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Biomedical subjects

K Griebenow

Publications and source records attributed to K Griebenow.

17 recordsLinked to original sources
PubMed2001

Non-aqueous encapsulation of excipient-stabilized spray-freeze dried BSA into poly(lactide-co-glycolide) microspheres results in release of native protein.

Encapsulation of the model protein bovine serum albumin (BSA) into poly(D,L lactide-co-glycolide) (PLG) microspheres was performed by a non-aqueous oil-in-oil (o/o) methodology. Powder formulations of BSA obtained by spray-freeze drying were first suspended in methylene chloride containing PLG followed by coacervation by adding silicon oil and microsphere hardening in heptane. The secondary structure of BSA was determined at relevant steps of the encapsulation procedure by employing Fourier-transform infrared (FTIR) spectroscopy. This fast and non-invasive method demonstrated the potential to rapidly screen pharmaceutically relevant protein delivery systems for their suitability. Structural perturbations in BSA were reduced during the spray-freeze drying step by employing the excipient trehalose. The protein was then encapsulated into PLG microspheres under various conditions without inducing significant structural perturbations. BSA released from these microspheres had a similar monomer content as unencapsulated BSA and also the same secondary structure. Upon blending of a poloxamer (Pluronic F-68) with the polymer phase, in vitro release was characterized by a small initial release and a prolonged and continuous sustained phase. In conclusion, the developed o/o methodology coupled with FTIR spectroscopic monitoring of protein structure is a powerful approach for the development of sustained release microspheres.

Chromatography, High Pressure Liquid↗
PubMed2001

Dihedral angles of trialanine in D2O determined by combining FTIR and polarized visible Raman spectroscopy.

We have measured the polarized visible Raman and FTIR spectra of trialanine and triglycine in D(2)O at acid, neutral, and alkaline pD. From the Raman spectra we obtained the isotropic and the anisotropic scattering. A self-consistent spectral analysis of the region between 1550 and 1800 cm(-1) was carried out to obtain the intensities, frequencies, and halfwidths of the respective amide I bands. A model was developed by means of which the intensity ratios of the amide I bands in all spectra and the respective frequency differences were utilized to determine the orientational angle theta between the peptide groups and the strength of excitonic coupling between the corresponding amide I modes. By exploiting results from a recent ab initio study on triglycine (Torii, H; Tasumi, M. J. Raman Spectrosc. 1998, 29, 81), we used these parameters to determine the dihedral angles phi and psi between the peptide groups. Our results show that trialanine adopts a 3(1)-helical structure in D(2)O for all of its three protonation states. The structure is insensitive to the carboxylate protonation and changes only slightly with N-terminal protonation. Triglycine is structurally more heterogeneous in the zwitterionic and the cationic state. Our spectral analysis suggests that 3(1)-helices coexist with right-handed alpha-helical and/or with beta-turn conformations. The N-terminal protonation stabilizes the 3(1)-structure. Our study provides compelling evidence that tripeptides adopt stable conformations in aqueous solution and that they are suitable model systems to investigate the initiation of secondary structure formation.

Alanine↗
PubMed2001

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V(0), ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38 degrees C and 56 degrees C, respectively). No relationship between this property and V(0) was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V(0) was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested.

Acetonitriles↗
PubMed2001

Reduction of structural perturbations in bovine serum albumin by non-aqueous microencapsulation.

Protein stability is a factor limiting the use of sustained-release devices in medical applications. The aim of this study was to reduce structural perturbations occurring in the frequently used model protein, bovine serum albumin (BSA), upon microencapsulation in poly(D,L-lactide-co-glycolide) (PLG) microspheres. Spray freeze-dried BSA was encapsulated into PLG microspheres by a completely non-aqueous oil-in-oil encapsulation procedure. FTIR spectroscopy was used as a non-invasive method to quantify procedure-induced structural perturbations in BSA. Spray-freeze drying of BSA caused significant structural perturbations that were minimized by co-spray freeze-drying BSA with trehalose. BSA-containing microspheres were produced by suspension of the powder by homogenization in methylene chloride containing PLG, followed by formation of coacervate droplets by the addition of silicon oil and hardening using the solvent heptane. Resulting microspheres had dimensions of approximately 100 microm and the encapsulation efficiency for BSA was > 90%. FTIR data showed that the structure of the BSA-trehalose formulation encapsulated into PLG microspheres was less perturbed than that of BSA obtained from buffer alone. The results demonstrate that the structure-guided encapsulation approach introduced for non-aqueous casting encapsulation procedures can be extended to the non-aqueous production of pharmaceutically relevant PLG microspheres involving a complex encapsulation procedure.

