SEDNTERP: a calculation and database utility to aid interpretation of analytical ultracentrifugation and light scattering data.

Proper interpretation of analytical ultracentrifugation (AUC) data for purified proteins requires ancillary information and calculations to account for factors such as buoyancy, buffer viscosity, hydration, and temperature. The utility program SEDNTERP has been widely used by the AUC community for this purpose since its introduction in the mid-1990s. Recent extensions to this program (1) allow it to incorporate data from diffusion as well as AUC experiments; and (2) allow it to calculate the refractive index of buffer solutions (based on the solute composition of the buffer), as well as the specific refractive increment (dn/dc) of proteins based on their composition. These two extensions should be quite useful to the light scattering community as well as helpful for AUC users. The latest version also adds new terms to the partial specific volume calculations which should improve the accuracy, particularly for smaller proteins and peptides, and can calculate the viscosity of buffers containing heavy isotopes of water. It also uses newer, more accurate equations for the density of water and for the hydrodynamic properties of rods and disks. This article will summarize and review all the equations used in the current program version and the scientific background behind them. It will tabulate the values used to calculate the partial specific volume and dn/dc, as well as the polynomial coefficients used in calculating the buffer density and viscosity (most of which have not been previously published), as well as the new ones used in calculating the buffer refractive index.

The Glucagon-Like Peptide 2 Analog Teduglutide Reversibly Associates to Form Pentamers.

Glucagon-like peptide 1 and 2 and their analog peptide therapeutics are known to reversibly associate to form oligomers. Here we report the association properties of the glucagon-like peptide 2 analog teduglutide at concentrations up to ∼15 mg/mL. Both sedimentation equilibrium (SE-AUC) and sedimentation velocity (SV-AUC) show that teduglutide dissociates completely to monomers below 0.1 mg/mL. SE-AUC shows that the apparent weight-average molar mass increases substantially between 0.1 and 1 mg/mL, reaching a maximum of ∼14.5 kDa (∼3.9-mer) near 2 mg/mL, and then falling at higher concentrations because of strong solution nonideality effects (highly positive second virial coefficient). Circular dichroism spectra over the range from 0.1 to 2 mg/mL show that self-association is accompanied by significant increases in alpha-helix content, and that the associated state has a distinct tertiary structure. The SV-AUC data up to 2.2 mg/mL are fitted fairly well by an ideal rapidly reversible monomer-pentamer association. The SE-AUC modeling included thermodynamic nonideality effects. SE-AUC data up to ∼15 mg/mL imply a monomer-pentamer association at lower concentrations, but the pentamers also appear to weakly associate to form decamers. These results illustrate the importance of directly modeling the solution nonideality effects, which if neglected would lead to an incorrect preferred stoichiometry.

The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution.

Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in controlling growth and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, there is limited information on the quaternary structure of LRAP. Here, sedimentation velocity analytical ultracentrifugation (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP at various pH values, ionic strengths, and concentrations. We found that the monomer is the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7-4.1, pH 4.5-8, 50 mmol/L(mM) to 200 mM NaCl, 0.065-2 mg/mL). The monomer is also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and for LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregates in a narrow pH range near the isoelectric point of pH 4.1. SV and SANS show that the LRAP monomer has a radius of ∼2.0 nm and an asymmetric structure, and solution NMR studies indicate that the monomer is largely unstructured. This work provides new insights into the secondary, tertiary, and quaternary structure of LRAP in solution and provides evidence that the monomeric species may be an important functional form of some amelogenins.

Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G(s)α to C2 and the ensuing 7° rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.

Limiting the sedimentation coefficient for sedimentation velocity data analysis: partial boundary modeling and g(s) approaches revisited.

