Continuous monitoring of a monoclonal antibody by size exclusion chromatography reveals a correlation between system suitability parameters and column aging.

Size exclusion chromatography (SEC) is a foundational analytical method to assess product purity of biological molecules. To ensure accurate and reproducible data that meet regulatory agency standards, it is critical to monitor the chromatographic column with efficient and continuous approaches. In this study, 19 SEC columns (Waters Acquity BEH200) were evaluated using an in-house monoclonal antibody made at Regeneron. System suitability parameters (SSPs) were used to monitor the performance of the SEC assay, including USP resolution, USP plate count, USP tailing factor, asymmetry factor, elution time, peak width, and peak height. A general linear model was built and revealed that elution time, peak width, asymmetry factor, and tailing factor increased with injection number, while peak height, resolution, and plate count decreased. After 1000 injections, tailing factor and peak width increased by more than 10%, while resolution and plate count decreased by more than 10% from their respective starting values.

Low pKa of Lys promotes glycation at one complementarity-determining region of a bispecific antibody.

Protein glycation is a common, normally innocuous, post-translational modification in therapeutic monoclonal antibodies. However, when glycation occurs on complementarity-determining regions (CDRs) of a therapeutic monoclonal antibody, its biological activities (e.g., potency) may be impacted. Here, we present a comprehensive approach to understanding the mechanism of protein glycation using a bispecific antibody. Cation exchange chromatography and liquid chromatography-mass spectrometry were used to characterize glycation at a lysine residue within a heavy chain (HC) CDR (HC-CDR3-Lys98) of a bispecific antibody. Thermodynamic analysis revealed that this reaction is reversible and can occur under physiological conditions with an apparent affinity of 8-10 mM for a glucose binding to HC-CDR3-Lys98. Results from kinetic analysis demonstrated that this reaction follows Arrhenius behavior in the temperature range of 5°C-45°C and can be well predicted in vitro and in a non-human primate. In addition, this glycation reaction was found to be driven by an unusually low pKa on the ε-amino group of HC-CDR3-Lys98. Van't Hoff analysis and homology modeling suggested that this reaction is enthalpically driven, with this lysine residue surrounded by a microenvironment with low polarity. This study provides, to our knowledge, new insights toward a mechanistic understanding of protein glycation and strategies to mitigate the impact of protein glycation during pharmaceutical development.

Physical and Functional Characterization of a Viral Genome Maturation Complex.

Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of the λ-genome maturation complex. The λ-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence, cos. The Escherichia coli integration host factor protein (IHF) is required for efficient λ-development in vivo and a specific IHF recognition sequence is found within cos. We show that IHF and the terminase protomer cooperatively assemble at the cos site and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at the cos site. Tetramer assembly activates the cos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.

Trimerization Dictates Solution Opalescence of a Monoclonal Antibody.

Opalescence, sometimes observed in antibody solutions, is thought to be mediated by light scattering of soluble oligomers or insoluble particulates. However, mechanistic features, such as stoichiometry and self-association affinity of oligomeric species related to opalescence, are poorly understood. Here, opalescence behavior of a monoclonal antibody (mAb-1) solution was studied over a wide range of solution conditions including different protein concentrations, pH, and in the presence or absence of salt. Hydrodynamic and thermodynamic properties of mAb-1 solutions were studied by analytical ultracentrifugation and dynamic light scattering. Opalescence in mAb-1 solutions is pH and concentration dependent. The degree of opalescence correlates with reversible monomer-trimer equilibrium detected by analytical ultracentrifugation. Increased trimer formation corresponds to increased opalescence in mAb-1 solutions at higher pH and protein concentrations. Addition of NaCl shifts this equilibrium toward monomer and reduces solution opalescence. This study demonstrates that opalescence in mAb-1 solutions does not arise from the light scattering of monomer or random molecular self-associations but is strongly correlated with a specific self-association stoichiometry and affinity. Importantly, at pH 5.5 (far below isoelectric point of mAb-1), the solution is not opalescent and with nonideal behavior. This study also dissects several parameters to describe the hydrodynamic and thermodynamic nonideality.

Thermodynamic Interrogation of the Assembly of a Viral Genome Packaging Motor Complex.

