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Continuous matrix assisted refolding (MAR) can be achieved on a solid support by using a continuous chromatographic system. Recycling the aggregate fraction, simultaneously formed during a refolding reaction, can further increase the refolding yield. Due to the nature of this reaction, aggregates are the main reason for a refolding yield below stoichiometric conversion. A preparative continuous annular chromatographic system (P-CAC) equipped with an ion exchange resin was used to continuously refold the model protein alpha-lactalbumin. For this purpose, this protein was denatured, reduced and adsorbed on the ion exchange resin. Elution was performed with or without redox reagents in the buffer system permitting fast formation of the native disulfide bonds. In the case redox reagents were present, the protein refolds then during its residence time on the matrix. However, aggregate formation is also increased and refolding yields are lower. Tightly bound aggregates were removed from the column by 2M guanidinium hydrochloride. In order to increase the system yield, this aggregate fraction was recycled after lowering the conductivity by ultradiafiltration and adjustment of the protein concentration by dilution. For on-column refolding, recycling of aggregates at a recycling rate of 0.17 increased the system yield from 25% to 30%. An algorithm was developed to show interdependencies of the single influencing parameters. The operability of the system was demonstrated but limitations due to instability of the P-CAC, especially inhomogeneous flow and peak wobbling, have to be considered.

Hydrophobic interaction chromatography (HIC) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. In contrast to reversed-phase chromatography, this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. Hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. When proteins are injected on HIC columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. The higher the salt concentration, the lower is the amount of eluted protein. The rest can be desorbed with a buffer of low salt concentration or water. It has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. This has been also corroborated by physicochemical measurements. The retention data of 5 different model proteins and 10 different stationary phases were evaluated. Partial unfolding of proteins upon adsorption on surfaces of HIC media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. Furthermore, we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain.

Two different approaches of matrix assisted refolding have been evaluated and compared to conventional refolding by dilution. Bovine alpha-lactalbumin was used for the studies as model protein. It was adsorbed under denaturing conditions on an ion exchange matrix and refolding was completed on the column prior to elution or, depending on the buffer system, in the eluate. Agarose based chromatography matrices showed high capacities for the denatured alpha-lactalbumin. A positive effect on the yield of refolded protein by the matrix could be observed for Fractogel EMD DEAE and a negative for Toyopearl DEAE 650M, DEAE Sepharose FF and Q Sepharose FF. In the case of Fractogel EMD DEAE the ion exchange surface might act as a folding helper. This property may be caused by the grafted polymers. For Source 30Q only a marginal negative influence on the refolding kinetics was observed, thus the ion exchanger is only a mean for removal of chaotropic agents. Refolding on the column is characterized by a low yield but high productivity due to significant reduction of refolding time.

Objectives: The following paper presents the results of a post-treatment examination on inpatients with personality disorders who received psychodynamic psychotherapy. The examination was carried out approximately 3.3 years after the patients were discharged., Methods: Of a group of 110 patients with personality disorders (ICD-10), 72 patients underwent follow-up examination. A comprehensive catamnesis was compiled for 59 former patients; 13 former patients answered by post. Treatment success was evaluated on the basis of the Symptom Checklist 90-R, Inventory of Interpersonal Problems and Questionnaire of Social Support. These surveys show no significant statistical difference between the data of the subgroups of participants and non-participants of the post-treatment examination at the start and end of therapy (Mann-Whitney-U-Test, p > 0.05). Treatment success was also examined using the Global Assessment of Functioning Scale, the Impairment-Score as well as an analysis of the patient's use of psychiatric and inpatient psychotherapeutic care and the medication received., Results: Due to the reduction in general mental stress and interpersonal problems, and the increase in perceived social support, the patients showed a positive development. After being discharged 41% of the patients received outpatient psychotherapy without further psychiatric and inpatient psychotherapeutic care., Conclusions: The above results demonstrate that patients with personality disorders benefit from inpatient psychodynamic psychotherapy.

A refolding reactor was developed for continuous matrix-assisted refolding of proteins. The reactor was composed of an annular chromatography system and an ultrafiltration system to recycle aggregated proteins produced during the refolding reaction. The feed solution containing the denatured protein was continuously fed to the rotating bed perfused with buffer promoting folding of the protein. As the protein passed through the column, it was separated from chaotropic and reducing agents and the refolding process took place. Native proteins and aggregates could be continuously separated due to different molecular size. The exit stream containing aggregates was collected, concentrated by ultrafiltration and recycled to the feed solution. The high concentrations of chaotropic and reducing agents in the feed solution enabled dissociation of the recycled aggregates and consequently were fed again to the refolding reactor. When the initial feed mixture of denatured protein is used up, only buffer-containing chaotropic agents and recycled aggregates are fully converted to native protein. This process resulted in a stoichiometric conversion from the denatured protein to its correctly folded native state. The system was tested with bovine alpha-lactalbumin as model protein. Superdex 75 PrepGrade was used as size-exclusion medium. The yield of 30% active monomer in the batch process was improved to 41% at a recycling rate of 65%. Assuming that the aggregates can be redissolved and recycled into the feed stream in a quantitative manner, a refolding yield close to 100% is possible. The method can be also applied to other chromatographic principles suited for the separation of aggregates.

Hydrophobic interaction chromatography media suited for large scale separations were compared regarding dynamic binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins alpha-lactalbumin, lysozyme, beta-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding binding capacity affected by salt. These sorbents exhibited a disproportional increase of binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.

Currently, the selection of a hydrophobic interaction chromatography (HIC) sorbent for protein separation purposes is entirely based on empirical means. An attempt was made to characterize different HIC sorbents from various manufacturers. The selectivity was determined by isocratic pulse experiments of a set of reference proteins and an algorithm was developed to classify the sorbents according to their selectivity and hydrophobicity. The obtained semi-quantitative parameters take into account the dependence of salt on adsorption. The sorbent characteristics evaluated with the model proteins were compared to the separation of a real feedstock. A good agreement was achieved between the developed evaluation procedure and the separation behaviour of the real feed stock.

The increasing interest in research on burden and health risks of family caregivers was furthered by the increasing prevalence rate of dementias in the elderly and by the fact that 80-90% of the demented are cared for by near family members. The present paper presents some results of a study investigating stress-related symptoms of family caregivers. From a clinical perspective these results point to needed interventions for supporting the caregivers. Specific burden components, depression, and body complaints were assessed in n = 70 family caregivers of dementia patients. A substantial degree of social isolation, depressive disorders, and physical complaints could be found. The level of burden experienced was not directly related to severity of dementia and to impairment of the demented or to structural conditions of the care (degree and length of care). Differences were observed between spouse caregivers and other relatives; spouses suffer more psychosomatic complaints and depression.