Autophagy and Akt-Stimulated Cellular Proliferation Synergistically Improve Antibody Production in CHO Cells.

Over the past decade, engineered producer cell lines have led 10-fold increases in antibody yield, based on an improved understanding of the cellular machinery influencing cell health and protein production. With prospects for further production improvements, increased antibody production would enable a significant cost reduction for life-saving therapies. In this study, we strategized methods to increase cell viability and the resulting cell culture duration to improve production lifetimes.

Electrophoretically Snagging Viral Genomes in Wormlike Micelle Networks Using Peptide Nucleic Acid Amphiphiles and dsDNA Oligomers.

We demonstrate that the attachment of 30-170 bp dsDNA oligomers to ssDNA viral genomes gives a significant additional mobility shift in micelle-tagging electrophoresis (MTE). In MTE, a modified peptide nucleic acid amphiphile is attached to the viral genome to bind drag-inducing micelles present in capillary electrophoresis running buffers. Further attachment of 30-170 bp dsDNA oligomers drastically shifts the mobility of the 5.

Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway.

Rosmarinic acid (RA) has gained attraction in bioprocessing as a media supplement to improve cellular proliferation and protein production. Here, we observe up to a two-fold increase in antibody production with RA-supplementation, and a concentration-dependent effect of RA on cell proliferation for fed-batch Chinese hamster ovary (CHO) cell cultures. Contrary to previously reported antioxidant activity, RA increased the reactive oxygen species (ROS) levels, stimulated endoplasmic reticulum (ER) stress, activated the unfolded protein response (UPR), and elicited DNA damage.

Use of single analytic tool to quantify both absolute N-glycosylation and glycan distribution in monoclonal antibodies.

Recombinant proteins represent almost half of the top selling therapeutics-with over a hundred billion dollars in global sales-and their efficacy and safety strongly depend on glycosylation. In this study, we showcase a simple method to simultaneously analyze N-glycan micro- and macroheterogeneity of an immunoglobulin G (IgG) by quantifying glycan occupancy and distribution. Our approach is linear over a wide range of glycan and glycoprotein concentrations down to 25 ng/mL.

Rapid In-Process Measurement of Live Virus Vaccine Potency Using Laser Force Cytology: Paving the Way for Rapid Vaccine Development.

Vaccinations to prevent infectious diseases are given to target the body's innate and adaptive immune systems. In most cases, the potency of a live virus vaccine (LVV) is the most critical measurement of efficacy, though in some cases the quantity of surface antigen on the virus is an equally critical quality attribute. Existing methods to measure the potency of viruses include plaque and TCID50 assays, both of which have very long lead times and cannot provide real time information on the quality of the vaccine during large-scale manufacturing.

Rapid, multiplexed detection of the let-7 miRNA family using γPNA amphiphiles in micelle-tagging electrophoresis.

miRNA is a promising class of biomarkers whose levels can be assayed to detect various forms of cancer and other serious diseases. These short, noncoding nucleic acids are difficult to detect due to their low abundance and the marginal stability of their duplexes with DNA probes. In addition, miRNAs within the same family have high sequence homology, and often, related miRNA differ in sequence by only a single base.

Viral adventitious agent detection using laser force cytology: Intrinsic cell property changes with infection and comparison to in vitro testing.

Adventitious agent testing in biomanufacturing requires assays of broad detection capability to screen for as many infectious agents as possible. The current gold standard for general infectious adventitious virus screening is the in vitro assay in which test articles are cultured onto a panel of different cell lines and observed for cytopathic effect (CPE). However, this assay is inherently subjective due to the nature of visual observation of cell morphology and labor and time intensive, requiring highly trained personnel to identify CPE.

Determination of the zeta potential of planar solids in nonpolar liquids.

ZetaSpin determines zeta potential by measuring the streaming potential generated by rotating a disk-shaped sample about its axis while submerged in the liquid. The apparatus and procedure developed for ZetaSpin in aqueous solutions was adapted for use in highly nonpolar fluids like surfactant-doped alkanes. Perhaps most unexpected is the need for up to 10 min (instead of a fraction of one second for aqueous solutions) for the electrometer to display changes in streaming potential in response to changes in rotation speed.

