Preparation and characterisation of spray-dried and crystallised trypsin: FT-Raman study to detect protein denaturation after thermal stress.

The production of stable protein formulations is difficult due to unique properties of proteins. Accordingly, spray drying and crystallisation techniques were assessed for their effects on trypsin, a model protein. Samples were investigated using polarising microscopy, thermogravimetry, differential scanning calorimetry (DSC), FT-Raman spectroscopy and enzymatic assay. Unprocessed, spray-dried and crystallised trypsin were evaluated in solution for secondary structure in low and high protein concentrations using aqueous state FT-Raman spectroscopy and for folding reversibility employing high sensitivity differential scanning calorimetry. Spray-dried trypsin showed FT-Raman spectral changes and less biological activity, after rehydration, compared with unprocessed and crystallised trypsin. Crystals maintained activity better than did the spray-dried form and retained a higher folding reversibility compared to unprocessed and spray-dried protein. Proteins may denature with structural changes under thermal stress and lose their activities. Thus, this research studied the effect of heating solid unprocessed, spray-dried and crystallised trypsin samples on their secondary structures, using FT-Raman spectroscopy, to identify the influence of the initial solid form on its propensity for thermal denaturation and whether this can be correlated with catalytic activity. DSC heated protein samples to two temperatures, one before the apparent denaturation temperature (T(m)) and the other after the T(m). Samples heated below their T(m) showed some perturbations of the secondary structure and some activity, whilst materials rose to the higher temperature were insoluble with complete loss of activity.     

Solid state chemistry of proteins: I. glass transition behavior in freeze dried disaccharide formulations of human growth hormone (hGH)

Although freeze dried formulations are commonly characterized using differential scanning calorimetry (DSC), a protein-rich system behaves as a "strong glass", and the glass transition temperature, T(g), cannot be directly determined by DSC. A strong glass means a small heat capacity change at T(g), triangle upC(p), and a very broad glass transition region, or a large triangle upT(g). However, direct experimental evidence for a small triangle upC(p) and a large triangle upT(g) have been lacking. Here, we utilize extrapolation of thermal analysis data in protein:disaccharide mixtures to evaluate T(g), triangle upT(g), and triangle upC(p) for "pure" human growth hormone (hGH) from low to moderate residual water. We find that triangle upT(g) is indeed large and triangle upC(p) is very small. Also, the T(g) for pure hGH decreases from a value of about 136 degrees C when dry to around 25 degrees C at 12% water. This glass transition is not the onset of mobility within the protein molecule but rather signals onset of whole molecule rotation and translation. We also observe complex pre-T(g) thermal events in the DSC data, which are interpreted as consequences of relaxation events, largely due to the disaccharide, and are characteristic of freeze dried systems having a broad distribution of relaxing substates.     

Granulocyte-colony stimulating factor maintains a thermally stable,compact, partially folded structure at pH 2

At acidic pH many proteins exist in a partially unfolded form, called the “A” state. This is defined as a flexible, expanded structure with well-defined, usually native-like secondary structure, but no unique tertiary structure, and showing no cooperativity during thermal-induced denaturation. Granulocyte-colony stimulating factor (G-CSF), a four-helix bundle cytokine, maintains both thermal stability and tertiary structure at pH 2.O. We therefore examined the conformation and thermal unfolding of G-CSF at pH 2.0, 4.0 and 7.0 using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). The secondary structure of the molecule remains highly helical as the pH is lowered from 7.0 to 2.0. The tertiary structure of the protein is slightly different at each pH value, but even at pH 2.0 G-CSF maintains a regular three-dimensional structure. The structure is hydrodynamically compact at these different pH values, with no increase in Stake's radius even at pH 2.0.The thermal-induced denaturation of G-CSF was determined by monitoring changes in the CD or FTIR spectra. At pH 2.0 the temperature at which thermal-induced denaturation begins is higher than it is at pH 4.0 or 7.0, the thermal unfolding transition remains cooperative and some α-helical structure persists even at 86°C. At pH 4.0 and 7.0, secondary and tertiary structures disappear simultaneously during thermal denaturation, whereas at pH 2.0 small changes in the far-UV CD region begin to occur first, followed by the simultaneous cooperative loss of tertiary structure and much of the remaining secondary structure. The structure of G-CSF at pH 2.0 is thus revealed as compact, with a unique, three-dimensional structure, highly helical secondary structure, and most importantly, a cooperative thermal unfolding transition. G-CSF at acid pH thus does not adopt the “A” state.     

