Strategy for Generating Sequence-Defined Aptamer Reagent Sets for Detecting Protein Contaminants in Biotherapeutics.

Biologic drugs are typically manufactured in mammalian host cells, and it is critical from a drug safety and efficacy perspective to detect and remove host cell proteins (HCPs) during production. This is currently achieved with sets of polyclonal antibodies (pAbs), but these suffer from critical shortcomings because their composition is inherently undefined, and they cannot detect nonimmunogenic HCPs. In this work, we report a high-throughput screening and array-based binding characterization strategy that we employed to generate a set of aptamers that overcomes these limitations to achieve sensitive, broad-spectrum detection of HCPs from the widely used Chinese hamster ovary (CHO) cell line.

Decomposing Dynamical Couplings in Mutated scFv Antibody Fragments into Stabilizing and Destabilizing Effects.

Conformational fluctuations within scFv antibodies are characterized by a novel perturbation-response decomposition of molecular dynamics trajectories. Both perturbation and response profiles are stratified into stabilizing and destabilizing conditions. The linker between the VH and VL domains exhibits the dominant dynamical response by being coupled to nearly the entire protein, responding to both stabilizing and destabilizing perturbations.

Mutations in Antibody Fragments Modulate Allosteric Response Via Hydrogen-Bond Network Fluctuations.

A mechanical perturbation method that locally restricts conformational entropy along the protein backbone is used to identify putative allosteric sites in a series of antibody fragments. The method is based on a distance constraint model that integrates mechanical and thermodynamic viewpoints of protein structure wherein mechanical clamps that mimic substrate or cosolute binding are introduced. Across a set of six single chain-Fv fragments of the anti-lymphotoxin-β receptor antibody, statistically significant responses are obtained by averaging over 10 representative structures sampled from a molecular dynamics simulation.

Modulation of the Hydration Water Around Monoclonal Antibodies on Addition of Excipients Detected by Terahertz Time-Domain Spectroscopy.

Terahertz time-domain spectroscopy (THz-TDS) has been shown to detect overlapping extended hydration layers around proteins. Here, we used THz-TDS to detect modulation of the extended hydration layer around monoclonal antibodies (mAbs) by the introduction of commonly used excipients. Proline and sucrose altered the hydration layer around a mAb (mAb1), which was observed as a negative shift in the plateau in absorbance above ~100 mg/mL mAb1 (~70,000 water molecules per mAb); arginine had no effect.

Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants.

Characterisation of Stress-Induced Aggregate Size Distributions and Morphological Changes of a Bi-Specific Antibody Using Orthogonal Techniques.

A critical step in monoclonal antibody (mAb) screening and formulation selection is the ability of the mAb to resist aggregation following exposure to environmental stresses. Regulatory authorities welcome not only information on the presence of micron-sized particles, but often any information on sub-visible particles in the size range obtained by orthogonal sizing techniques. The present study demonstrates the power of combining established techniques such as dynamic light scattering (DLS) and micro-flow imaging (MFI), with novel analyses such as raster image correlation spectroscopy (RICS) that offer to bridge existent particle sizing gaps in this area.

Universal method for the determination of nonionic surfactant content in the presence of protein.

A new analytical method has been developed for the quantitative determination of ethylene glycol-containing nonionic surfactants, such as polyethylene glycol 8000, polysorbate 80, and Pluronic F-68. These surfactants are commonly used in pharmaceutical protein preparations, thus, testing in the presence of protein is required. This method is based on the capillary gas chromatographic analysis of ethylene glycol diacetate formed by hydrolysis and acetylation of surfactants that contain ethylene glycol.

Mechanism of reversible self-association of a monoclonal antibody: role of electrostatic and hydrophobic interactions.

Reversible self-association of protein therapeutics, the phenomenon of formation of native reversible oligomeric species as a result of noncovalent intermolecular interactions, can add additional manufacturing, stability, delivery, and safety complexities in biopharmaceutical development. Its early detection, characterization, and mitigation can therefore contribute to the success of drug development. A variety of structural and environmental factors can contribute to the modulation of self-association with mechanisms still elusive in some cases due to the inherent structural complexity of proteins.

The effect of palmitoylation on the conformation and physical stability of a model peptide hormone.

Peptides are ideal drug candidates due to their potency and specificity, but suffer from a short half-life and low membrane permeability. Acylation can overcome these limitations but the consequences to stability under different formulation conditions and stresses are largely unreported. Using synchrotron radiation circular dichroism (SRCD), we show that palmitoylation of a 28 amino acid peptide hormone (pI 9.

Redistribution of flexibility in stabilizing antibody fragment mutants follows Le Châtelier's principle.

Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e.

Thermodynamic stability and flexibility characteristics of antibody fragment complexes.

Free energy landscapes, backbone flexibility and residue-residue couplings for being co-rigid or co-flexible are calculated from the minimal Distance Constraint Model (mDCM) on an exploratory dataset consisting of VL, scFv and Fab antibody fragments. Experimental heat capacity curves are reproduced markedly well, and an analysis of quantitative stability/flexibility relationships (QSFR) is applied to a representative VL domain and several complexes in the scFv and Fab forms. Global flexibility in the denatured ensemble typically decreases in the larger complexes due to domain-domain interfaces.

A systematic multitechnique approach for detection and characterization of reversible self-association during formulation development of therapeutic antibodies.

