Analysis of antibody self-interaction by bio-layer interferometry as tool to support lead candidate selection during preformulation and developability assessments.

Developability assessment of therapeutic mAb candidates before entering CMC development mitigates the risk of later failure because of manufacturing and stability issues. For mAbs derived from library based screenings, such evaluation starts with the first panning and ends with the selection of a lead candidate. This candidate should show, amongst others, high affine target binding and beneficial conformational as well as chemical stability. In addition, colloidal stability, reflected by the self-interaction propensity, should be superior in order to reduce aggregate formation and unacceptably high viscosity at elevated protein concentrations. Here, we present a study demonstrating the application of self-interaction bio-layer interferometry (SI-BLI) in a developability assessment, including the evaluation of preformulations. We reveal that the formulation rankings based on SI-BLI, DLS and viscosity measurements correlate. SI-BLI provides a deeper understanding of influencing factors on mAb self-interaction such as ionic strength or cation species. The attractive mAb self-interaction propensity was significantly more suppressed by Mg2+ compared to Na+. SI-BLI can be performed in high throughput with minimal material and sample preparation needs. Therefore, it can be applied in early stages of developability assessment going beyond the use of a platform formulation and a small number of analysis, to screen more parameters before proceeding with candidate selection and further extensive development.

Porosity and mechanically optimized PLGA based in situ hardening systems.

Goal of the present study was to develop and to characterize in situ-hardening, porous PLGA-based systems for their future application as bone grafting materials. Therefore, we investigated the precipitation behavior of formulations containing PLGA and a water-miscible solvent, DMSO, PEG 400, and NMP. To increase porosity, a pore forming agent (NaCMC) was added and to enhance mechanical properties of the system, an inorganic filler (α-TCP) was incorporated. The behavior upon contact with water and the influence of the prior addition of aqueous media on the morphology of the corresponding hardened implants were investigated. We proved cell-compatibility by live/dead assays for the hardened porous polymer/ceramic-composite scaffolds. The IsHS formulations can therefore be used to manufacture hardened scaffolds ex vivo by using molds with the desired shape and size. Cells were further successfully incorporated into the IsHS by precultivating the cells on the α-TCP-powder prior to their admixing to the formulation. However, cell viability could not be maintained due to toxicity of the tested solvents. But, the results demonstrate that in vivo cells should well penetrate, adhere, and proliferate in the hardened scaffolds. Consequently, we consider the in situ hardening system being an excellent candidate as a filling material for non-weight-bearing orthopedic indications, as the resulting properties of the hardened implant fulfill indication-specific needs like mechanical stability, elasticity, and porosity.

Residual transglutaminase in collagen - effects, detection, quantification, and removal.

In the present study, we developed an enzyme-linked immunosorbent assay (ELISA) for microbial transglutaminase (mTG) from Streptomyces mobaraensis to overcome the lack of a quantification method for mTG. We further performed a detailed follow-on-analysis of insoluble porcine collagen type I enzymatically modified with mTG primarily focusing on residuals of mTG. Repeated washing (4 ×) reduced mTG-levels in the washing fluids but did not quantitatively remove mTG from the material (p < 0.000001). Substantial amounts of up to 40% of the enzyme utilized in the crosslinking mixture remained associated with the modified collagen. Binding was non-covalent as could be demonstrated by Western blot analysis. Acidic and alkaline dialysis of mTG treated collagen material enabled complete removal the enzyme. Treatment with guanidinium chloride, urea, or sodium chloride was less effective in reducing the mTG content.

Magnetic Drug Targeting as New Therapeutic Option for the Treatment of Biomaterial Infections.

