Pharmaceutical Technology Chair of Prof. Dr. Gerhard Winter - Faculty for Chemistry and Pharmacy
Pharmaceutical Technology Chair of Prof. Dr. Gerhard Winter - Faculty for Chemistry and Pharmacy
Over the last 12 years we have worked on many different projects. Major topics and highlights are summarizes in the following.
Together with Roche we were able to study very large scale freeze/thaw processes on protein bulk substance solutions, mainly antibodies. Different freezing systems were studied, the formulation aspects as well as the technology/process parameters were evaluated. Results can be found in the dissertation of Ralph Zippelius.
Already in the mid 90ies we have worked on vacuum drying of sensitive pharmaceutical protein drug solutions together with G. Lee ( Erlangen and R. Rudolph( Roche/later Halle). In Munich we continued working on the subject applying vacuum concentrators for drying larger volumes and we dried small volumes after application of droplets via nozzle printer on surfaces. Results can be found in the thesis of M. Willmann and A. Stabenau and pertaining publications and patent applications.
Such technologies were also combined with another project carried out in cooperation with Switch Biotec, where we formulated xero-gels or dry films for delivery of wound healing factors to the injured skin. Formulations were optimized in a way that they allow storage of the moisture sensitive groth factor and topical release after rehydration of the carrier immediately before application to the wound. Results can be found in the thesis of R. Schmidt and in pertaining patent applications.
To improve the current practice in early pharmaceutics in industry to dissolve and apply sparingly soluble drug substances in DMSO, we developed and tested in vivo microemulsions for oral application in preclinical studies. The project has been realized together with Merck, Grafing, result can be found in the thesis of S. Effer.
More recently we took another approach towards formulation of sparingly soluble drugs. This time nanosuspensions, prepared by precipitation from organic ( alcoholic) suspensions, were the method of choice, not nanomilling. K. Freitag, supported by Prof. Peukter (Erlangen) and in cooperation with a large pahramceutical company studied many basic aspects of supersaturation , crystallization and crystal growth
To improve the process technology of freeze drying in its main time consuming step, the primary drying, I.Presser, together with Boehringer Ingelheim, Biberach applied new technologies for endpoint determination and some other process related topics. MS, a microbalance and NIR based moisture determination were the main subjects of the work published by Presser at al.
With the work of W. Fraunhofer we pushed ahead the use of flow field flow fractionation (FFF) analytics for characterizing protein drug solutions and nanoparticulate formulations, later including also VLP dispersions. With at least 2 different systems on the market since the year 2000, FFF or better AF4 has made a leap forward and must be considered as a valuable alternative to SEC. Its real strength lies in the ability to allow direct injection of unfiltered samples carrying aggregated macromolecules in the presence of monomers and even fragment and on the high end even particles of colloidal carriers like nanoparticles, liposomes and the like. K. Mathis used FFF in a second generation thesis in comparison to SEC and for Special applications. Publications from Fraunhofer, Coester, later Lang and Mathis et al. have reported on FFF. Some smaller work packages can also be found at “Wyatt FFF application notes”.
A large formulation project of translational impact has been carried out by F. Gruber, S. Waibler as Ph.D. students and C. Welz as a Post doc. The project was dedicated to the formulation of paclitaxel- cationic liposomes. According to the pre-work of McDonald et al. at the UCSF and the development work lead by K. Naujoks and U. Michaelis at MBT, later Medigene in Martinsried, the soft targeting of activated endothelia by cationic liposomes for tumor targeting and angiogenesis inhibition, we provided formulation work for different aspects of liposome production and characterization. Publications and Patent applications by the authors named above are available. The formulations developed together were used by the industrial partner in Phase II clinical studies. Later M. Wiggenhorn concentrated on scale up and process transfer issues regarding such liposomal formulations. The standard lyophilisation should be replaced by spray drying or other scalable process technologies. In the course of his studies M. Wiggenhorn applied supercritical drying, spray freeze drying and spray drying, the latter including an elegant in line liposome sizing step. Several patent emerged out of this thesis.
With Silke Mohls thesis and H. Reithmeiers Post doc work we started a long lasting research on lipid based protein depot systems. Starting with alpha interferon as the first model drug and compressed triglyceride disks we studied the basic aspects of release mechanism, lipid degradation or erosion, relevance of pore formers and other excipients. It soon became clear that the systems have a high potential to be used as a valid alternative to polymer based implants. E. Wolf helped us to present the first and preliminary in vivo study with the new platform technology. G. Reich at Heidelberg University applied NIR spectroscopy on the protein loaded systems and we are able to directly determine the protein load in a non-destructive manner. An unexpected, but obvious effect was found for PEG in its double role as a pore former and precipitant for proteins within the pores of the release systems. With S. Schulze/Herrmann and M. Schwab we extended the work on the subject including help from our partners in Lille (J. Siepmann for mathematical modelling of release) and Otago (T. Rades et al, for in vivo enzymatic biodegradation). More recently G. Sax continued the work on lipid based implants as he, together with S. Schulze moved ahead from the unhandy disk implants to extruded rods that can be easily applied in vivo via a trocar needle. Meanwhile we have data on biodegradation of such rods in vivo, more release kinetics in vitro, demonstrating that there is a second, not pore bound release pathway through the molten lipid part of the incubated implant. To elucidate such pathways the group of C. Bräuchle applied their wide field microscopy and allowed us to calculated diffusion coefficients of labelled protein drugs inside lipid implants. For antibodies we have found extremely long release periods of more than half a year. The results are published in a larger number of papers and a patent has been issued on the platform technology. At the moment we are actively pursuing such systems for special applications in local drug delivery. On the process side we have improved our technical capabilities with a downscale mini-extruder allowing a further reduction of batch sizes and optimal process control.