Viral terminase enzymes serve as genome packaging motors in many complex double-stranded DNA viruses. The functional motors are multiprotein complexes that translocate viral DNA into a capsid shell, powered by a packaging ATPase, and are among the most powerful molecular motors in nature. Given their essential role in virus development, the structure and function of these biological motors is of considerable interest. Bacteriophage λ-terminase, which serves as a prototypical genome packaging motor, is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. This protomer assembles into a functional higher-order complex that excises a unit length genome from a concatemeric DNA precursor (genome maturation) and concomitantly translocates the duplex into a preformed procapsid shell (genome packaging). While the enzymology of λ-terminase has been well described, the nature of the catalytically competent nucleoprotein intermediates, and the mechanism describing their assembly and activation, is less clear. Here we utilize analytical ultracentrifugation to determine the thermodynamic parameters describing motor assembly and define a minimal thermodynamic linkage model that describes the effects of salt on protomer assembly into a tetrameric complex. Negative stain electron microscopy images reveal a symmetric ring-like complex with a compact stem and four extended arms that exhibit a range of conformational states. Finally, kinetic studies demonstrate that assembly of the ring tetramer is directly linked to activation of the packaging ATPase activity of the motor, thus providing a direct link between structure and function. The implications of these results with respect to the assembly and activation of the functional packaging motor during a productive viral infection are discussed.

Analytical Ultracentrifugation as a Tool to Study Nonspecific Protein-DNA Interactions.

Analytical ultracentrifugation (AUC) is a powerful tool that can provide thermodynamic information on associating systems. Here, we discuss how to use the two fundamental AUC applications, sedimentation velocity (SV), and sedimentation equilibrium (SE), to study nonspecific protein-nucleic acid interactions, with a special emphasis on how to analyze the experimental data to extract thermodynamic information. We discuss three specific applications of this approach: (i) determination of nonspecific binding stoichiometry of E. coli integration host factor protein to dsDNA, (ii) characterization of nonspecific binding properties of Adenoviral IVa2 protein to dsDNA using SE-AUC, and (iii) analysis of the competition between specific and nonspecific DNA-binding interactions observed for E. coli integration host factor protein assembly on dsDNA. These approaches provide powerful tools that allow thermodynamic interrogation and thus a mechanistic understanding of how proteins bind nucleic acids by both specific and nonspecific interactions.

Integration host factor assembly at the cohesive end site of the bacteriophage lambda genome: implications for viral DNA packaging and bacterial gene regulation.

Integration host factor (IHF) is an Escherichia coli protein involved in (i) condensation of the bacterial nucleoid and (ii) regulation of a variety of cellular functions. In its regulatory role, IHF binds to a specific sequence to introduce a strong bend into the DNA; this provides a duplex architecture conducive to the assembly of site-specific nucleoprotein complexes. Alternatively, the protein can bind in a sequence-independent manner that weakly bends and wraps the duplex to promote nucleoid formation. IHF is also required for the development of several viruses, including bacteriophage lambda, where it promotes site-specific assembly of a genome packaging motor required for lytic development. Multiple IHF consensus sequences have been identified within the packaging initiation site (cos), and we here interrogate IHF-cos binding interactions using complementary electrophoretic mobility shift (EMS) and analytical ultracentrifugation (AUC) approaches. IHF recognizes a single consensus sequence within cos (I1) to afford a strongly bent nucleoprotein complex. In contrast, IHF binds weakly but with positive cooperativity to nonspecific DNA to afford an ensemble of complexes with increasing masses and levels of condensation. Global analysis of the EMS and AUC data provides constrained thermodynamic binding constants and nearest neighbor cooperativity factors for binding of IHF to I1 and to nonspecific DNA substrates. At elevated IHF concentrations, the nucleoprotein complexes undergo a transition from a condensed to an extended rodlike conformation; specific binding of IHF to I1 imparts a significant energy barrier to the transition. The results provide insight into how IHF can assemble specific regulatory complexes in the background of extensive nonspecific DNA condensation.

Characterization of the non-specific DNA binding properties of the Adenoviral IVa2 protein.

Human Adenovirus (Ad) is a non-enveloped, icosahedral virus with a linear, double-stranded DNA genome. The Ad IVa2 protein is involved in multiple viral processes including viral late gene transcription and virus assembly. Previous studies have shown that IVa2 loads additional viral proteins onto conserved DNA elements within the Ad genome to regulate these viral processes. IVa2 also possesses strong non-specific DNA binding activity, and it is likely it uses this activity to recruit proteins to the conserved DNA elements. Here we have investigated the non-specific DNA binding activity of IVa2 using nitrocellulose/DEAE filter binding and sedimentation equilibrium techniques. We have analyzed our data using the McGhee and Von Hippel approach [1], and find that IVa2 binds with strong, positive nearest-neighbor cooperativity. In addition, we describe how to apply the McGhee and von Hippel approach to directly analyze sedimentation equilibrium data using non-linear least-squares methods. We discuss the implications of these results with respect to current virus assembly mechanisms.