Biocompatible Materials↗
PubMed2001

Encapsulation of bovine serum albumin in poly(lactide-co-glycolide) microspheres by the solid-in-oil-in-water technique.

Non-aqueous protocols to encapsulate pharmaceutical proteins into biocompatible polymers have gained much attention because they allow for the minimization of procedure-induced protein structural perturbations. The aim of this study was to determine if these advantages could be extended to a semi-aqueous encapsulation procedure, namely the solid-in-oil-in-water (s/o/w) technique. The model protein bovine serum albumin (BSA) was encapsulated into poly(lactide-co-glycolide) (PLG) microspheres by first suspending lyophilized BSA in methylene chloride containing PLG, followed by emulsification in a 1% aqueous solution of poly(vinyl alcohol). By variation of critical encapsulation parameters (homogenization intensity, BSA:PLG ratio, emulsifier concentration, ratio of organic to aqueous phase) an encapsulation efficiency of > 90% was achieved. The microspheres obtained showed an initial burst release of < 20%, a sustained release over a period of about 19 days, and a cumulative release of at least 90% of the encapsulated BSA. Different release profiles were observed when using different encapsulation protocols. These differences were related to differences in the microsphere erosion observed using scanning electron microscopy. Release of BSA was mainly due to simple diffusion or to both diffusion and microsphere erosion. Fourier-transform infrared studies were conducted to investigate the secondary structure of BSA during the encapsulation. Quantification of the alpha-helix and beta-sheet content as well as of overall structural changes showed that the secondary structure of encapsulated BSA was not more perturbed than in the lyophilized powder used initially. Thus, the encapsulation procedure did not cause detrimental structural perturbations in BSA. In summary, the results demonstrate that the s/o/w technique is an excellent alternative to the water-in-oil-in-water technique, which is still mainly used in the encapsulation of proteins in PLG microspheres.

Drug Compounding↗
PubMed2001

Prevention of structural perturbations and aggregation upon encapsulation of bovine serum albumin into poly(lactide-co-glycolide) micropheres using the solid-in-oil-in water technique.

Bovine serum albumin (BSA) was encapsulated into poly(lactide-co-glycolide) (PLG) microspheres by a solid-in-oil-in-water (s/o/w) technique. We tested whether perturbations in BSA secondary structure could be minimized during encapsulation by using trehalose and how this would influence BSA aggregation and release. BSA secondary structure was monitored noninvasively by Fourier-transform infrared spectroscopy. When BSA was co-lyophilized with trehalose, lyophilization-induced structural perturbations were significantly reduced. The formulation obtained (BSA-Tre) was encapsulated into PLG microspheres and, by optimizing critical encapsulation parameters, a loading efficiency of 85% was achieved. However, due to the loss of the excipient in the o/w emulsion step, the structure of BSA-Tre was more perturbed than before encapsulation. Excipient-loss and encapsulation-induced structural perturbations could be prevented by saturating the aqueous phase in the o/w step with trehalose and by using the organic solvent chloroform. This in turn reduced the formation of soluble BSA aggregates. BSA was released from PLG microspheres using the improved formulations with an initial release in 24 h of not more than 22%, followed by a sustained release over at least 2 weeks. In summary, optimization of the encapsulation conditions in the s/o/w procedure resulted in the encapsulation of BSA without procedure-induced structural perturbations and minimized the release of aggregated protein. This demonstrates that the s/o/w technique is an excellent alternative to the most common encapsulation procedure, namely the water-in-oil-in-water technique.

Chromatography, High Pressure Liquid↗
PubMed2001

Improved activity and stability of lysozyme at the water/CH2Cl2 interface: enzyme unfolding and aggregation and its prevention by polyols.

