John S. Philo

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.

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.

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.

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°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°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. Proteins 2007.

Binding of Neu Differentiation Factor with the Extracellular Domain of Her2 and Her3

The interaction of neu differentiation factor (NDF) with the extracellular domains of Her2 (sHer2) and Her3 (sHer3) have been studied using native gels, light scattering, and sedimentation equilibrium. The full-length NDFβ2 was shown to bind sHer3 with a dissociation constant of 26 ± 9 nM, while it showed a 1000-fold weaker binding to sHer2. Taken together, these results demonstrate that NDF is a high affinity ligand for Her3, but not for Her2. No increase in affinity of the NDFβ2 for sHer3 was observed upon addition of sHer2 to the NDFβ2-sHer3 mixture. Binding of NDFβ2 to sHer3 did not induce receptor dimerization or oligomerization, the stoichiometry being one sHer3 per one NDF molecule. This finding suggests that transmembrane and/or intracellular domains of receptor family members or perhaps additional unidentified components may be involved in NDF induced dimerization and autophosphorylation, or alternatively, that dimerization is not the mechanism for Her3 autophosphorylation and signal transduction.

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 Gsα 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.

Reversibility of Heat-Induced Denaturation of the Recombinant Human Megakaryocyte Growth and Development Factor

AbstractPurpose. The present study was performed to examine the effect of solution conditions on the reversibility of the thermal denaturation of megakaryocyte growth and development factor (rHuMGDF). Methods. Changes in the far U V CD spectra of rHuMGDF with temperature were used to monitor the thermal denaturation of the protein, and the recovery of folded protein following a return to room temperature. The effect of protein concentration, scan rate, and buffer composition on thermal denaturation and on the reversibility were determined. Surface tension measurements were used to determine the effect of this unfolding reaction on the surface adsorption of the protein. Sedimentation velocity was used to assess recovery of native monomer and the size of soluble aggregates. In addition, monomeric protein remaining in solution after incubation at 37°C for 2 weeks in either 10 mM imidazole of 10 mM phosphate was determined. Results. In phosphate buffer the rHuMGDF irreversibly precipitates upon unfolding under all the conditions examined. In imidazole the unfolding is at least partially reversible, with no visible precipitate seen; the degree of reversibility increased by lowering both protein and salt concentrations, and the amount of time spent at elevated temperature. In order to compare thermal unfolding occuring with different degrees of reversibility, the melting temperature was defined as the temperature at which melting begins. The melting temperature itself is relatively independent of the buffer composition, or experimental conditions. At low protein concentrations the protein stabilizer sucrose had a marginal effect on the thermal transition of rHuMGDF, while at protein concentrations of about 2 mg/ml the inclusion of sucrose increased the apparent melting temperature by about 4°C, to that seen at low protein concentrations, but had little effect on the reversibility of denaturation. Inclusion of 1 or 2 M urea did not affect the reaction. Surface tension measurements of rHuMGDF solutions showed little difference before and after melting, and in the presence or absence of sucrose. When unfolding is irreversible, the MGDF appears to form soluble aggregates of tetramers to 14-mers, while under reversible conditions native monomer is recovered. More monomeric MGDF remained in solution following storage for 2 weeks at 37°C in imidazole than in phosphate, in both the presence and absence of sucrose. Conclusions. These results can be explained by assuming that thermal denaturation proceeds as a two-step reaction, the first step being the equilibrium between folded and unfolded states, while the second step is a slow irreversible aggregation. The different buffer systems affect the rate of the aggregation step, but not the intrinsic thermal stability nor the rate of the unfolding step.

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.

Densimetric determination of equilibrium binding of sucrose octasulfate with basic fibroblast growth factor.

Fibroblast growth factors (FGFs) strongly bind to heparin and are thereby stabilized against deactivation and proteolytic cleavage. Sucrose octasulfate (SOS), which has a chemical structure resembling the repeating unit of heparin, has also been shown to enhance stability of basic FGF against thermal denaturation and to induce a small conformational change. We have examined SOS binding to bFGF using equilibrium dialysis. The difference in SOS concentration across the dialysis membrane was measured using a precision density meter, since the density of SOS differs greatly from that of water. With care, this densimetric technique can measure binding with a precision of ± 0.1 mol/mol using about 2 mg/ml of protein. These results show that the binding saturates at 2 mol of SOS per mole of bFGF as the SOS concentration increases to 3.6 mM or higher. The effect of SOS on the thermal stability of bFGF was examined using denaturation at a constant heating rate, by both turbidity and differential scanning calorimetry. Since the thermal denaturation is irreversible, the temperature where aggregation abruptly increases was taken to indicate the onset of denaturation. This temperature increased by ∼12°C as the SOS concentration increased from 0.018 to 3.6 mM and remained constant above 3.6 mM, consistent with our binding data if the binding is specific to the native state.

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.