Cooperative heteroassembly of the adenoviral L4-22K and IVa2 proteins onto the viral packaging sequence DNA.

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus. Viral DNA packaging refers to the process whereby the viral genome becomes encapsulated by the viral particle. In Ad, activation of the DNA packaging reaction requires at least three viral components: the IVa2 and L4-22K proteins and a section of DNA within the viral genome, called the packaging sequence. Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the packaging sequence and that these interactions are absolutely required for the observation of DNA packaging. However, the equilibrium mechanism for assembly of IVa2 and L4-22K onto the packaging sequence has not been determined. Here we characterize the assembly of the IVa2 and L4-22K proteins onto truncated packaging sequence DNA by analytical sedimentation velocity and equilibrium methods. At limiting concentrations of L4-22K, we observe a species with two IVa2 monomers and one L4-22K monomer bound to the DNA. In this species, the L4-22K monomer is promoting positive cooperative interactions between the two bound IVa2 monomers. As L4-22K levels are increased, we observe a species with one IVa2 monomer and three L4-22K monomers bound to the DNA. To explain this result, we propose a model in which L4-22K self-assembly on the DNA competes with IVa2 for positive heterocooperative interactions, destabilizing binding of the second IVa2 monomer. Thus, we propose that L4-22K levels control the extent of cooperativity observed between adjacently bound IVa2 monomers. We have also determined the hydrodynamic properties of all observed stoichiometric species; we observe that species with three L4-22K monomers bound have more extended conformations than species with a single L4-22K bound. We suggest this might reflect a molecular switch that controls insertion of the viral DNA into the capsid.

Thermodynamic linkage of large-scale ligand aggregation with receptor binding.

There are many examples in the literature that deal explicitly with the coupling of ligand oligomerization with receptor binding. For example, many transcription factors dimerize and this plays a fundamental role in sequence specific DNA recognition. However, many biological macromolecules undergo reversible, large scale aggregation processes, some of which are indefinite. The thermodynamic coupling of these aggregation processes to other processes, such as protein-protein and protein-DNA interactions, has not been explored in depth. Here we consider the thermodynamic consequences of large scale ligand aggregation on the determination of fundamental thermodynamic parameters, such as equilibrium binding constants and ligand-receptor stoichiometries. We find that a fundamental consequence of an aggregating ligand is that the free ligand concentration (ligand that is not found in aggregates) is buffered over a wide total ligand concentration range. In general, the larger the size of the aggregates, the wider the range over which the free ligand concentration is buffered. An additional consequence of this observation is that an upper limit is set on the fractional occupancy of the ligand's receptor, such that even if the ligand is over-expressed to very high levels in the cell, this will not necessarily ensure that 100% of the ligand's receptors will be occupied. The implications of these results for sequence specific DNA binding proteins will be discussed.

Self-association of the adenoviral L4-22K protein.

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus that causes infections of the respiratory tract, urinary tract, and gastrointestinal tract. Assembly of virus particles requires condensation and encapsidation of the linear viral genome. This process requires sequence specific binding of two viral proteins, called IVa2 and L4-22K, to a conserved sequence located at the left end of the viral genome, called the packaging sequence (PS). IVa2 and an alternatively spliced form of L4-22K, called L4-33K, also function as transcriptional activators of the major late promoter (MLP), which encodes viral structural and core proteins. IVa2 and L4-33K bind to identical conserved DNA sequences downstream of the MLP, called the downstream element (DE), to activate transcription. To begin to dissect how the IVa2, L4-22K, and L4-33K proteins simultaneously function as transcriptional activators and DNA packaging proteins, we need to understand the thermodynamics of assembly of these proteins on DNA that contains the PS as well as the DE. Toward this end, we have characterized the self-assembly properties of highly purified, recombinant L4-22K protein. We show that L4-22K reversibly assembles into higher-order structures according to an indefinite, isodesmic assembly scheme. We show that the smallest polymerizing unit is likely the L4-22K monomer (s(20,w) = 2.16 ± 0.04 S) and that the monomer assembles with itself and/or other aggregates with an equilibrium association constant, L, of 112 (102, 124) µM(-1) (0.1 M NaCl, pH 7, 25 °C). A mechanistic consequence of an isodesmic, indefinite assembly process is that the free concentration of the smallest polymerizing unit cannot exceed 1/L. We discuss the implications of this observation with respect to the thermodynamics of assembly of L4-22K and IVa2 on the PS.