Brown and coworkers (Eur. Biophys. J. 38 (2009) 1079-1099) introduced partial boundary modeling (PBM) to simplify sedimentation velocity data analysis by excluding species outside the range of interest (e.g., aggregates, impurities) via restricting the sedimentation coefficient range being fitted. They strongly criticized the alternate approach of fitting g(s) distributions using similar range limits, arguing that (i) it produces "nonoptimal fits in the original data space" and (ii) the g(s) data transformations lead to gross underestimates of the parameter confidence intervals. It is shown here that neither of those criticisms is valid. These two approaches are not truly fitting the same data or in equivalent ways; thus, they should not actually give the same best-fit parameters. The confidence limits for g(s) fits derived using F statistics, bootstrap, or a new Monte Carlo algorithm are in good agreement and show no evidence for significant statistical distortion. Here 15 g(s) measurements on monoclonal antibody samples gave monomer mass estimates with experimental standard deviations of less than 1%, close to the confidence limit estimates. Tests on both real and simulated data help to clarify the strengths and drawbacks of both approaches. New algorithms for computing g(s) and a scan-differencing approach for PBM are introduced.

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone-binding domain: possible allosteric regulation and a conserved structural motif for the chloride-binding site.

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.

The critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals.

Size exclusion chromatography (SEC) is the most widely used method for aggregation analysis of pharmaceutical proteins. However SEC analysis has a number of limitations, and one of the most important ones is protein adsorption to the resin. This problem is particularly severe when using new columns, and often column preconditioning protocols are required. This review focuses on the role that addition of various cosolvents to the mobile phase plays in suppressing that protein adsorption. Cosolvents such as salt, amino acids, and organic solvents are often used for this purpose. Because the protein interaction with the resin surface is highly heterogeneous, different cosolvents affect the protein adsorption differently. We will summarize the various effects of cosolvents on protein adsorption and retention and describe the mechanism of the cosolvent effects.

Mechanisms of protein aggregation.

Aggregation or reversible self-association of protein therapeutics can arise through a number of different mechanisms. Five common aggregation mechanisms are described and their relations to manufacturing processes to suppress and remove aggregates are discussed.

A critical review of methods for size characterization of non-particulate protein aggregates.

Although size exclusion chromatography (SEC) has been, and will continue to be, the primary analytical tool for characterization of the content and size distribution of non-particulate aggregates in protein pharmaceuticals, regulatory concerns are driving increased use of alternative and complementary methods such as analytical ultracentrifugation and light scattering techniques. This review will highlight and critically review the capabilities, advantages, and drawbacks of SEC, analytical ultracentrifugation, and light scattering methods for characterizing aggregates with sizes below about 0.3 microns. The physical principles of the biophysical methods are briefly described and examples of data for real samples and how that data is interpreted are given to help clarify capabilities and weaknesses.

Development of Calcitonin Salmon Nasal Spray: similarity of peptide formulated in chlorobutanol compared to benzalkonium chloride as preservative.

The similarity of an intranasal salmon calcitonin (sCT) employing chlorobutanol as preservative (Calcitonin Salmon Nasal Spray) was compared to the reference listed drug (RLD) employing benzalkonium chloride as preservative (Miacalcin Nasal Spray). Various orthogonal methods assessed peptide structuring, dynamics, and aggregation state. Mass spectrometry, amino acid analysis, and N-terminal sequencing all demonstrated similarity in primary structure. Near- and far-UV circular dichroism (CD) data supported similarity in secondary and tertiary sCT structure. Nuclear magnetic resonance studies further supported similarity of three-dimensional structure and molecular dynamics of the peptide. Other methods, such as sedimentation velocity and size exclusion chromatography, demonstrated similarity in peptide aggregation state. These latter methods, in addition to reversed phase chromatography, were also employed for monitoring stability under forced degradation, and at the end of recommended shelf storage and patient use conditions. In all cases and for all methodologies employed, similarity to the RLD was observed with respect to extent of aggregation and other degradation processes. Finally, ELISA and bioassay data demonstrated similarity in biological properties. These investigations comprehensively demonstrate physicochemical similarity of Calcitonin Salmon Nasal Spray and the RLD, and should prove a useful illustration to pharmaceutical scientists developing alternative and/or generic peptide or protein products.

Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation.

This study explores the capability of modern analytical ultracentrifugation (AUC) to characterize the homogeneity, under product formulation conditions, of preparations of adenovirus vectors used in gene therapy and to assess the lot-to-lot consistency of this unique drug product. We demonstrate that a single sedimentation velocity run on an adenovirus sample can detect and accurately quantify a number of different forms of virus particles and subvirus particles. These forms include (a) intact virus monomer particles, (b) virus aggregates, (c) empty capsids (ECs), and (d) smaller assembly intermediates or subparticles formed during normal or aberrant virus assembly (or as a result of damage to the intact adenovirus or EC material during all phases of virus production). This information, which is collected on adenovirus samples under the exact formulation conditions that exist in the adenovirus vial, is obtained by direct boundary modeling of the AUC data generated from refractometric and/or UV detection systems using the computer program SEDFIT developed by Peter Schuck. Although both detectors are useful, refractometric detection using the Rayleigh interferometer offers a key advantage for providing accurate concentration information due to the similar response factors for both protein and DNA and its insensitivity to light scattering effects. Additional AUC data obtained from analytical band sedimentation velocity and density gradient sedimentation equilibrium experiments in CsCl with UV detection were also generated. These results further support conclusions concerning the solution properties of adenovirus, the identity of the different virus species, and the overall capability of boundary sedimentation velocity analysis.

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies.

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.

Is any measurement method optimal for all aggregate sizes and types?

Protein-based pharmaceuticals exhibit a wide range of aggregation phenomena, making it virtually impossible to find any one analytical method that works well in all cases. Aggregate sizes cover a range from small oligomers to visible "snow" and precipitates, and generally only the smaller species are reversible. It is less widely recognized that aggregates also exhibit a broad spectrum of lifetimes, and the lifetime has important consequences for detection methods. The fact that the measurement itself may destroy or create aggregates poses a major analytical challenge and is a key determinant for method selection. Several examples of some interesting aggregation phenomena and the analytical approaches we have used are presented. In one case, an "aggregate" seen by SEC in stressed samples was shown to actually be a partially denatured monomer using both size-exclusion chromatography with online multiangle laser light scattering (SEC-MALLS) and sedimentation velocity. In a second case, freeze/thaw stress generates transient, metastable oligomers that are extremely sticky and difficult to measure by SEC. By using sedimentation velocity as the "gold standard" a much improved SEC method was developed and used to investigate the temperature-dependent dissociation of these oligomers. For problems with visible particulates, dynamic light scattering has been effective, in our hands, at detecting the precursors to the large, visible particles and tracking the source of stress or damage to particular manufacturing steps.

Improved methods for fitting sedimentation coefficient distributions derived by time-derivative techniques.

Time-derivative approaches to analyzing sedimentation velocity data have proven to be highly successful and have now been used routinely for more than a decade. For samples containing a small number of noninteracting species, the sedimentation coefficient distribution function, g(s *), traditionally has been fitted by Gaussian functions to derive the concentration, sedimentation coefficient, and diffusion coefficient of each species. However, the accuracy obtained by that approach is limited, even for noise-free data, and becomes even more compromised as more scans are included in the analysis to improve the signal/noise ratio (because the time span of the data becomes too large). Two new methods are described to correct for the effects of long time spans: one approach that uses a Taylor series expansion to correct the theoretical function and a second approach that creates theoretical g(s *) curves from Lamm equation models of the boundaries. With this second approach, the accuracy of the fitted parameters is approximately 0.1% and becomes essentially independent of the time span; therefore, it is possible to obtain much higher signal/noise when needed. This second approach is also compared with other current methods of analyzing sedimentation velocity data.

Role of arginine in protein refolding, solubilization, and purification.

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.

Elution of antibodies from a Protein-A column by aqueous arginine solutions.

