Abstract 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 i...

Dielectrophoresis (DEP) cytometry was used to investigate changes in the membrane capacitance, membrane conductance, and cytoplasm conductivity of single Chinese hamster ovary (CHO) cells subjected to thermal stress. CHO cells were exposed to temperatures ranging from 37 °C to 50 °C for 15 min and measured immediately after exposure. Heat-treated c...

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 measure...

Antibodies are synthesized in mammalian cell culture as heterogeneous mixtures of glycoforms. Production of single glycoforms remains a challenge despite their value as therapeutics. We report a method of sequential enzymatic-based changes to antibodies while immobilized on an affinity column. Various antibodies (monoclonal and polyclonal) were iso...

Humans infected with H7N9 avian influenza viruses can result in severe pneumonia and acute respiratory syndrome with an approximately 40% mortality rate, and there is an urgent need to develop an effective vaccine to reduce its pandemic potential. In this study, we used a novel PELC/CpG adjuvant for recombinant H7HA (rH7HA) subunit vaccine developm...

Human interferon gamma (hIFNγ) affects tumour cells and modulates immune responses, showing promise as an anti-cancer biotherapeutic. This study investigated the effect of glycosylation and expression system of recombinant hIFNγ in ovarian carcinoma cell lines, PEO1 and SKOV3. The efficacy of E. coli- and mammalian-expressed hIFNγ (hIFNγ-CHO and HE...

An integrated dielectrophoresis cytometer has been designed and implemented using a standard 0.35 μm CMOS technology. This design makes use of differential ring oscillators. The frequency difference between the two oscillators, in presence of a cell, is detected by an XOR gate. The frequency difference is then measured by a frequency counter. This...

Cation exchange (CEX) chromatography is widely used for large-scale separation of monoclonal antibody (mAb) aggregates. The aggregates bind more strongly to CEX media and hence elute after the monomeric mAb in a salt gradient. However, monomer-aggregate resolution that is typically obtained is poor, which results in low product recovery. In the cur...

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 operati...

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 microfluidicdielectrophoresis cytometry technique to simultaneously electroporate and measure the time-dependent dielectric response of single Chinese hamster ovary cells. Using ex...

Glycosylation and intracellular assembly of monoclonal antibodies (MAbs) is important for glycan profile consistency. To better understand how these factors may be influenced by a lower redox potential, an IgG1-producing NS0 cell line was grown in the presence of varying concentrations of dithiothreitol (DTT). Cultures were monitored for growth and...

Glycoengineering of mAbs has become common practice in attempts to generate the ideal mAb candidate for a wide range of therapeutic applications. The effects of these glycan modifications on the binding affinity of IgG mAbs for FcγRIIIa and their cytotoxicity is well known. However, little is understood about the effect that these modifications hav...

Yeast and plant hydrolysates are used as media supplements to support the growth and productivity of CHO cultures for biopharmaceutical production. Through fractionation of a yeast lysate and metabolic analysis of a fraction that had bioactivity equivalent to commercial yeast extract (YE), bioactive components were identified that promoted growth a...

Importance: Influenza viruses trigger seasonal epidemics or pandemics with mild-to-severe consequences for human and poultry populations. DCs are the most potent professional antigen-presenting cells, which play a crucial role for the link of innate and adaptive immunity. In this study, we obtained stable-expression CHO cells to produce rH1HA, rH5...

We present a dielectric model and its parameters for Chinese hamster ovary (CHO) cells based on a double-shell structure which includes the cell membrane, cytoplasm, nuclear envelope, and nucleoplasm. Employing a dielectrophoresis(DEP) based technique and a microfluidic system, the DEP response of many single CHO cells is measured and the spectrum...

We present a microfluidic device for measuring the dielectrophoresis (DEP) response of single Chinese hamster ovary cells while in flow. The device measures the sign and amplitude of the DEP induced translation of single cells at two frequencies by sensing the change in impedance at microwave frequency as the cell passes over a multi-electrode tran...

The presence of a2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized a2,6-sialylated IgGs would help elucidate...

Glycosylation affects many general functional factors of a glycoprotein, such as stability, inhibition of proteolysis, solubility, aggregation, as well as other attributes critical to its use as a biotherapeutic. Therefore our understanding of the glycosylation pathway, factors that affect it, and our ability to manipulate glycosylation are essenti...

Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-man...

Rationale In the expression of recombinant proteins, an important parameter to control or influence is their level of sialylation. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric (MS) methods tend to either underestimate (positive mode) or overestimate (negative mode) the content of sialylated vs. neutral g...

Studying biological phenomena at the cellular level allows understanding the basics of cellular function and the mechanism of their interactions with internal or external stimuli. Among available single cell analysis techniques such as, fluorescent labeling, magnetic labeling, mechanical, and electrical approaches, electrical ones are of special in...

N-glycosylation is one of the critical quality attributes for the therapeutic monoclonal antibodies. Characterization of N-glycans of monoclonal antibodies provides valuable information about its therapeutic efficacy. We present a non-invasive method of isolating N-glycans from Immunoglobulin G based antibodies for glycosylation analysis. The metho...