Aqueous two-phase systems as a formulation concept for spray-dried protein

This study investigates to what extent an aqueous two-phase system (ATPS) can encapsulate and protect the secondary structure of a protein during spray drying. The ATPSs contained polyvinyl alcohol (PVA) and dextran solutions, in different proportions. A model protein, bovine serum albumin (BSA) and, in some experiments, trehalose were added to the ATPS prior to spray drying. Electron spectroscopy for chemical analysis (ESCA), differential scanning calorimetry (DSC), UV spectrophotometry, size exclusion high-performance liquid chromatography (SEC-HPLC) and Fourier transform infrared spectroscopy (FTIR) were used for analysis of solid and reconstituted samples. The anticipated function of the ATPS was to improve the stability of the protein by preventing interactions with the air-liquid interface during drying and by improving the encapsulation of the protein in the dried powder. BSA was found to preferentially partition to the dextran phase and in the absence of PVA, BSA dominated the powder surface. In samples containing PVA, the polymer mainly covered the powder surface, even though the dextran-rich phase was continuous, thus preventing protein surface interactions and providing improved encapsulation. However, PVA was found to cause partial loss of the native structure of BSA although the protein was well encapsulated during spray drying.     

In Vitro Simulation of Solid-Solid Dehydration, Rehydration, and Solidification of Trehalose Dihydrate Using Thermal and Vibrational Spectroscopic Techniques

PURPOSE: The processes of dehydration, rehydration, and solidification of trehalose dihydrate were examined to simulate it in the drying and wetting states. METHODS: Techniques included differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and Fourier transform infrared (FT-IR) microspectroscopy combined with thermal analysis. Trehalose dihydrate was pressed on one KBr pellet (IKBr method) or sealed within two KBr pellets (2KBr method) for FT-IR measurement. RESULTS: On the DSC thermogram, the shoulder between 60 degrees C and 80 degrees C represented a transitional change because no weight loss occurred in this area of the TGA curve. The endothermic peak at 100 degrees C represented dehydration of trehalose dihydrate to anhydrous trehalose; a 9.5% weight loss in the TGA curve occurred from 81 degrees C. The thermal-dependent FT-IR spectra for trehalose dihydrate prepared by the IKBr method changed markedly with temperature in the 1800-1500 cm(-1) region during dehydration. IR peak intensity at 1687 cm(-1) assigned to the bending vibrational mode of solid-like water declined with temperature and decreased sharply at 67 degrees C. Another IR peak at 1640 cm(-1) associated with the bending of liquid water increased at 67 degrees C but disappeared at 79 degrees C as a result of water evaporation. Both peaks for the sample prepared by the 2KBr method changed dramatically at 64 degrees C; peak intensity at 1640 cm(-1) remained constant above 64 degrees C. CONCLUSIONS: A new polymorphic transition of trehalose dihydrate was first evidenced at 64-67 degrees C from both DSC curves and thermal-related FT-IR spectra. This transitional temperature reflected the thermal-dependent transformation from solid-like water to liquid water in the trehalose dihydrate structure during dehydration. During rehydration, trehalose anhydrate was rehydrated to the dihydrate, and liquid water in the dihydrate structure was again transformed to solid-like water. The polymorphic transition within this temperature range seems to correlate with the bioprotective effect of trehalose dihydrate in preserving protein stability.     

Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage

Few researches on the protein stabilization of recombinant human serum albumin (rHSA) have been done. In the present study, we assessed the impact of sugar lyoprotectants on the protein stability of lyophilized rHSA (65 KDa) in the solid state. For the assessment, rHSA was formulated with sucrose and trehalose, respectively, alone or in combination with mannitol, which were lyophilized and stored at 35 degrees C. Degradation and aggregation of the resulting lyophilized formulations was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Induction of amorphous state by the lyophilactants with rHSA was determined by differential scanning calorimetry (DSC). The protein secondary structure of the rHSA in the formulations was analyzed by Fourier transform infrared spectroscopy (FT-IR). Results from SDS-PAGE analysis displayed that mannitol formulation caused aggregation resulting in a few bands that were greater than 65 KDa, whereas sucrose and trehalose formulations revealed no such aggregation. However, the aggregation of the protein decreased when mannitol was combined with sucrose or trehalose. DSC measurement supported the electrophoresis data showing that sucrose and trehalose formed complete amorphous state, but mannitol induced a partial amorphous state. These data indicate during lyophilization the most effective protein protection against aggregation was provided by sucrose and trehalose. The protection lasted during 4 months storage at 35 degrees C. FT-IR analysis displayed that the sucrose formulation inhibited deamidation. In conclusion, our data suggest that sucrose and trehalose as additives seems to be sufficient to protect from lyophilization of rHSA protein and also maintain its stability in the solid state during storage.     

Solid state chemistry of proteins: II. The correlation of storage stability of freeze-dried human growth hormone (hGH) with structure and dynamics in the glassy solid

This research presents storage stability of human growth hormone, hGH, in lyophilized di-saccharide formulations. Stability via HPLC assay was assessed at 40 and 50 degrees C. Structure of the protein in the solids was assessed by infrared spectroscopy. Molecular mobility was characterized by structural relaxation times estimated from DSC data and by measurement of atomic motion on a nanosecond time scale by neutron scattering. Very large stability differences were observed among the various formulations, with both chemical and aggregation stability showing the same qualitative trends with formulation. Near the T(g), T(g) appeared to be a relevant stability parameter, but for storage well below T(g), stability seems unrelated to T(g). Stability (chemical and aggregation) was weakly correlated with secondary structure of the protein, and there was a partial quantitative correlation between degradation rate and the structural relaxation time. However, at equivalent levels of disaccharide relative to protein, sucrose systems were about a factor of two more stable than trehalose formulations, but yet had greater mobility as measured by structural relaxation time. Secondary structure was equivalent in both formulations. Neutron scattering results documented greater suppression of fast dynamics by sucrose than by trehalose, suggesting that well below T(g), fast dynamics are important to stability.     

Prediction of the bitterness of single,binary- and multiple-component amino acid solutions using a taste sensor

Upon freeze-drying in the absence of lyoprotectants, Fourier transform infrared (FTIR) spectroscopy has detected changes in the secondary structures of proteins. Such FTIR studies have been typically conducted using protein/KBr pellets, where additional protein degradation could potentially occur due to pressure effects and partial dissolution of the chaotropic KBr. Diffuse reflectance FTIR spectroscopy, in which no sample preparation is necessary, was evaluated as an alternative spectroscopic method to examine protein structure upon freeze-drying. The therapeutic proteins recombinant human deoxyribonuclease I (rh-DNase) and recombinant human insulin like growth factor I (rh-IGF-I) were freeze-dried with mannitol, sucrose, trehalose, and two molecular weight dextrans (69 and 503 kDa) separately, at concentrations ranging from 0 to 100% (w/w). Upon freeze-drying, rh-DNase and rh-IGF-I underwent significant changes in their secondary structure. For both proteins, the presence of intermolecular beta-sheets due to aggregation was detected and the alpha-helix content decreased significantly. The addition of carbohydrates to the formulations inhibited the protein secondary structure rearrangement in a concentration-dependent manner. Sucrose and trehalose appeared to be the most efficient excipients in preventing secondary structure changes. The conformational changes observed for both proteins appeared to be reversible upon rehydration.     