Implant-associated infections are a challenging problem in surgery. Bacteria in biofilms are difficult to treat as they are less susceptible to antibiotics or antiseptics which require high drug concentrations at the site of infection. We present a novel strategy to concentrate high antibiotic doses systemically at the target site using newly developed antibiotic-functionalized nanoparticles directed by a magnetic drug-targeting system. The important and effective antibiotic gentamicin served as antimicrobial substance and was ionically or covalently attached to magnetic nanoparticles. Subsequently, the particles were characterized thoroughly. Anti-infective properties with regard to Staphylococcus aureus and the degree of cytotoxicity concerning human umbilical vein endothelial cells were determined. The enrichment of the magnetic nanoparticles at the surface of model tubes in circulatory experiments was investigated. We describe a promising technique for the loading of magnetic nanoparticles to treat systemic infections. Gentamicin-coated magnetic nanoparticles reduced bacterial growth even beyond pathologically relevant concentrations within 24 h. Excellent concentration independent biocompatibility was found for the nanoparticles themselves and we demonstrate that the magnetic nanoparticles can be navigated and concentrated on surfaces of model implants using a permanent magnetic field.

Augmentation of antibiotic activity by low-frequency electric and electromagnetic fields examining Staphylococcus aureus in broth media.

Systemic treatment of biomaterial-associated bacterial infections with high doses of antibiotics is an established therapeutic concept. The purpose of this in vitro study was to determine the influence of magnetic, electromagnetic, and electric fields on gentamicin-based, antibiotic therapy. It has been previously reported that these fields are successful in the treatment of bone healing and reducing osteitis in infected tibia-pseudarthroses. Four separate experimental setups were used to expose bacterial cultures of Staphylococcus aureus both in Mueller-Hinton broth (MHB) and on Mueller-Hinton agar (MHA), in the presence of gentamicin, to (1) a low-frequency magnetic field (MF) 20 Hz, 5 mT; (2) a low-frequency MF combined with an additional alternating electric field (MF + EF) 20 Hz, 5 mT, 470 mV/cm; (3) a sinusoidal alternating electric field (EF AC) 20 Hz, 470 mV/cm; and (4) a direct current electric field (EF DC) 588 mV/cm. No significant difference between samples and controls was detected on MHA. However, in MHB each of the four fields applied showed a significant growth reduction of planktonically grown Staphylococcus aureus in the presence of gentamicin between 32% and 91% within 24 h of the experiment. The best results were obtained by a direct current EF, decreasing colony-forming units (CFU)/ml more than 91%. The application of electromagnetic fields in the area of implant and bone infections could offer new perspectives in antibiotic treatment and antimicrobial chemotherapy.

Optimization of a pharmaceutical freeze-dried product and its process using an experimental design approach and innovative process analyzers.

The aim of the present study was to examine the possibilities/advantages of using recently introduced in-line spectroscopic process analyzers (Raman, NIR and plasma emission spectroscopy), within well-designed experiments, for the optimization of a pharmaceutical formulation and its freeze-drying process. The formulation under investigation was a mannitol (crystalline bulking agent)-sucrose (lyo- and cryoprotector) excipient system. The effects of two formulation variables (mannitol/sucrose ratio and amount of NaCl) and three process variables (freezing rate, annealing temperature and secondary drying temperature) upon several critical process and product responses (onset and duration of ice crystallization, onset and duration of mannitol crystallization, duration of primary drying, residual moisture content and amount of mannitol hemi-hydrate in end product) were examined using a design of experiments (DOE) methodology. A 2-level fractional factorial design (2(5-1)=16 experiments+3 center points=19 experiments) was employed. All experiments were monitored in-line using Raman, NIR and plasma emission spectroscopy, which supply continuous process and product information during freeze-drying. Off-line X-ray powder diffraction analysis and Karl-Fisher titration were performed to determine the morphology and residual moisture content of the end product, respectively. In first instance, the results showed that - besides the previous described findings in De Beer et al., Anal. Chem. 81 (2009) 7639-7649 - Raman and NIR spectroscopy are able to monitor the product behavior throughout the complete annealing step during freeze-drying. The DOE approach allowed predicting the optimum combination of process and formulation parameters leading to the desired responses. Applying a mannitol/sucrose ratio of 4, without adding NaCl and processing the formulation without an annealing step, using a freezing rate of 0.9°C/min and a secondary drying temperature of 40°C resulted in efficient freeze-drying supplying end products with a residual moisture content below 2% and a mannitol hemi-hydrate content below 20%. Finally, using Monte Carlo simulations it became possible to determine how varying the factor settings around their optimum still leads to fulfilled response criteria, herewith having an idea about the probability to exceed the acceptable response limits. This multi-dimensional combination and interaction of input variables (factor ranges) leading to acceptable response criteria with an acceptable probability reflects the process design space.

