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. Despite such stressful events, RA appeared to maintained cell health via mammalian target of rapamycin (mTOR) pathway activation; both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) were stimulated in RA-supplemented cultures. By reversing such mTOR pathway activity through either chemical inhibitor addition or siRNA knockdown of genes regulating the mTORC1 and mTORC2 complexes, antibody production, UPR signaling, and stress-induced DNA damage were reduced. Further, the proliferative effect of RA appeared to be regulated selectively by mTORC2 activation and have reproduced this observation by using the mTORC2 stimulator SC-79. Analogously, knockdown of mTORC2 strongly reduced X-box binding protein 1 (XBP1) splicing, which would be expected to reduce antibody folding and secretion, sugging that reduced mTORC2 would correlate with reduced antibody levels. The crosstalk between mTOR activation and UPR upregulation may thus be related directly to the enhanced productivity. Our results show the importance of the mTOR and UPR pathways in increasing antibody productivity, and suggest that RA supplementation may obviate the need for labor-intensive genetic engineering by directly activating pathways favorable to cell culture performance.

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. Additionally, we present a case study demonstrating the effect of small molecule metabolic regulators on glycan heterogeneity using this approach. In particular, sodium oxamate (SOD) decreased Chinese hamster ovary (CHO) glucose metabolism and reduced IgG glycosylation by 40% through upregulating reactive oxygen species (ROS) and reducing the UDP-GlcNAc pool, while maintaining a similar glycan profile to control cultures. Here, we suggest glycan macroheterogeneity as an attribute should be included in bioprocess screening to identify process parameters that optimize culture performance without compromising antibody quality.

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. Here, we report the evaluation of LumaCyte's Radiance Laser Force Cytology platform as a new way to measure the potency of LVVs in upstream biomanufacturing process in real time and compare this to traditional TCID50 potency. We also assess this new platform as a way to detect adventitious agents, which is a regulatory expectation for the release of commercial vaccines. In both applications, we report the ability to obtain expedited and relevant potency information with strong correlation to release potency methods. Together, our data propose the application of Laser Force Cytology as a valuable process analytical technology (PAT) for the timely measurement of critical quality attributes of LVVs.

Progress toward rapid, at-line N-glycosylation detection and control for recombinant protein expression.

Proteins continue to represent a large fraction of the therapeutics market, reaching over a hundred billion dollars in market size globally. One key feature of protein modification that can affect both structure and function is the addition of glycosylation following protein folding, leading to regulatory requirements for the accurate assessment of protein attributes, including glycan structures. The non-template-driven, innately heterogeneous N-glycosylation process thus requires accurate detection to robustly generate protein therapies. A challenge exists in the timely detection of protein glycosylation without labor-intensive manipulation. In this article, we discuss progress toward N-glycoprotein control, focusing on novel control strategies and the advancement of rapid, high-throughput analysis methods.

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. Laser force cytology (LFC) is an alternative, automated analytical method that uses a combination of optical and fluidic forces along with imaging to objectively and quantitatively assess CPE in cell culture. Importantly, because LFC uses no labels or antibodies, the assay is appropriate for general adventitious agent testing. Using LFC, changes in cellular features associated with virally infected cells were identified using principal component analysis. Using these features of infected cells, the sensitivity and earliness of detection with LFC was directly compared with the in vitro assay for a diverse panel of viruses incubated with chinese hamster ovary (CHO), Vero, and Medical Research Council cell strain 5 (MRC-5) cells. LFC detected viral infection with a sensitivity equal to the in vitro assay on average, but in certain virus and cell combinations including mouse minute virus (MMV) and reovirus 3 in CHO cells, detection was 4 days earlier and for MMV, the limit of detection was 10-fold lower. Overall, these results demonstrate the ability of LFC to serve as a biopharmaceutical adventitious agent testing methodology with sensitivity equivalent to the in vitro assay, but in an objective and automated manner.

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. In this report, we demonstrate an independent detection seven members of the let-7 family of miRNA in a single run. Key to success is the use of mini-PEG-substituted PNA amphiphiles (γPNAA) and highly fluorescent DNA nanotags in micelle tagging electrophoresis (MTE). Multiplexed detection is accomplished in capillary electrophoresis (CE) using oligomeric nanotags of pre-programmed lengths where the presence of a specific miRNA links its nanotag to a micelle drag-tag, which shifts the nanotag elution time to a defined region for detection. We further demonstrate that the peak shape and elution time are unaffected by the presence of up to 10 mg/ml of serum protein in the sample, with a total runtime of less than 4 min and a LOD of 10-100 pM. We discuss efforts to substantially decrease the detection limit using nanotags that are >1000 bp in length.