Martin Schwab also started our work on loading drugs on formulated spider silk proteins ( the latter was received from T. Scheibel ( TUM / Bayreuth). In a first step we set the basis how to manufacture nanoparticles, microparticles, films and to load them with drug molecules of different charge and polarity. M. Hofer, together with J. Myschik/Engert continued the work by Schwab and Lammel and a robust process for spider silk NP production and loading with protein molecules has been set up. Several papers describe the state of our research in this field. More recently the use of spider silk protein is subject of further BMBF funded research projects carried out together with AMsilk and T. Scheibel in Bayreuth.
Under the guidance and supervision of Conrad Coester who led a research group during his habilitation at the LMU, gelatin based nanoparticles ( GNPs) were intensively studied. Besides the usual two step desolvation method a one-step method was developed, together with gelatine providers. Loading of GNPs with different types of nucleic acids and oligonucleotides was studied and optimized. In the course of this work it turned out that long term storage of such systems needs stabilization via freeze drying, and together with T. Anchordoquy at Denver J. Zillies established the pertaining protocols. Furthermore, field flow fractionation turned out to be ideal tool to analyze GNPs, and to better determine their loading. Later, S. Fuchs applied Ultrasound resonator technology, a seldom used method to analyze GNPs and their swelling as well as their loading. Biological applications, showing uptake in different cell cultures and first studies in vivo were carried out by K. Zwiorek mostly in cooperation with C. Bourquin from the group of Prof. Endres, LMU Großhadern. Finally controlled/targeted immune-stimulation via CPG oligonucleotides after s.c. application could be reached and significant successes in the area of tumor vaccination were achieved. Intravasally applied GNPs can on the other hand be applied for Kupffer cell targeting in the liver, for NF-kappaB inhibition ( together with A. Vollmar et al). In a Humboldt-Project C. Coester and K. Ofokansi used matrix loading of GNPs to apply highly loaded insulin GNPs for oral protein delivery. In rats it worked well, the studies were carried out and published together with G. Fricker at the University of Heidelberg. A. Fuchs studied body distribution after i.v. application with PET spectroscopy provided by Prof. Wester from the nuclear medicine. S. Schultes on the other hand, in cooperation with Medigene, funded by the BFS, studies the fate of such particles in in vivo capillary systems using the Cremaster model established by the group of Prof. Krombach, LMU Großhadern and with a self built in vitro flow through systems. Finally a breakthrough application was found when CPG loaded GNPs were aerosolized and inhaled by asthmatic horses. The group of Prof. Gehlen and Dr. Klier at the veterinary hospital of the LMU could improve the disease status in horse patients drastically by using our co-developed GNPs. The studies are ongoing and other branches of the successful use of immune-modulating GNPs are used on dogs now.
A large number of publications on GNPs has been issued along with a few patent applications.
In cooperation with Cytos, Zürich - a leader in the area of VLP based immunotherapy - we developed one of the first clinical formulations for such modern vaccination drugs. The resulting lyophilisate was part of the clinical supplies package when the project was licensed from Cytos to Novartis. R. Lang has published the essentials of the formulation work.
Crystallisation of protein drugs like antibodies has been carried out by S. Gottschalk and C. Hildebrandt in a two-step Pd.D. program over many years together with a large pharmaceutical company. The results are in the process of being published and will soon be available. Mass crystallization of protein drugs has not the purpose to receive a single large crystal for x-ray structure analysis but aims on the potential benefits of crystals vs dissolved proteins. Theoretically a suspension can have a much lower viscosity compared to a highly concentrated protein solution. Furthermore, stability could be improved when the protein molecule is immobilized in its optimal, natural structure. A crystalline structure could provide such an optimal environment. Drying of protein crystals is almost impossible, as they contain high amounts of water in the crystal lattice and removing this water would destroy the crystal.
Microbubbles (MPs) are gas filled microparticles with a thin shell made from polymers or lipids. Due to their flexibility and their inert nature when manufactured from liposomes or HSA, they have been used as intravenous contrast agents. The gas core provides an excellent contrast for imaging via ultrasound. S. Tinkov and C. Coster, together with R. Bekeredjian from the University hospital in Heidelberg (group of Prof. Katus) performed excellent work on in vitro quality design of drug loaded MPs, especially for doxorubicin carriers. Such systems allow for almost perfect physical drug targeting, when a focused ultrasound beam is used to excite the MPs above a critical energy and they disrupt at the site of excitation. If ultrasound can be focused on a solid tumor, release of the cytotoxic drug on the site of action can be achieved. After comprehensive preparation studies, finally in vivo studies showed the proof of concept in vivo on tumor bearing mice. S. Tinkov, C. Coester et al. have published numerous papers on this work.