Synthesis, characterization and assessment of suitability of trehalose fatty acid esters as alternatives for polysorbates in protein formulation.

Nonionic polyethylene glycol-derived surfactants are today's choice as surfactants in protein formulations. Different groups discovered that although surface-induced stresses are reduced by these excipients, the long-term stability of different proteins decreased due to polyethylene glycol-related induction of oxidation processes under static storage conditions. In this paper, the potential of polyoxyethylene-free surfactants for protein formulation was evaluated. Three different sugar-based surfactants, 6-O-monocaprinoyl-α,α-trehalose, 6-O-monolauroyl-α,α-trehalose and 6-O-monopalmitoyl-α,α-trehalose, were synthesized in four reaction steps. These substances lack polyethylene glycol residues and can be produced from renewable resources. The chemical and physical properties of these three surfactants were investigated and compared with polysorbate 20 and 80. 6-O-monopalmitoyl-α,α-trehalose was insoluble in water at room temperature and was hence excluded from some of the further tests. The critical micellar concentration of all surfactants is in a comparable range of approximately 0.001-0.01% (m/V). The sugar-based surfactants showed slightly higher hemolytic activity than the polysorbate references. The surfactants with shorter chain length proved to be comparable to polysorbates in regard to physicochemical properties. Finally for human growth hormone, the protein-stabilizing properties against shaking-induced stress were tested and compared to polysorbate-containing formulations. Whereas in the absence of surfactant, dramatic monomer loss and aggregate formation occurred, it was found that 100% monomer content was maintained when 0.1% (m/V) 6-O-monocaprinoyl-α,α-trehalose or 6-O-monolauroyl-α,α-trehalose was added to the formulation. Polysorbate 80 at a concentration of 0.1% (m/V) also significantly stabilized the protein. Lower amounts of surfactants result in only partial stabilization. Furthermore, adsorption of human growth hormone to the container surface is reduced in the presence of the surfactants. Thus, the new sugar-based surfactants offer a promising alternative and have potential for application in protein formulations.

Importance of using complementary process analyzers for the process monitoring, analysis, and understanding of freeze drying.

The aim of the present paper is to demonstrate the importance of using complementary process analyzers (PAT tools) for the process monitoring, analysis, and understanding of freeze drying. A mannitol solution was used as a model system. Raman spectroscopic, near-infrared (NIR) spectroscopic, plasma emission spectroscopic, and wireless temperature measurements (TEMPRIS) were simultaneously performed in-line and real-time during each freeze-drying experiment. The combination of these four process analyzers to monitor a freeze-drying process is unique. The Raman and NIR data were analyzed using principal component analysis (PCA) and multivariate curve resolution (MCR), while the plasma emission spectroscopic and wireless temperature measurement data were analyzed using univariate data analysis. It was shown that the considered process analyzers do not only complement but also mutually confirm each other with respect to process step end points, physical phenomena occurring during freeze drying (process understanding), and product characterization (solid state). Furthermore and most important, the combined use of the process analyzers helped to identify flaws in previous studies in which these process analyzers were studied individually. Process analyzers might wrongly indicate that some process steps are fulfilled. Finally, combining the studied process analyzers also showed that more information per process analyzer can be obtained than previously described. A combination of Raman and plasma emission spectroscopy seems favorable for the monitoring of nearly all critical freeze-drying process aspects.

New anti-infective coatings of surgical sutures based on a combination of antiseptics and fatty acids.