Simultaneous differential scanning calorimetry,X-ray diffraction and FTIR spectrometry in studies of ovalbumin denaturation

The new application of differential scanning calorimetry (DSC) and the susceptibility of ovalbumin to α-chymotrypsin gave a quantitative estimation of protein denaturation in solid ovalbumin. Solid ovalbumin in granules with 11% of water was heated at 100 °C in closed and nonclosed ampules. In order to compare effects of size and crystal structure, two proteins (bovine albumin and γ-globulin) were examined at similar conditions for the extent of denaturation. Ovalbumin and bovine albumin showed similar extents of denaturation, but γ-globulin, with a very different molecular mass, showed the maximal conformational changes. The enthalpy of denaturation was measured to elucidate the conformational changes in solid proteins. Its value was used for calculation of the degree of denaturation. The thermodynamic data associated with transition were calculated and the number of bonds broken during denaturation was determined. Intrinsic fluorescence was utilized in order to compare these two methods. Moreover, X-ray diffraction and FTIR spectrometry were applied to native and denatured proteins.     

Study of protein conformational stability and integrity using calorimetry and FT-Raman spectroscopy correlated with enzymatic activity.

Maintaining protein conformational stability and integrity during formulation is critical for developing protein pharmaceuticals. Accordingly, high sensitivity differential scanning calorimetry (HSDSC) and Fourier transform (FT)-Raman spectroscopy were employed to assess conformational stabilities (thermal stability and folding reversibility) and structural integrities, respectively, for three model proteins: lysozyme, deoxyribonuclease I (DNase I) and lactate dehydrogenase (LDH) in lyophilised (as received) and spray-dried forms. Enzymatic assay after cooling of thermally denatured protein solutions from HSDSC determined if thermal transition reversibility was related to biological activity. HSDSC data showed that molecules from lyophilised lysozyme were able to refold better than the spray-dried form. This was confirmed by enzymatic assay. Moreover, enzymatic assay results revealed that lysozyme folding reversibility was related to the native structure of the protein that is essential for the biological activity. Thermal denaturation of DNase I and LDH samples in HSDSC was not reversible upon cooling of thermally denatured proteins (in contrast to lysozyme). Hence, it was decided to identify the effect of protein initial structures on its propensity to thermal denaturation via FT-Raman spectroscopy. In other words, proteins may denature with structural alterations due to stresses such as heat and the protein loses its enzymatic activity. Consequently, FT-Raman investigated the effects of spray drying and heating of solid DNase I and LDH samples, from differential scanning calorimetry, on protein conformational integrities. Lyophilised and spray-dried DNase I and LDH solid samples were heated to two temperatures, one before the apparent denaturation temperatures (Tm) and the other after the Tm. Samples heated below their Tm showed some alterations of the secondary structure and some enzymatic activity. HSDSC and FT-Raman spectroscopy are useful techniques to study protein conformations and their results correlate with those of enzymatic activity.     

"Prevention of structural perturbation and aggregation of hepatitis B surface antigen: screening of various additives"

The instability of protein and antigen(s) during encapsulation in biodegradable polymers by water-in-oil-in-water (w/o/w) encapsulation is well established. The aim of present study is to screen various additives to prevent the inactivation and loss of immunogenicity of HBsAg upon its exposure to the water/CH(2)Cl(2) (methylene chloride) interface by simulating the formulation steps involved in the preparation of microspheres. The secondary structure of HBsAg, recovered under different conditions after primary emulsification, was investigated by FTIR spectroscopy and Circular Dichorism. Subsequently, PLGA microspheres were formulated and characterized for their size, shape, incorporation efficiency, antigen integrity, and immunogenicity. The immunogenicity and the HBsAg recovery under different conditions were tested in BALB/c mice. Inulin and trehalose were found to be better stabilizing agents to prevent the aggregation, the structural perturbations and immunogenicity of HBsAg. This study substantiated that inulin could overcome the aggregation and denaturing effects of the water/CH(2)Cl(2) interface upon HBsAg during emulsification step and upon encapsulation.     