Protein inactivation and aggregation at the water/CH2Cl2 interface is one of the most detrimental events hindering the encapsulation of structurally unperturbed proteins into poly(lactide-co-glycolide) (PLG) microspheres for their clinical application as sustained release dosage forms. We have investigated the inactivation and aggregation of the model protein hen egg-white lysozyme at this interface and devised methods to prevent both events. When lysozyme was exposed to a large water/CH2Cl2 interface achieved by homogenization, lysozyme aggregation occurred. Fourier-transform infrared (FTIR) spectroscopic data demonstrated that the aggregates formed contained intermolecular beta-sheets. The aggregates were of a noncovalent nature because they slowly dissolved in D2O and the IR spectral bands typical for the intermolecular beta-sheets disappeared at approximately 1617 and 1690 cm(-1). The observed loss in specific enzyme activity of soluble lysozyme was caused by the irreversible formation of an unfolded lysozyme species, which was found to be monomeric, and was able to leave the water/CH2Cl2 interface and accumulate in the aqueous phase. Polyols were, in a concentration dependent fashion, efficient in ameliorating lysozyme unfolding and aggregation. However, prevention of lysozyme aggregation and activity loss in the various samples were unrelated. Thus, polyols must work by more than one mechanism preventing the two events. For the first time, an excipient effect on the conformational stability of lysozyme has been excluded from contributing to the prevention of lysozyme unfolding and aggregation.

Delayed-Action Preparations↗
PubMed2000

Protein spray-freeze drying. Effect of atomization conditions on particle size and stability.

PURPOSE: To investigate the effect of atomization conditions on particle size and stability of spray-freeze dried protein. METHODS: Atomization variables were explored for excipient-free (no zinc added) and zinc-complexed bovine serum albumin (BSA). Particle size was measured by laser diffraction light scattering following sonication in organic solvent containing poly(lactide-co-glycolide) (PLG). Powder surface area was determined from the N2 vapor sorption isotherm. Size-exclusion chromatography (SEC) was used to assess decrease in percent protein monomer. Fourier-transform infrared (FTIR) spectroscopy was employed to estimate protein secondary structure. PLG microspheres were made using a non-aqueous, cryogenic process and release of spray-freeze dried BSA was assessed in vitro. RESULTS: The most significant atomization parameter affecting particle size was the mass flow ratio (mass of atomization N2 relative to that for liquid feed). Particle size was inversely related to specific surface area and the amount of protein aggregates formed. Zinc-complexation reduced the specific surface area and stabilized the protein against aggregation. FTIR data indicated perturbations in secondary structure upon spray-freeze drying for both excipient-free and zinc-complexed protein. CONCLUSIONS: Upon sonication, spray-freeze dried protein powders exhibited friability, or susceptibility towards disintegration. For excipient-free protein, conditions where the mass flow ratio was > -0.3 yielded sub-micron powders with relatively large specific surface areas. Reduced particle size was also linked to a decrease in the percentage of protein monomer upon drying. This effect was ameliorated by zinc-complexation, via a mechanism involving reduction in specific surface area of the powder rather than stabilization of secondary structure. Reduction of protein particle size was beneficial in reducing the initial release (burst) of the protein encapsulated in PLG microspheres.

Biocompatible Materials↗
PubMed2000

Relationship between conformational stability and lyophilization-induced structural changes in chymotrypsin.

The relationship between protein conformational stability in aqueous solution and the magnitude of lyophilization-induced structural changes was investigated employing alpha- and gamma-chymotrypsin. As a measure of the conformational stability the melting temperature T(m) was determined in distilled water at various pH values. The proteins were then lyophilized from those pH values where the conformational stability was maximum (pH 4.5) and minimum (pH 7.8). Protein secondary structure was quantitatively determined utilizing Fourier-transform infrared spectroscopy employing two regions sensitive to protein structure, the amide-I (1600-1700 cm(-1)) and amide-III (1215-1335 cm(-1)). Lyophilization induced significant structural alterations in both proteins, characterized by a slight decrease in the alpha-helix and a significant increase in the beta-sheet content. However, regardless of the pH from which the proteins were lyophilized, the secondary structures in the solid state were indistinguishable. This result shows that there is no relationship between the conformational stability in aqueous solution and the magnitude of lyophilization-induced structural changes. We also investigated whether lyoprotectants could minimize lyophilization-induced structural changes by increasing protein conformational stability in aqueous solution. After having identified trehalose as being efficient in largely preventing lyophilization-induced structural alterations, we conducted co-lyophilization experiments from various pH values. The results obtained exclude any contribution from increased protein conformational stability caused by the additive in aqueous solution to the beneficial structural preservation upon lyophilization. This can be understood because the dehydration and not the freezing process, as shown in an air-drying experiment, mainly causes protein structural alterations.

Chymotrypsin↗
PubMed1999

FTIR characterization of the secondary structure of proteins encapsulated within PLGA microspheres.