Wound infection is a complication feared in surgery. The aim of this study was to develop new anti-infective coatings of surgical sutures and to compare the anti-microbial effectiveness and biocompatibility to the well-established Vicryl Plus. Synthetic absorbable PGA surgical sutures were coated with three different chlorhexidine concentrations and two different octenidine concentrations in combination with palmitic acid and lauric acid. Drug-release kinetics lasting 96 h were studied in phosphate-buffered saline at 37 degrees C. Anti-infective characteristics were determined by measuring the change in optical density of Staphylococcus aureus suspensions charged with coated sutures over time. Microorganisms adsorbed at the surface of coated sutures were assessed on blood agar plates and coated sutures eluted for 24 h were placed on bacterial lawns cultured on Mueller-Hinton plates to prove retained anti-microbial potency. A cell proliferation assay was performed to assess the degree of cytotoxicity. Anti-infective characteristics and biocompatibility were compared to Vicryl Plus. A coating technology for slow-release drug-delivery systems on surgical sutures could be developed. All coatings showed a continuous drug release within 96 h. Individual chlorhexidine and octenidine coated sutures showed superior anti-infective characteristics but inferior biocompatibility in comparison to Vicryl Plus. We conclude that the developed anti-infective suture coatings consisting of lipid-based drug-delivery systems in combination with antiseptics are highly effective against bacterial colonization in vitro; however, drug doses have to be adjusted to improve biocompatibility.

New anti-infective coatings of medical implants.

Implantable devices are highly susceptible to infection and are therefore a major risk in surgery. The present work presents a novel strategy to prevent the formation of a biofilm on polytetrafluoroethylene (PTFE) grafts. PTFE grafts were coated with gentamicin and teicoplanin incorporated into different lipid-like carriers under aseptic conditions in a dipping process. Poly-d,l-lactic acid, tocopherol acetate, the diglyceride Softisan 649, and the triglyceride Dynasan 118 were used as drug carriers. The drug release kinetics, anti-infective characteristics, biocompatibility, and hemocompatibility of the coatings developed were studied. All coatings showed an initial drug burst, followed by a low continuous drug release over 96 h. The dimension of release kinetics depended on the carrier used. All coated prostheses reduced bacterial growth drastically over 24 h, even below pathologically relevant concentrations. Different cytotoxic levels could be observed, revealing tocopherol acetate as the most promising biocompatible carrier. A possible reason for the highly cytotoxic effect of Softisan 649 could be assessed by demonstrating incorporated lipids in the cell soma with Oil Red O staining. Tromboelastography studies, enzyme-linked immunosorbent assays, and an amidolytic substrate assay could confirm the hemocompatibility of individual coatings. The development of the biodegradable drug delivery systems described here and in vitro studies of those systems highlight the most important requirements for effective as well as compatible anti-infective coatings of PTFE grafts. Through continuous local release, high drug levels can be produced at only the targeted area and physiological bacterial proliferation can be completely inhibited, while biocompatibility as well as hemocompatibility can be ensured.

Zinc gluconate in the treatment of dysgeusia--a randomized clinical trial.

In the treatment of dysgeusia, the use of zinc has been frequently tried, with equivocal results. The aim of the present randomized clinical trial, which involved a sufficiently large sample, was therefore to determine the efficacy of zinc treatment. Fifty patients with idiopathic dysgeusia were carefully selected. Zinc gluconate (140 mg/day; n=26) or placebo (lactose; n=24) was randomly assigned to the patients. The patients on zinc improved in terms of gustatory function (p <0.001) and rated the dysgeusia as being less severe (p <0.05). Similarly, signs of depression in the zinc group were less severe (Beck Depression Inventory, p <0.05; mood scale, p <0.05). With the exception of the salivary calcium level, which was higher in the zinc patients (p <0.05), no other significant group differences were found. In conclusion, zinc appears to improve general gustatory function and, consequently, general mood scores in dysgeusia patients.

Collagen sponges for bone regeneration with rhBMP-2.