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. There...

Controlled feeding of glucose has been employed previously to enhance the productivity of recombinant glycoproteins but there is a concern that low concentrations of glucose could limit the synthesis of precursors of glycosylation. Here we investigate the effect of glucose depletion on the metabolism, productivity and glycosylation of a chimeric hu...

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 subattofarad sensitivity, for detecting the DEP-induced tr...

A microwave frequency reflectometer with atto-farad capacitance sensitivity has been designed and tested for detection of particles and cells in a microfluidic channel. The design is implemented on a printed circuit board. Capacitance sensitivity up to 1.25 aF is achieved in a 50 Hz bandwidth. The sensor was used for detection of 5.7 μm polystyrene...

The article is now open source! Find it at http://bit.ly/1ESjp79. The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated acces...

The characterization of the N-glycan portion of antibodies has been the subject of several studies involving mass spectrometry. In this article, a workflow is presented that starts with the expression of a monoclonal antibody (EG2-hFc) in Chinese hamster ovary cells and continues with Protein A purification of the antibody. Then the protocol contin...

For many decades bovine serum has been used as an essential component for the growth of animal cells in culture. However, the combined disadvantages of variability in composition, cost but particularly the potential for contamination with viruses or prions has been the driver for the substitution of serum with a more defined and animal-component fr...

Glycosylation is a critical quality attribute of biotherapeutics produced from mammalian cells. Small changes in the glycan structures may have profound effects on the efficacy and functions of the glycoprotein, such as fucosylation of the IgG glycan reducing the ability of the IgG to bind to the FcϒRIIIA receptor. Chinese hamster ovary produced gl...

One of the main uses of adenosine triphosphate (ATP) within mammalian cells is powering the Na +/K+ ATPase pumps used to maintain ion concentrations within the cell. Since ion concentrations determine the cytoplasm conductivity, ATP concentration is expected to play a key role in controlling the cytoplasm conductivity. The two major ATP production...

Modulating autophagy provides a new method to increase CHO cell protein production. A fed-batch protocol using the autophagy inhibitor 3-methyl adenine (3-MA), developed for a tissue-plasminogen activator (t-PA) expressing DHFR based CHO cell line, was successfully adapted to a monoclonal antibody (MAb) expressing CHOK1-SV based CHO cell line. By o...

In this work the dielectric response evaluated by dielectrophoresis (DEP) cytometer was used to track cytoplasm conductivity and therefore ion concentrations during apoptosis induced by inhibition of mitochondrial ATP production. Oligomycin was used to inhibit mitochondrial ATP production in Chinese hamster ovary (CHO) cells. During induction healt...

Electroporation affects the dielectric properties of cells by creating pores in their membrane which allow transport of ions. Dielectrophoresis (DEP) is a powerful technique to investigate electroporation of single biological cells. Here we study the frequency dependent response of Chinese hamster ovary (CHO) cells and investigate the effect of dif...

Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, t...

Peptide hydrolysate supplements in mammalian cell cultures provide enhanced growth and productivity. The objective of this study was to compare the bioactivity of ten different commercially available hydrolysates from plant, microbial and animal sources. The peptide hydrolysates were tested as supplements to cultures of Chinese hamster ovary (CHO)...

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 fixe...

The glycosylation pattern of a chimeric heavy chain antibody (EG2) produced from CHO cells was affected by the glucose concentration (0-25mM) of cultures established at high density (>10(6)/ml) over 24h. The resulting proportion of non-glycosylated Mab was directly correlated to the exposure time of cells to media depleted of glucose. Deprivation o...

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 i...

Highly pathogenic avian influenza H5N1 viruses can result in poultry and occasionally in human mortality. A safe and effective H5N1 vaccine is urgently needed to reduce the pandemic potential. Hemagglutinin (HA), a major envelope protein accounting for approximately 80% of spikes in influenza virus, is often used as a major antigen for subunit vacc...

T-cell stimulation by antigen-presenting dendritic cells. Pre-treated DCs with LPS and recombinant HA proteins were co-incubated with splenocytes from mice immunized with different recombinant HA proteins for 2 d. Antigen presentation was determined by measuring (A) IFN-γ- and (B) IL-2-secreting T cells using ELISPOT assays. DCs pretreated with LPS...

Fetuin binding assays. 96-well plates were coated with 100 µg/ml fetuin and incubated with serially diluted recombinant HA pre-complexed with HRP-linked anti-His tag antibodies for 60 min at room temperature. HA binding was subsequently detected by ELISAs (450 nm OD). Data represent mean ± standard deviation. (TIFF)

Glycan array analyses. Recombinant HA proteins were added to a 98-glycan array and incubated for 1 h at 37°C. Rabbit anti-H5HA antibodies were added and incubated for 1 h. Cy3-conjugated goat anti-rabbit IgG antibodies were added next, followed by incubation for another 1 h. Binding activity is shown as fluorescence intensity detected by scanning w...