The Effect of Stabilizers and Denaturants on the Cold Denaturation Temperatures of Proteins and Implications for Freeze-Drying

Purpose The aim of the study is to investigate the effects of stabilizers and denaturants on the thermal and cold denaturation temperatures of selected proteins in systems of interest to freeze-drying.Methods β-Lactoglobulin and phosphoglycerate kinase (PGK) were chosen as model proteins. Protein thermal and cold denaturation temperatures were determined by both conventional and modulated differential scanning calorimetry and verified by tryptophan emission spectroscopy in selected systems.Results The cold denaturation of β-lactoglobulin was reversible, whereas the thermal denaturation was only reversible at high scanning rate (10°C/min). The cold denaturation temperatures of β-lactoglobulin decreased with an increase in protein concentration (self-stabilization). The cold denaturation temperature increased with increases in pH (from pH 2 to 7) with about 4.6°C increase per unit pH change. All stabilizers studied (i.e., sucrose, trehalose and glycerol) increased the thermal denaturation temperature of the proteins studied and decreased the cold denaturation temperature. The effect of sucrose in decreasing the PGK cold denaturation temperature [40°C per molar concentration increase (40°C/M)] was of the same magnitude as for β-lactoglobulin (36°C/M). The effect of stabilizers on cold denaturation temperatures is much greater than the effect on thermal denaturation temperatures. With sucrose, the β-lactoglobulin thermal denaturation temperature increases only about 5°C from 0 to 2.7 M, whereas the decrease in cold denaturation temperature was more than 35°C even at sucrose concentrationsas low as 0.9 M. Denaturants (urea and guanidine hydrochloride) increased the cold denaturationtemperatures of proteins and thereby destabilized protein; the magnitudes were 9°C/M (urea on T_cd of β-lactoglobulin) and 65°C/M (guanidine hydrochloride on PGK) compared with literature data of 16°C/M (guanidine hydrochloride on β-lactoglobulin). The cold denaturation temperatures of β-lactoglobulinand PGK extrapolated to zero concentration of denaturants were −14 and −26°C, respectively.Conclusions The protein cold denaturation temperature was pH-, protein concentration-, and additive-dependent. Stabilizers, such as sugars and/or polyols, can stabilize both protein thermal and cold denaturation, whereas the denaturants destabilize protein cold denaturation. The stabilization effect on protein cold denaturation is much larger than on thermal denaturation, a result of great importance in protein freeze-drying.     

A discussion of the principles and applications of Modulated Temperature DSC (MTDSC)

The benefits of Modulated Temperature DSC (MTDSC) over conventional differential scanning calorimetry (DSC) for studying thermal transitions in materials are reviewed by means of examples. These include the separation of overlapping phenomena such as melting/recrystallization in semi-crystalline materials, the heat capacity variation and enthalpic relaxation at the glass transition, and transitions from the different components of a blend. In addition, examples are presented demonstrating the ability of MTDSC to detect subtle transitions more readily and without loss of resolution. The possibility of measuring heat capacity in quasi-isothermal conditions and the evaluation of the thermal conductivity of a material are explained.     

The Stability of bFGF Against Thermal Denaturation

Abstract— The influence of sulphated ligand and pH on thermal denaturation of basic fibroblast growth factor (bFGF) was investigated by differential scanning calorimetry (DSC), and verified by fluorescence spectrophotometry. Purity of bFGF before and after heat denaturation was assessed by SDS-PAGE analysis. In DSC studies the samples were heated to 95°C. The midpoint of the temperature change in the thermogram was designated as T_m. Sulphated ligand experiments were undertaken in potassium phosphate (pH 6·5) and sodium acetate buffers. Control thermograms (with no ligand) showed a T_m at 59°C in potassium phosphate buffer. Higher T_m values were noted as sulphated ligand concentration was increased. Similarly when heparin was added, the T_m moved to a higher temperature. A ratio as low as 0·3:1 of heparin to bFGF, increased the T_m to 90°C, which is a 31°C shift in T_m. The effect of pH on thermal denaturation of bFGF was studied in a citrate-phosphate-borate buffer system. A shift in T_m from 46 to 65°C was observed as the pH is changed from 4 to 8. Changes in protein conformation as a function of pH were monitored by fluorescence spectroscopy. It was found that a pH range from 5 to 9 is optimal for the stability of bFGF formulations. In a stability study it was noted that heparin protected bFGF from thermal denaturation only at high temperature.     