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. Four tests (suggested by theory) confirm that the potential finally reported by the electrometer was indeed the streaming potential. Compared to electrophoresis, ZetaSpin does not require a value for the Debye length, avoids the complication caused by the electric-field-dependence of electrophoretic mobility and can be used with planar samples as well as colloidal particles.

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 λB to the ion-pair size a. Fuoss & Kraus experimentally confirmed the dependence on λB of the solvent, while more recent experiments explored the dependence on a. We also present an in-depth review of the charge-fluctuation theory used to explain the sharp increase in conductivity with added water for water-in-oil microemulsions stabilized by ionic surfactants. Water swells the droplets making a greater fraction of them charged. At least for low-water content, the same exponential dependence on λB/a is predicted, provided a is chosen as the size of the polar core of the droplet or inverted micelle. Potential electrolytes like alcohols acquire charge by exchanging a proton. The dissociation equilibrium of the resulting ion-pair in mixtures of toluene and alcohol appears to be well modelled by the Fuoss theory. Solutions of inverted micelles are also thought to acquire charge by exchanging a small ion between two net-neutral micelles. Except for the dissociation of true electrolytes, all of the charging scenarios described above can be represented by a two-reaction sequence: 1) the disproportionation of charge between two neutral molecules, inverted micelles or droplets; followed by 2) the dissociation of the "ion"-pair intermediates. (The dissociation of true electrolytes involves only the second.) For each of the above charging theories, the extent of the second reaction decays exponentially with λB/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). Runtime in a 40 cm capillary (30 kV) was 35 min for elution of all products down to the 26-base primer. We also show that smaller Triton X-100 micelles give a read length of 103 bases in a 4 min run, so that a combined analysis of the Sanger products using the two buffers in separate capillaries could be completed in 14 min for the full range of lengths. A van Deemter analysis shows that resolution is limited by diffusion-based peak broadening and wall adsorption. Effects of drag-tag polydispersity are not observed, despite the inherent polydispersity of the wormlike micelles. We ascribe this to a stochastic size-sampling process that occurs as micelle size fluctuates rapidly during the runtime. A theoretical model of the process suggests that fluctuations occur with a time scale less than 10 ms, consistent with the monomer exchange process in nonionic micelles. The CiEj buffer has a low viscosity (2.7 cP) and appears to be semidilute in micelle concentration. The large drag-tag size of the CiEj buffers leads to steric segregation of the DNA and tag for short fragments and attendant mobility shifts.

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 2.8 min.

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. As a case study, we show how our approach is used to determine the optimal run conditions for micelle end-labeled free-solution electrophoresis and examine the trade-offs between a single capillary system and a parallel capillary system. Parallel capillaries are shown to only be beneficial for DNA lengths above 230 bases using a polydisperse micelle end-label otherwise single capillaries produce faster separations.

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. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.

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. Differences in the disjoining pressure between the two systems were expected to be due to variations in the strength of van der Waals interactions. Lifshitz theory calculations suggest that this substrate switch will lead to relatively small changes in disjoining pressure as compared to the more pronounced effects reported due to changes in lubricant chemistry. We demonstrate the sensitivity of our AFM method by distinguishing between these similar systems.

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. We have recently proposed the use of micellar drag-tags in conjunction with sequence-specific hybridization of peptide nucleic acid amphiphiles (PNAAs). This work investigates the effect of multiple PNAA attachment on DNA resolution using MEKC. Simultaneous PNAA hybridization allows for the separation of long DNA targets, up to 1012 bases, using micellar drag-tags. Each PNAA handle independently interacts with the micellar phase, reducing the overall mobility of this complex relative to individual PNAA binding. The sequence- and size-based dependence of this separation technique is maintained with multiple PNAA binding over a range of DNA sizes. Results are accurately described by ELFSE theory, yielding alpha=54 for single-micelle tagging and alpha=142 for dual-micelle tagging. This method is the first example of a non-covalent drag-tag used to separate DNA of 1000 bases based on both size and sequence.