A commonly used technique for protein encapsulation in microspheres is the double-emulsion method wherein an initial water-in-oil (w/o) emulsion of protein and polymer is formed via sonication, and then a second emulsion (w/o)/w is formed by dispersion in an aqueous phase via homogenization. This approach is often used to produce microspheres of biodegradable poly(lactic-co-glycolic acid) (PLGA). The harsh processing associated with this method can cause denaturation of the encapsulated protein. Herein, we have used Fourier transform infrared (FTIR) spectroscopy to determine the secondary structures of two model proteins, bovine serum albumin (BSA) and chicken egg-white lysozyme, within PLGA microspheres. The alpha-helix content of both proteins in the microspheres was about a third lower than in the lyophilized state, indicating conformational changes upon protein entrapment within the microspheres. BSA microspheres containing the stabilizing excipient trehalose have a higher alpha-helix content than those without excipient, suggesting that trehalose partially prevents the denaturing effects incurred during processing. In addition, BSA released from microspheres is improved by incorporation of trehalose: analysis of the protein released from the microspheres indicates that there is less BSA dimer formation in the trehalose-containing microspheres than in those without trehalose.

Animals↗
PubMed1999

On the structural preservation of recombinant human growth hormone in a dried film of a synthetic biodegradable polymer.

In this work we describe the structural investigation of the model protein recombinant human growth hormone (rhGH) under conditions relevant to polymeric sustained-delivery depots, including the dried protein entrapped in a film of poly(DL-lactic-co-glycolic)acid. At each step of the procedure, dehydration of rhGH by lyophilization, suspension in methylene chloride, and drying from that suspension, the structure of rhGH was probed noninvasively using Fourier transform infrared (FTIR) spectroscopy. We found that the structure of rhGH was significantly changed by the dehydration process as indicated by a marked drop in the alpha-helix content and increase in the beta-sheet content. Subsequent suspension of this powder in methylene chloride, drying from that suspension, and drying from a methylene chloride/PLGA solution introduced only minor additional structural changes when using appropriate conditions. This result is likely due to the limited molecular mobility of proteins in nonprotein-dissolving organic solvents. Finally, when rhGH was co-lyophilized with the lyoprotectant trehalose, which preserves the secondary structure, the rhGH entrapped in the PLGA matrix also had a nativelike secondary structure.

Freeze Drying↗
PubMed1998

Effect of excipients on the stability and structure of lyophilized recombinant human growth hormone.

We have investigated the effect of mannitol, sorbitol, methyl alpha-D-mannopyranoside, lactose, trehalose, and cellobiose on the stability and structure of the pharmaceutical protein recombinant human growth hormone (rhGH) in the lyophilized state. All excipients afforded significant protection of the protein against aggregation, particularly at levels to potentially satisfy water-binding sites on the protein in the dried state (i.e., 131:1 excipient-to-protein molar ratio). At higher excipient-to-protein ratios, X-ray diffraction studies showed that mannitol and sorbitol were prone to crystallization and afforded somewhat less stabilization than at lower ratios where the excipient remained in the amorphous, protein-containing phase. The secondary structure of rhGH was determined using Fourier transform infrared (FTIR) spectroscopy. rhGH exhibited a decrease in alpha-helix and increase in beta-sheet structures upon drying. Addition of excipient stabilized the secondary structure upon lyophilization to a varying extent depending on the formulation. Samples with a significant degree of structural conservation, as indicated by the alpha-helix content, generally exhibited reduced aggregation. In addition, prevention of protein-protein interactions (indicated by reduced beta-sheet formation) also tended to result in lower rates of aggregation. Therefore, in addition to preserving the protein structure, bulk additives that do not crystallize easily and remain amorphous in the solid state can be used to increase protein-protein distance and thus prevent aggregation.

Drug Stability↗
PubMed1996

The secondary structure and aggregation of lyophilized tetanus toxoid.

Tetanus toxoid (TT), the vaccine for tetanus, is an important protein antigen and candidate for sustained release from polymeric matrices. During administration from the latter, the solid (e.g., lyophilized) protein will be exposed to elevated levels of temperature and moisture, conditions which trigger its aggregation. To examine the connection between this aggregation and the structure of the TT molecule in the solid state, Fourier-transform infrared (FTIR) spectroscopy was employed to determine the secondary structure of TT in the presence of various excipients. We found that excipient-free TT undergoes a significant alteration (mostly reversible) in the secondary structure during lyophilization. Specifically, more than half the total alpha-helix content was lost with a concomitant increase in beta-sheet structure. The extent of structural alterations in the presence of 1:5 (g:g protein) NaCl, sorbitol, or poly-(ethylene glycol), did not correlate with stability conferred towards moisture-induced aggregation. These results suggest that the degree of retention of the native protein structure in the dry state is not a general predictor of stability for the "wetted" solid within polymer controlled-release vehicles.