Solid state chemistry of proteins IV. what is the meaning of thermal denaturation in freeze dried proteins?

This research addresses the thermodynamic significance of the denaturation endotherm observed during differential scanning calorimetry (DSC) scans of proteins in dry formulations, such as freeze dried solids. Human growth hormone formulations are the chosen representative examples. We employ observations of denaturation temperature, glass transition temperature, and the differences between estimated molecular mobilities to argue that unfolding is under partial thermodynamic control. Further, unfolding during a DSC scan is simulated using a three state kinetic model, which is a two state unfolding model followed by aggregation. Kramers-type rate constants are used, where the preexponential term is dominated by viscous forces. Simulation results are in qualitative agreement with experiment, and clearly show that while the denaturation endotherm is impacted by irreversibility, caused by nonzero scan rate and aggregation, the position of the endotherm peak is changed only slightly. Thus, the denaturation peak is a good approximation for the thermodynamic denaturation temperature. Using data for denaturation temperature, heat of denaturation, and heat capacity of denaturation, free energy versus temperature curves were calculated. We find that even formulations with added saccharides are thermodynamically unstable near ambient temperature; significant denaturation in the solid state is prevented by low mobility.     

The Impact of Thermal Treatment on the Stability of Freeze-Dried Amorphous Pharmaceuticals: II. Aggregation in an IgG1 Fusion Protein

The objective of this research was to investigate the impact of thermal treatment on storage stability of an IgG1 fusion protein. IgG1 protein formulations were prepared by freeze-drying the protein with sucrose. Some samples were used as controls, and others were subjected to a further heat treatment (annealing). The protein structure was investigated with Fourier transform infrared spectroscopy (FTIR), and protein aggregation was monitored with size exclusion HPLC. Enthalpy recovery was studied using DSC, and global mobility represented by the structural relaxation time constant (τ^β) was characterized by a thermal activity monitor (TAM). The local mobility of the protein system was monitored by both ¹³C solid-state NMR and neutron backscattering. Annealing increased the storage stability of the protein, as shown by the smaller aggregation rate and less total aggregation at the end of a storage period. The structural relaxation time constant of an annealed sample was significantly higher than the unannealed control sample, suggesting a decrease in global mobility of the protein system upon annealing. However, annealing does not significantly impact the protein secondary structure or the local mobility. Given the similar protein native structure and specific surface area, the improved stability upon annealing is mainly a result of reduced global molecular mobility. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:683–700, 2010     

The glass transition and sub-T(g)-relaxation in pharmaceutical powders and dried proteins by thermally stimulated current

The main goal of the study was to evaluate the applicability of thermally stimulated current (TSC) as a measure of molecular mobility in dried globular proteins. Three proteins, porcine somatotropin, bovine serum albumin, and immunoglobulin, as well as materials with a strong calorimetric glass transition (T(g)), that is, indomethacin and poly(vinypyrrolidone) (PVP), were studied by both TSC and differential scanning calorimetry (DSC). Protein/sugar colyophilized mixtures were also studied by DSC, to estimate calorimetric T(g) for proteins using extrapolation procedure. In the majority of cases, TSC detected relaxation events that were not observed by DSC. For example, a sub-T(g) TSC event (beta-relaxation) was observed for PVP at approximately 120 degrees C, which was not detected by the DSC. Similarly, DSC did not detect events in any of the three proteins below the thermal denaturation temperature whereas a dipole relaxation was detected by TSC in the range of 90-140 degrees C depending on the protein studied. The TSC signal in proteins was tentatively assigned as localized mobility of protein segments, which is different from a large-scale cooperative motions usually associated with calorimetric T(g). TSC is a promising method to study the molecular mobility in proteins and other materials with weak calorimetric T(g).     