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. The mobility of the tagged DNA is established by its extent of partitioning to the micelle phase as well as its size relative to the attached micelle. A model is presented that can be used to determine the length of an unknown oligomer given an experimentally obtained mobility. We find that the collective action of micelles that transiently attach to the tagged DNA impart about the same hydrodynamic drag as covalently bound "drag-tags" of a similar size. With the use of the PNAA-MEKC method, PCR products of 88, 134, 216, and 447 bases are clearly resolved in less than 5 min. To our knowledge, this work represents the first use of surfactant micelles as drag-tags to separate DNA in capillary electrophoresis. Furthermore, the PNAA tag only attaches to DNA containing a target sequence, helping ensure that only the desired PCR products are analyzed.

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. The extent of uptake for net negatively charged bovine serum albumin (BSA) scaled linearly with the surface mass concentration of grafted PDMAEMA, regardless of grafting density. BSA was bound at a constant ratio of 120 DMAEMA monomer units per protein molecule for all brushes examined. The equivalent three-dimensional concentration of BSA bound in the brush (i.e., the bound BSA surface excess concentration divided by the brush thickness) decreased monotonically with decreasing grafting density. The concentration of BSA bound within brushes prepared at higher grafting densities was comparable with the aqueous protein solubility limit. BSA desorption from the brush required changes in solution pH and/or ionic strength to eliminate its net electrostatic attraction to PDMAEMA. Net positively charged lysozyme was completely rejected by the PDMAEMA brushes.

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). Use of alkyl-modified HIC media (butyl and octyl sepharose) over phenyl-modified media (phenyl sepharose) reduced the elution time of unbound DNA while not affecting the elution time of the PNAA/DNA complex. Modifying the alkane tail length for PNAA from C(12) to C(18) increased slightly the retention of PNAA/DNA duplexes. By combining these two refinements, we show that sequence-specific purifications of DNA oligomers 60 bases in length or more can be achieved with high resolution, even when the PNAA alkane is attached to the center of the target strand. The insensitivity of the PNAA/DNA duplex binding to choice of HIC media appears to be due to a surface-induced aggregation phenomenon that does not occur in the case of untagged DNA. We also report on the use of batch HIC as an adequate predictor of elution profiles in linear gradient HIC, and its potential to considerably reduce purification times by applying step gradients.

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. Fits of the theory to quasi-equilibrium force-extension curves yield the effective meniscus curvature and, by extension, the disjoining pressure of the underlying film. AFM force curves collected at varying film thicknesses compare very well with predictions from Lifshitz theory of dispersive interactions in thin films, with no adjustable parameters. This complete description of meniscus deformation during atomic force microscopy force-extension experiments makes possible the measurement of unknown disjoining pressures as required for screening of lubricant-overcoat combinations required for next-generation data storage systems.

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 5.91nm. In low-ionic-strength buffer (50mM Tris-HCl, pH 8.0), the FRET signal was maintained in the presence of a stoichiometric amount of 10- and 20-mers DNA complements, but the signal attenuated for 40-mer complements, indicating that DNA first binds the PNAA before the PNAA/DNA duplex desorbs from the lipid bilayer. The FRET signal was maintained in the presence of 10-, 20-, 40-, and 60-mer DNA in high ionic-strength buffer, showing that the driving force for the desorption is electrostatic repulsion between the bound DNA oligomer and the liposome surface. This conclusion is corroborated by comparison of the PNA/DNA binding energy, the energy of adsorption of the di-alkyl PNAA to the lipid bilayer, and a calculation of the DNA/lipid bilayer electrostatic repulsion using the linearized Poisson-Boltzmann equation.

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 = 5.15 nm; b = 3.20 nm) above its critical micelle concentration (CMC) of 110.9 microM. On addition of a stoichiometric amount of complementary DNA, PNAA hybridizes to DNA, suppressing the formation of PNAA micelles. At a ratio of 19:1 SDS/PNAA (total concentration = 20 mM), spherical micelles are formed with outer radius Rs = 2.67 nm, slightly larger than spherical micelles of pure SDS. Capillary electrophoresis studies show that PNAA/DNA duplexes do not comicellize with SDS micelles. No such effects are observed using noncomplementary DNA. The shape and size of the PNAA micelles is also verified by dynamic light scattering (DLS) studies. These results provide an interesting case study with competing electrostatic, hydrophobic, and hydrogen-bonding interactions in micellar systems and make possible the use of PNAA in micellar separations of DNA oligomers.