Excipients↗
PubMed1995

Lyophilization-induced reversible changes in the secondary structure of proteins.

Changes in the secondary structure of some dozen different proteins upon lyophilization of their aqueous solutions have been investigated by means of Fourier-transform infrared spectroscopy in the amide III band region. Dehydration markedly (but reversibly) alters the secondary structure of all the proteins studied, as revealed by both the quantitative analysis of the second derivative spectra and the Gaussian curve fitting of the original infrared spectra. Lyophilization substantially increases the beta-sheet content and lowers the alpha-helix content of all proteins. In all but one case, proteins become more ordered upon lyophilization.

Amides↗
PubMed1995

Fourier-transform infrared spectroscopic investigation of protein stability in the lyophilized form.

Upon the removal of water, proteins undergo a major, reversible rearrangement of their secondary structure, as revealed by FTIR spectroscopy. We have found herein that for recombinant human albumin (rHA) the extent of this structural change does not depend significantly either on the composition of the aqueous solution prior to lyophilization (protein concentration, pH, and the presence of excipients such as dextran or NaCl) or on the mode of dehydration (lyophilization, spray drying, or rotary evaporation), even though these factors profoundly affect rHA's solid-state stability against moisture-induced aggregation. In all cases, the alpha-helix content of rHA drops from 58% in solution to 25-35% in the dehydrated state, the beta-sheet content rises from 0 to 10-20%, and unordered structures increase from 40% to 50-60%. We have also investigated another model protein, hen egg-white lysozyme, and confirmed that it too undergoes a significant alteration of the secondary structure upon lyophilization. The extent of this structural reorganization has been found to be insensitive to the pH of the aqueous solution prior to lyophilization from pH 1.9 to 5.1, even though the thermal transition temperature (Tm) in aqueous solution over this range varies by 30 degrees C.

Dextrans↗
PubMed1993

Picosecond energy transfer and trapping kinetics in living cells of the green bacterium Chloroflexus aurantiacus.

The excitation energy transfer and trapping processes in intact cells of Chloroflexus aurantiacus were studied by picosecond time-resolved fluorescence spectroscopy. The fluorescence decay kinetics is investigated over the near infrared emission range between 730 nm and 920 nm using various excitation wavelengths and excitation intensities. The data were analyzed by global decay analysis and are presented as decay-associated spectra (DAS). The specific dependence of the decay kinetics on the excitation wavelength and on the photochemical redox state of the reaction center (RC) allows the identification of the energy transfer and trapping components. The DAS provide evidence for two chlorosomal energy transfer processes. The first one occurs between the chlorosomal bacteriochlorophyll (BChl)-c and the BChl-a792 complex (B792) in the chlorosomal baseplate with an equilibration time constant of 15-16 ps, while the second one occurs from the B792 pigments to the BChl-a806 pigments in the B806-866 complex with a time constant of 35-40 ps. The overall energy trapping process in whole cells is mainly determined by the kinetics of the primary charge separation process in the RCs. With open RCs (QA oxidized) the trapping time constant is 70-90 ps, while the trapping process with closed RCs (QA reduced) takes as long as 180-200 ps. The results on whole cells reported here are interpreted in conjunction with those reported earlier for the various isolated complexes, i.e., two different chlorosome preparations (Holzwarth, A.R., Müller, M.G. and Griebenow, K. (1990) J. Photochem. Photobiol. B 5, 457-465), the B806-866 complex (Griebenow, K., Müller, M.G. and Holzwarth, A.R. (1991) Biochim. Biophys. Acta 1059, 226-232) and isolated reaction centers (Müller, M.G., Griebenow, K. and Holzwarth, A.R. (1991) Biochim. Biophys. Acta 1098, 1-12). Based on these data, a unified and self-consistent scheme for the primary processes in the whole photosynthetic system of C. aurantiacus is presented. The BChl antenna pigment groups are arranged to form a linear energy transfer cascade with four energy transfer steps from shorter-wavelength- to longer-wavelength-absorbing antenna pools. The overall fluorescence decay kinetics of the photosynthetic system of C. aurantiacus turns out to be 'trap-limited' by the reaction center rather than 'diffusion-limited' by the energy transfer processes.

Cyanobacteria↗