Formation of Charge Carriers in Liquids.

After presenting a brief historical overview of the classic contributions of Faraday, Arrhenius, Kohlrausch, Bjerrum, Debye, Hückel and Onsager to understanding the conductivity of true electrolytes in aqueous solutions, we present an in-depth review of the 1933 work of Fuoss & Kraus who explored the effect of the solvent on electrolyte dissociation equilibria in either polar or nonpolar media. Their theory predicts that the equilibrium constant for dissociation decays exponentially with the ratio of the Bjerrum length λ to the ion-pair size a. Fuoss & Kraus experimentally confirmed the dependence on λ of the solvent, while more recent experiments explored the dependence on a.

A 502-Base Free-Solution Electrophoretic DNA Sequencing Method Using End-Attached Wormlike Micelles.

We demonstrate that the use of wormlike nonionic micelles as drag-tags in end-labeled free-solution electrophoresis ("micelle-ELFSE") provides single-base resolution of Sanger sequencing products up to 502 bases in length, a nearly 2-fold improvement over reported ELFSE separations. "CiEj" running buffers containing 48 mM C12E5, 6 mM C10E5, and 3 M urea (32.5 °C) form wormlike micelles that provide a drag equivalent to an uncharged DNA fragment with a length (α) of 509 bases (effective Rh = 27 nm).

Optimization of ELFSE DNA sequencing with EOF counterflow and microfluidics.

We present a nonlinear optimization study of different implementations of the DNA electrophoretic method "End-labeled Free-solution Electrophoresis" in commercial capillary electrophoresis systems and microfluidics to improve the time required for readout. Here, the effect of electro-osmotic counterflows and snap-shot detection are considered to allow for detection of peaks soon after they are electorphoretically resolved. Using drag tags available in micelle form, we identify a design capable of sequencing 600 bases in 2.

Modeling and Global Optimization of DNA separation.

We develop a non-convex non-linear programming problem that determines the minimum run time to resolve different lengths of DNA using a gel-free micelle end-labeled free solution electrophoresis separation method. Our optimization framework allows for efficient determination of the utility of different DNA separation platforms and enables the identification of the optimal operating conditions for these DNA separation devices. The non-linear programming problem requires a model for signal spacing and signal width, which is known for many DNA separation methods.

High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.

Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets.

Quasi-equilibrium AFM measurement of disjoining pressure in lubricant nanofilms II: Effect of substrate materials.

Atomic force microscopy (AFM) was used to measure the disjoining pressures of perfluoropolyether lubricant films (0.8-4.3 nm of Fomblin Z03) on both silicon wafers and hard drive disks coated with a diamondlike carbon overcoat.

Length-dependent DNA separations using multiple end-attached peptide nucleic acid amphiphiles in micellar electrokinetic chromatography.

End-labeled free-solution electrophoresis (ELFSE) is an alternative approach to gel-based methods for size-based electrophoretic separation of DNA. In ELFSE, an electrically neutral "drag-tag" is appended to DNA to add significant hydrodynamic drag, thereby breaking its constant charge-to-friction ratio. Current drag-tag architecture relies on covalent attachment of polymers to each DNA molecule.

Identification of PCR products using PNA amphiphiles in micellar electrokinetic chromatography.

We present a method to identify single-stranded PCR products of varying lengths by hybridization of n-alkylated peptide nucleic acids (PNA amphiphiles) to the products, followed by separation with micellar electrokinetic chromatography (MEKC). These end-attached PNA amphiphiles (PNAA) partition to nonionic micelles in the running buffer (Triton X-100), linking the tagged DNA to the micellar drag-tag. This linkage shifts the electrophoretic mobility of a tagged component away from both untagged DNA and tagged DNA of different lengths.

High capacity, charge-selective protein uptake by polyelectrolyte brushes.

Surface plasmon resonance was used to measure binding of proteins from solution to poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes end-grafted from gold surfaces by atom transfer radical polymerization (ATRP). PDMAEMA brushes were prepared with a variety of grafting densities and degrees of polymerization. These brushes displayed charge selective protein uptake.