Integrity of crystalline lysozyme exceeds that of a spray-dried form

The development of proteins as therapeutic agents is challenging partly due to their inherent instabilities. Consequently, crystallisation and spray drying techniques were assessed to determine their effects on protein integrity using lysozyme as a model protein. Unprocessed, crystallised and spray-dried lysozyme were characterised by: thermal analysis using hot stage microscopy (HSM), differential scanning calorimetry (DSC), high sensitivity differential scanning calorimetry (HSDSC) and thermogravimetry (TGA); and spectroscopic analysis employing Fourier transform Raman (FT-Raman). Moisture contents were determined by TGA and Karl Fisher titration (KFT). Enzymatic assay measured biological activity. HSM showed no changes in crystals until complete melting. TGA and KFT indicated that spray-dried lysozyme contained a lower moisture content than crystals, hence the higher apparent thermal stability was shown by DSC. HSDSC revealed that crystallisation and spray drying did not affect the denaturation temperature of lysozyme in solution when compared with unprocessed material. However, in the solid state, FT-Raman spectra showed perturbation of the conformational structure of spray-dried sample, whereas crystal conformation remained intact. Enzymatic assay revealed increased activity retention of crystals compared with spray-dried powder. Hence, crystals maintained the conformational integrity and activity of lysozyme in solution.     

Biophysical characterisation of GlycoPEGylated recombinant human factor VIIa

The effects of GlycoPEGylation on the structural, kinetic and thermal stability of recombinant human FVIIa were investigated using rFVIIa and linear 10 kDa and branched 40 kDa GlycoPEGylated(?) recombinant human FVIIa derivatives. The secondary and tertiary structure of rFVIIa measured by circular dichroism (CD) was maintained upon PEGylation. In contrast, the thermal and kinetic stability of rFVIIa was affected by GlycoPEGylation, as the apparent unfolding temperature T(m) measured by differential scanning calorimetry (DSC) and the temperature of aggregation, T(agg), measured by light scattering (LS) both increased with GlycoPEGylation. Both T(m) and T(agg) were independent of the molecular weight and the shape of the PEG chain. From the present biophysical characterisation it is concluded that after GlycoPEGylation, rFVIIa appears to be unaffected structurally (secondary and tertiary structure), slightly stabilised thermally (unfolding temperature) and stabilised kinetically (temperature of aggregation).     

Effect of polyanions on the structure and stability of repifermin (keratinocyte growth factor-2)

The interaction of several of the fibroblast growth factors (FGFs) with polyanions is thought to be of physiological significance and has been exploited to create more stable pharmaceutical formulations of FGF-1 and -2. The extent of such phenomena throughout the 23-member FGF family is, however, unknown. In these studies, we examine the effect of several polyanions on the structure and stability of keratinocyte growth factor 2 (KGF-2, FGF-10), a candidate for use as a wound-healing agent. Employing a variety of methods sensitive to the protein's structure including circular dichroism (CD), intrinsic fluorescence, derivative near-UV absorption spectroscopy, bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid) fluorescence, differential scanning calorimetry (DSC), and dynamic light scattering (DLS), we find that a variety of polyanions (e.g., heparin, sucrose octasulfate (SOS), and inositol hexaphosphate (IHP)) stabilize KGF-2 by increasing the thermal-unfolding temperature by approximately 9-15 degrees C. Negatively charged liposomes produce a similar effect, arguing for relatively nonspecific interactions of polyanions with KGF-2. Unlike some other FGFs, no evidence for the presence of a molten globule state is found during thermal perturbation of this growth factor. The generality of this polyanion/protein interaction is discussed as well as its potential role in various cellular events such as protein folding and transport.