Interaction of the adenoviral IVa2 protein with a truncated viral DNA packaging sequence.

Adenoviral (Ad) infection typically poses little health risk for immunosufficient individuals. However, for immunocompromised individuals, such as AIDS patients and organ transplant recipients, especially pediatric heart transplant recipients, Ad infection is common and can be lethal. Ad DNA packaging is the process whereby the Ad genome becomes encapsulated by the viral capsid. Specific packaging is dependent upon the packaging sequence (PS), which is composed of seven repeated elements called A repeats. The Ad protein, IVa2, which is required for viral DNA packaging, has been shown to bind specifically to synthetic DNA probes containing A repeats I and II, however, the molecular details of this interaction have not been investigated. In this work we have studied the binding of a truncated form of the IVa2 protein, that has previously been shown to be sufficient for virus viability, to a DNA probe containing A repeats I and II. We find that the IVa2 protein exists as a monomer in solution, and that a single IVa2 monomer binds to this DNA with high affinity (K(d)< approximately 10 nM), and moderate specificity, and that the trIVa2 protein interacts in a fundamentally different way with DNA containing A repeats than it does with non-specific DNA. We also find that at elevated IVa2 concentrations, additional binding, beyond the singly ligated complex, is observed. When this reaction is modeled as representing the binding of a second IVa2 monomer to the singly ligated complex, the K(d) is 1.4+/-0.7 microM, implying a large degree of negative cooperativity exists for placing two IVa2 monomers on a DNA with adjacent A repeats.

Acrolein inhibits cytokine gene expression by alkylating cysteine and arginine residues in the NF-kappaB1 DNA binding domain.

Cigarette smoke is a potent inhibitor of pulmonary T cell responses, resulting in decreased immune surveillance and an increased incidence of respiratory tract infections. The alpha,beta-unsaturated aldehydes in cigarette smoke (acrolein and crotonaldehyde) inhibited production of interleukin-2 (IL-2), IL-10, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha by human T cells but did not inhibit production of IL-8. The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) in cigarette smoke were inactive. Acrolein inhibited induction of NF-kappaB DNA binding activity after mitogenic stimulation of T cells but had no effect on induction of NFAT or AP-1. Acrolein inhibited NF-kappaB1 (p50) binding to the IL-2 promoter in a chromatin immunoprecipitation assay by >99%. Using purified recombinant p50 in an electrophoretic mobility shift assay, we demonstrated that acrolein was 2000-fold more potent than crotonaldehyde in blocking DNA binding to an NF-kappaB consensus sequence. Matrix-assisted laser desorption/ionization time-of-flight and tandem mass spectrometry demonstrated that acrolein alkylated two amino acids (Cys-61 and Arg-307) in the DNA binding domain. Crotonaldehyde reacted with Cys-61, but not Arg-307, whereas the saturated aldehydes in cigarette smoke did not react with p50. These experiments demonstrate that aldehydes in cigarette smoke can regulate gene expression by direct modification of a transcription factor.

Cost of depression of adults in Taiwan.

OBJECTIVE: To estimate the direct cost of depression in Taiwanese adults for the years 2000-2002. METHODS: The medical claims database of the National Health Bureau was analyzed and the cost of treating adults (>15 years of age) with the diagnosis of depression was calculated. RESULTS: The total direct medical costs of adult depression in the three years 2000, 2001, and 2002 were approximately US dollars 93 million, US dollars 117 million, and US dollars 140million, respectively. CONCLUSION: The cost of depression increased continuously over the period from 2000-2002. However, the percentage of patients receiving treatment did not increase steadily over the same time period with treatment rates of 1.5% in 2000, 2.3% in 2001, and 2.0% in 2002. The recent annual prevalence of depression in Taiwan has been estimated at 4-5%. Thus, the Taiwanese health authority spends an annual average of US dollars 116.6 million to treat depression (1.2% of total national expenses). In sum, the treatment of depression, while costly, deserves greater attention by public health officials in order to avoid the already significant burden of this disease on both patients and society. Future research will therefore require more accurate statistical data in order to assess the effects of depression-related burdens on individuals and society, especially with respect to the capacity to work.