In the US alone, approximately 500,000 patients annually undergo surgical procedures to treat bone fractures, alleviate severe back pain through spinal fusion procedures, or promote healing of non-unions. Many of these procedures involve the use of bone graft substitutes. An alternative to bone grafts are the bone morphogenetic proteins (BMPs), which have been shown to induce bone formation. For optimal effect, BMPs must be combined with an adequate matrix, which serves to prolong the residence time of the protein and, in some instances, as support for the invading osteoprogenitor cells. Several factors involved in the preparation of adequate matrices, specifically collagen sponges, were investigated in order to test the performance in a new role as an implant providing local delivery of an osteoinductive differentiation factor. Another focus of this review is the current system consisting of a combination of recombinant human BMP-2 (rhBMP-2) and an absorbable collagen sponge (ACS). The efficacy and safety of the combination has been clearly proven in both animal and human trials.

Effects of processing conditions on the rheological behavior of collagen dispersions.

Biomedical collagen preparations are mainly based on liquid aqueous preparations either used directly as injectables or transferred for example into solid implants or porous devices. In all cases the viscosity of the dispersion or solution has to be controlled or adjusted by pH, temperature, collagen concentration or crosslinking. We tested these effects on the rheological and structural properties of collagen fiber dispersions focusing on oscillatory rheometry. With increasing difference between pH and pI viscosity increased and went through a maximum with less rigidity of the fibers. The dispersions acted like a predominantly permanently linked network. This character changed to only partially linked at low collagen concentration due to isolation of the individual fibers. Up to 40 degrees C, temperature is a tool suitable for adjusting viscosity without changes in the network structure. At 50 degrees C, fourier transform - infrared spectroscopy (FT-IR) spectroscopy indicated the transition of the helical into random coil structure. Renaturation of the helices was found upon cooling but atomic force microscopy (AFM) indicated severe temperature induced damages of the fibers. Crosslinking with glutaraldehyde (GTA) leads to an increase in viscosity and the effect on the network structure depends on the processing conditions.

Delivery systems for BMPs: factors contributing to protein retention at an application site.

BACKGROUND: Recombinant human bone morphogenetic proteins (rhBMPs) are being tested in clinical studies for their capacity to elicit bone formation. Biomaterials used in delivery systems also play a critical role in supporting the osteoinductive activity of BMPs, attributable to the controlled presentation of the BMPs to target cells. Despite extensive preclinical studies, the factors contributing to local rhBMP pharmacokinetics remain to be elucidated. METHODS: The rhBMP pharmacokinetics were studied in a rat subcutaneous implant and in an intramuscular injection model. In situ levels of rhBMPs were quantitated with use of 125I-labeled tracers. The effects of protein structural features and the nature of the biomaterial implant were explored. Osteoinduction by biomaterial+rhBMP combinations was assessed by a semiquantitative, histology-based bone score. RESULTS: With the use of rhBMP-2, rhBMP-4, and an N-truncated rhBMP-2, the protein isoelectric point was found critical for the initial retention of rhBMPs in an implant. Osteoinduction studies carried out in parallel indicated that rhBMPs with a higher implant retention elicited more bone formation. In the clinically used collagen+rhBMP-2 device, collagen crosslinking and sterilization were most influential in rhBMP-2 retention. To increase retention at an application site, thermoreversible polymers were engineered and shown to enhance local rhBMP-2 retention, especially by injectable delivery. CONCLUSIONS: Two critical components of an osteoinductive device--namely, the biomaterial and the rhBMP--were shown to influence local protein pharmacokinetics and osteoinductive activity of the device. Designer biomaterials can provide an additional mechanism to modulate local protein pharmacokinetics. CLINICAL RELEVANCE: These studies form the foundation of next-generation osteoinductive devices with improved potency at sites of desired bone regeneration and reduced side effects at other sites.

Characterization of absorbable collagen sponges as rhBMP-2 carriers.