Quasi-equilibrium AFM measurement of disjoining pressure in lubricant nano-films I: Fomblin Z03 on silica.

We have identified conditions in which the atomic force microscope can be used to stretch a meniscus of a perfluoropolyether (PFPE) lubricant pinned between an AFM tip and a nanometer-thick PFPE film to obtain the disjoining pressure of the film. Under quasi-equilibrium conditions, the chemical potential of the film can be equated to that of the stretched meniscus. A theory is presented that provides a complete description of the capillary force of a stretched meniscus.

Sequence-specific purification of DNA oligomers in hydrophobic interaction chromatography using peptide nucleic acid amphiphiles: extended dynamic range.

We present improvements on a previously reported method (Vernille JP, Schneider JW. 2004. Biotechnol Prog 20(6):1776-1782) to purify DNA oligomers by attachment of peptide nucleic acid amphiphiles (PNAA) to particular sequences on the oligomers, followed by their separation from unbound oligomers using hydrophobic interaction chromatography (HIC).

Effect of electrostatic interactions on binding and retention of DNA oligomers to PNA liposomes assessed by FRET measurements.

A FRET-based method is used to observe the desorption of di-alkyl peptide nucleic acid amphiphiles (PNAA) from liposomes occurring on binding of complementary DNA oligomers. PNA liposomes were prepared containing fluorescein-labeled PNAA and rhodamine-labeled dipalmitoylphosphoethanolamine (DPPE). These liposomes showed efficient energy transfer from the fluorescein to rhodamine, with an average donor-to-acceptor distance of 5.

Morphological characterization of self-assembled peptide nucleic acid amphiphiles.

Peptide nucleic acid amphiphiles (PNAA) are a promising set of materials for sequence-specific separation of nucleic acids from complex mixtures. To implement PNAA in micellar separations, the morphology and size of PNAA micelles in the presence and absence of a sodium dodecyl sulfate (SDS) cosurfactant have been studied by small-angle X-ray scattering and dynamic light scattering. We find that a 6-mer PNAA with a 12-carbon n-alkane tail forms ellipsoidal micelles (a = 5.

Characterization of distance-dependent damping in tapping-mode atomic force microscopy force measurements in liquid.

We have used a spectral analysis method to characterize changes in the local damping coefficient for an acoustically driven cantilever as it approaches a hard surface in liquid. We show a significant distance dependence of the damping coefficient (and associated quality factor) that must be accounted for to achieve successful theoretical reproduction of experimental tapping-mode force curves. We model the cantilever dynamics using a forced damped harmonic oscillator model and solve the equation of motion using the method of finite differences.

Thermodynamic and structural characterization of amino acid-linked dialkyl lipids.

Using differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR), we determined some thermodynamic and structural parameters for a series of amino acid-linked dialkyl lipids containing a glutamic acid-succinate headgroup and di-alkyl chains: C12, C14, C16 and C18 in CHES buffer, pH 10. Upon heating, DSC shows that the C12, C14 and annealed C16 lipids undergo a single transition which XRD shows is from a lamellar, chain ordered subgel phase to a fluid phase. This single transition splits into two transitions for C18, and FTIR shows that the upper main transition is predominantly the melting of the hydrocarbon chains whereas the lower transition involves changes in the headgroup ordering as well as changes in the lateral packing of the chains.

Sequence-specific binding of DNA to liposomes containing di-alkyl peptide nucleic acid (PNA) amphiphiles.

We present a method to covalently attach peptide nucleic acid (PNA) to liposomes by conjugation of PNA peptide to charged amino acids and synthetic di-alkyl lipids ("PNA amphiphile," PNAA) followed by co-extrusion with disteroylphosphatidylcholine (DSPC) and cholesterol. Attachment of four Glu residues and two ethylene oxide spacers to the PNAA was required to confer proper hydration for extrusion and presentation for DNA hybridization. The extent of DNA oligomer binding to 10-mer PNAA liposomes was assessed using capillary zone electrophoresis.