Acidic pH is commonly used to elute antibodies from Protein-A affinity column, although low pH may result in aggregation of the proteins. As an alternative, here arginine was tested as an eluent and compared with a more conventional eluent of citrate. Using purified monoclonal antibodies, recovery of antibodies with 0.1M citrate, pH 3.8, was less than 50% and decreased further as the pH was increased to 4.3. At the same pH, the recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 31% recovery, although the elution under such condition showed extensive tailing. Such tailing was observed at pH 3.8 when 0.1M citrate was used. Size exclusion analysis indicated that the eluted antibodies were mostly monomeric whether eluted with citrate or arginine. This demonstrates the usefulness of arginine as an efficient eluent for Protein-A chromatography.

Re-examining the oligomerization state of macrophage migration inhibitory factor (MIF) in solution.

The state of oligomerization of macrophage migration inhibitory factor (MIF, also known as glycosylation inhibiting factor, GIF) in solution has been variously reported as monomer, dimer, trimer, or mixtures of all three. Several crystal structures show MIF to be a trimer. Sedimentation velocity shows a recombinant human MIF sample is quite homogeneous, with 98% as a species with s(20,w)=3.07 S and D(20,w)=8.29 x 10(-7) cm(2)/s. Using the partial specific volume calculated from the amino acid composition these values imply a mass of 33.56 kDa, well above that of dimer, but also 9% below the trimer mass of 37.035 kDa. Sedimentation equilibrium data at loading concentrations from 0.01 to 1 mg/ml show unequivocally that the self-association is extremely tight. However, the apparent mass is 33.53 kDa [95% confidence 33.25-33.82], again 9% below that expected for 100% trimer. To examine the possibility this protein has an unusual partial specific volume, sedimentation equilibrium was also done in H(2)O/D(2)O mixtures, giving 0.765+/-0.017 ml/g rather than the calculated 0.735 ml/g. With this revised partial specific volume, the equilibrium and velocity data each give M=37.9+/-2.8 kDa, fully consistent with a strongly-associated trimeric quaternary structure.

Structure of folding intermediates at pH 4.0 and native state of microbial transglutaminase.

Recombinant microbial transglutaminase has been expressed in Escherichia coli as insoluble inclusion bodies. After we searched for refolding conditions, refolding of the protein could be done by first dilution of the unfolded enzyme in a buffer at pH 4.0, and then by titration of the pH from 4.0 to 6.0. CD analysis showed that a burst of secondary structure formation occurred within the dead time of the experiment and accounted for 75% of the signal change in the far UV CD, with little tertiary structure being formed. This burst was followed by slow rearrangement of the secondary structure accompanied by formation of tertiary structure. The secondary and tertiary structures of the final sample at pH 4.0, corresponding to the folding intermediate, were different from these structures at pH 6.0. Once the native structure was obtained, acidification of the native protein to pH 4.0 did not lead to a structure like that of the folding intermediate. Sedimentation velocity analysis showed that the folding intermediate had an expanded structure and contained no other structure species including large aggregates.

Secondary and quaternary structural transition of the halophilic archaeon nucleoside diphosphate kinase under high- and low-salt conditions.

Most halophilic enzymes from extremely halophilic archaea are denatured immediately after transfer from high-salt to low-salt medium. However, nucleoside diphosphate kinase (HsNDK) from the extremely halophilic archaeon Halobacterium salinarum seems to be exceptional, since the enzyme exhibited catalytic activity even under the low-salt condition. Here we show the mechanism how HsNDK is active under both high- and low-salt conditions that the HsNDK hexamer in high-salt medium dissociates into a dimer in the low-salt medium without denaturation. The observed change of the subunit structure was accompanied by a large decrease of alpha-helical content and lowered thermal sensitivity, yet keeping the conformations. This novel hexamer to dimer conversion under high- and low-salt conditions, respectively, seems to be the mechanism by which HsNDK is avoided from the irreversible denaturation.