For clinical use recombinant human bone morphogenetic protein (rhBMP-2) is soaked onto an absorbable collagen sponge (ACS) for bone regeneration. Therefore, loss of rhBMP-2 upon mechanical handling during implantation and a potential effect of the carrier on in vivo retention is of interest. The interactions between drug and carrier were looked at from the application mode and the amount of protein which can be mechanically expressed from the combination was investigated. The results indicated that rhBMP-2 binds to the collagen system. The most hydrophilic double extended homodimer showed the least binding affinity to ACS. By extending the waiting time between soaking and implantation, protein incorporation could be increased. In addition, the amount of rhBMP-2 which could be expressed was reduced by heavier ACS material and allowed for a shorter waiting period, especially at lower rhBMP-2 concentration. Crosslinking of ACS with formaldehyde led to reduced binding of rhBMP-2 to collagen either by direct hindrance of binding or reduction in swelling and number of binding sites available. Higher product pH or anion concentration enabled to increase rhBMP-2 incorporation but was limited by the potential precipitation of rhBMP-2. Despite a variety of chemical changes of ACS by ethylene oxide sterilization incorporation was not changed significantly. The in vivo release kinetics of 125I-rhBMP-2 from the collagen sponge were studied using a rat ectopic implant model. The ACS/rhBMP-2 systems tested demonstrated small, but significant differences in the in vivo retention of rhBMP-2. Consequently, it is important to have as little variability in pH, anion concentration, crosslinking and ACS mass as possible to achieve consistent or maximum binding and to avoid rhBMP-2 precipitation. Furthermore, these characteristics can be important for other in vivo applications.

Bone regeneration with recombinant human bone morphogenetic protein-2 (rhBMP-2) using absorbable collagen sponges (ACS): influence of processing on ACS characteristics and formulation.

The effects of variability in three parameters (mass, cross-linking with CH2O, and EtO sterilization) of three surgically implantable absorbable collagen sponges (ACS) were studied. Sponges soaked with recombinant human bone morphogenetic protein-2 (rhBMP-2) solution were analyzed for pH, conductivity, and rhBMP-2 precipitation. A method using trinitrobenzenesulfonic acid was developed to quantify the free amino groups of the collagen sponge. With up to 240 min exposure to CH2O, the amount of free amino groups was reduced to 80%. In comparison, the denaturation temperature as determined by differential scanning calorimetry (DSC) after the sponges were soaked with phosphate-buffered saline, increased from 48 to 55 degrees C, indicating stronger interactions due to cross-linking. Subsequent sterilization with EtO caused a marked decrease in the amount of free amino groups (approximately 33% of nonsterilized controls) independent of previous CH2O treatment. However, the denaturation temperature was on average 5 degrees C lower in sterilized sponges than in nonsterilized material. In contrast to CH2O exposure, the strong reaction with EtO appeared to weaken the collagen structure. Resistance of the sponge to collagenase correlated with the degree of collagen cross-linking but was slightly reduced by sterilization. In addition, the pH of ACS soaked with water was substantially increased by sterilization. Protein precipitation was a function of pH and salt concentration but there was no effect due to collagen alone. Results indicated that ACS weight has to be limited to avoid rhBMP-2 precipitation.

Characterization of absorbable collagen sponges as recombinant human bone morphogenetic protein-2 carriers.