In addition to controlling typical instabilities such as physical and chemical degradations, understanding monoclonal antibodies' (mAbs) solution behavior is a key step in designing and developing process and formulation controls during their development. Reversible self-association (RSA), a unique solution property in which native, reversible oligomeric species are formed as a result of the noncovalent intermolecular interactions has been recognized as a developability risk with the potential to negatively impact manufacturing, storage stability, and delivery of mAbs. Therefore, its identification, characterization, and mitigation are key requirements during formulation development.

A systematic multitechnique approach for detection and characterization of reversible self-association during formulation development of therapeutic antibodies.

In addition to controlling typical instabilities such as physical and chemical degradations, understanding monoclonal antibodies' (mAbs) solution behavior is a key step in designing and developing process and formulation controls during their development. Reversible self-association (RSA), a unique solution property in which native, reversible oligomeric species are formed as a result of the noncovalent intermolecular interactions has been recognized as a developability risk with the potential to negatively impact manufacturing, storage stability, and delivery of mAbs. Therefore, its identification, characterization, and mitigation are key requirements during formulation development.

SYBR Green I: fluorescence properties and interaction with DNA.

In this study, we have investigated the fluorescence properties of SYBR Green I (SG) dye and its interaction with double-stranded DNA (dsDNA). SG/dsDNA complexes were studied using various spectroscopic techniques, including fluorescence resonance energy transfer and time-resolved fluorescence techniques. It is shown that SG quenching in the free state has an intrinsic intramolecular origin; thus, the observed >1,000-fold SG fluorescence enhancement in complex with DNA can be explained by a dampening of its intra-molecular motions.

Improved particle counting and size distribution determination of aggregated virus populations by asymmetric flow field-flow fractionation and multiangle light scattering techniques.

A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The method is based on the spherical particle counting approach given by Wyatt and Weida in 2004, with additional modifications. The new method was tested by analyzing polystyrene beads and adenovirus samples, both having a well-characterized particle size and concentration.

Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding.

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity.

Evaluation of a synthetic peptide as a replacement for the recombinant fusion protein of respiratory syncytial virus in a potency ELISA.

This report describes the development of a potency ELISA using a peptide derived from the motavizumab binding epitope of respiratory syncytial virus (RSV) F-protein. Motavizumab is an antibody therapeutic studied for the prevention of RSV disease. It binds to the RSV glycoprotein F (F-protein), blocking the ability of RSV to fuse with target cells.

Metal-enhanced PicoGreen fluorescence: application to fast and ultra-sensitive pg/ml DNA quantitation.

In this paper we provide both a theoretical and experimental analysis of the sensitivity of a DNA quantitation assay using a fluorescent chromophore which non-covalently binds dsDNA. It is well-known that the range of DNA concentrations available for fluorescence quantitation depends on the concentration of the chromophore, its affinity for nucleic acids, the binding site size on DNA and the ratio between the fluorescence intensity of the chromophore when bound to DNA compared to free chromophore in solution. We present experimental data obtained for a PicoGreen (PG)/DNA quantitation assay, which is in complete agreement with the results of our theoretical analysis.

Metal-enhanced PicoGreen fluorescence: application for double-stranded DNA quantification.

PicoGreen (PG) is a fluorescent probe for both double-stranded DNA (dsDNA) detection and quantification based on its ability to form a luminescent complex with dsDNA as compared with the free dye in solution. To expand the sensitivity of PG detection, we have studied the spectral properties of PG, both free and in complex with DNA in solution, when the fluorophore is in proximity to silver nanoparticles. We show that for a broad range of PG concentrations (20 pM-3.

Biophysical characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity.

Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation.

Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus.

We have identified a single tryptophan (Trp) residue responsible for loss of binding and biological activity upon ultraviolet (UV) light irradiation in MEDI-493, a humanized monoclonal antibody (MAb) against respiratory syncytial virus (RSV). This finding provides a better understanding of structure-function relationship in a 150-kDa protein. Irradiation of MEDI-493 with UV light resulted in spectral changes typical of Trp photoproducts and in a progressive loss of MEDI-493 binding and biological activity as measured by ELISA, Biacore, and cell-based assays.

Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins.

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments.

Validation of peptide mapping with electrospray mass spectrometry for recombinant proteins of biopharmaceutical interest and its applications as an identity test and a characterization tool.

In this paper, the steps required to validate a liquid chromatography peptide mapping method with mass spectrometric detection (LC-MS) for use as an identity test and characterization tool are presented. All aspects of peptide mapping are evaluated and optimized, including protein sample preparation (protein reduction, alkylation and enzymatic digestion), high performance liquid chromatography (HPLC) separation of the resulting peptides, and the use of a mass spectrometric detection. In addition, the validation of a single quadruple MS detector is described and the implementation of on-line electrospray ionization MS (ESI-MS) as an adjunct detector to support the investigation of peak differences is presented.

Characterization of a novel modification to monoclonal antibodies: thioether cross-link of heavy and light chains.

A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoclonal antibodies has been characterized. An additional band with an apparent molecular weight of 92 kDa was detected when monoclonal antibodies were analyzed by reducing capillary gel electrophoresis (rCGE) and reducing SDS-PAGE. To further investigate this observation, an early-eluting peak in the size exclusion chromatogram of a reduced and alkylated monoclonal antibody was collected and characterized by liquid chromatography, mass spectrometry, and gel electrophoresis.

Bisimidazoacridones: 2. Steady-state and time-resolved fluorescence studies of their diverse interactions with DNA.

Several bisimidazoacridones (BIA) are potent, selective antineoplastic agents, whereas others have potent anti-human immunodeficiency virus activity. BIA are bifunctional agents that consist of two imidazoacridone (IA) chromophores held together by various linkers. Interaction of BIA with DNA has been postulated to be required for their biological activity.