For clinical use recombinant human bone morphogenetic protein-2 (rhBMP-2) is soaked onto an absorbable collagen sponge (ACS) for bone regeneration. Therefore, loss of rhBMP-2 upon mechanical handling during implantation and a potential effect of the carrier on in vivo retention is of interest. The interactions between drug and carrier were looked at from the application mode and the amount of protein which can be mechanically expressed from the combination was investigated. The results indicated that rhBMP-2 binds to the collagen system. The most hydrophilic double extended homodimer showed the least binding affinity to ACS. By extending the waiting time between soaking and implantation, protein incorporation could be increased. In addition, the amount of rhBMP-2 which could be expressed was reduced by heavier ACS material and allowed for a shorter waiting period, especially at lower rhBMP-2 concentration. Crosslinking of ACS with formaldehyde led to reduced binding of rhBMP-2 to collagen either by direct hindrance of binding or reduction in swelling and number of binding sites available. Higher product pH or anion concentration enabled to increase rhBMP-2 incorporation but was limited by the potential precipitation of rhBMP-2. Despite a variety of chemical changes of ACS by ethylene oxide sterilization incorporation was not changed significantly. The in vivo release kinetics of (125)I-rhBMP-2 from the collagen sponge were studied using a rat ectopic implant model. The ACS/rhBMP-2 systems tested demonstrated small but significant differences in the in vivo retention of rhBMP-2. Consequently, it is important to have as little variability in pH, anion concentration, crosslinking, and ACS mass as possible to achieve consistent or maximum binding and to avoid rhBMP-2 precipitation. Furthermore, these characteristics can be important for other in vivo applications.

rhBMP-collagen sponges as osteoinductive devices: effects of in vitro sponge characteristics and protein pI on in vivo rhBMP pharmacokinetics.

Osteoinductive devices, comprised of biodegradable collagen scaffolds and recombinant human Bone Morphogenetic Proteins (rhBMPs), are being currently pursued for local bone induction. To better understand the biological performance of such devices, we have carried out a series of studies to investigate the effects of sponge properties and protein structural features on the pharmacokinetics of implanted rhBMPs. The results indicated little dependence of the rhBMP-2 pharmacokinetics on the in vitro determined sponge properties. The protein isoelectric point (pI), on the other hand, was found to significantly affect the initial implant retention of rhBMPs, but not the subsequent pharmacokinetics. A 100-fold difference in the implant-retained dose could be observed depending on the type of rhBMP implanted. We conclude that protein structural features are important variables controlling in vivo pharmacokinetics of rhBMPs, and possibly the osteoinductive potency of the devices.

Collagen--biomaterial for drug delivery.

The use of collagen as a biomaterial is currently undergoing a renaissance in the tissue engineering field. The biotechnological applications focus on the aspects of cellular growth or delivery of proteins capable of stimulating cellular response. However, basic knowledge about collagen biochemistry and the processing technology in combination with understanding of the physico-chemical properties is necessary for an adequate application of collagen for carrier systems. The purpose of this review article is to summarize information available on collagen dosage forms for drug delivery as well as to impart an overview of the chemical structures and the galenical properties including detailed description of the processing steps - extraction, purification, chemical crosslinking and sterilization. The most successful and stimulating applications are shields in ophthalmology, injectable dispersions for local tumor treatment, sponges carrying antibiotics and minipellets loaded with protein drugs. However, the scientific information about manipulating release properties or mechanistic studies is not as abundant as for some synthetic polymers.

Basic thermoanalytical studies of insoluble collagen matrices.

Insoluble collagen has been utilized as a base material for parenteral drug carrier systems. Information on its physicochemical properties was obtained by focussing on thermoanalytical methods. On the way from the raw material to the matrices, the acidic aqueous dispersion represents an important intermediate state. DSC and FTIR revealed its complete denaturation at 43 degrees C. Dense homogeneous collagen matrices were prepared by air-drying at 25 degrees C and became denatured at 103.5 degrees C, far above normal storage temperatures. Dielectrical Thermal Analysis demonstrated transitions in the dielectrical storage and loss moduli, reflecting the dissipation of electrical energy and increased molecular mobility caused by collapse of the triple helical structure. Cross-linking of the collagen dispersion with glutaraldehyde induced no alteration in the thermoanalytical properties of dry matrices. However, in the swollen state, after incubation of the devices in phosphate buffer the transition temperature increased from 50 to 70 degrees C as cross-linking was intensified. This indicated stronger interactions between the collagen fibre structures. Dissolution tests with cytochrome c-loaded matrices showed that higher amounts of the model protein were trapped inside the matrices as more glutaraldehyde was added.