Search

In recent years, the stability of biotherapeutics in vivo has received increasing attention. Assessing the stability of biotherapeutics in serum may support the selection of adequate molecule candidates. In our study, we compared the physical stability of 8 different monoclonal antibodies (mAbs) in phosphate-buffered saline (PBS) and human serum. mAbs were Alexa Fluor 488-labeled and characterized with respect to fragmentation, aggregation, and proteinaceous particle formation. Samples were analyzed using size-exclusion chromatography, light obscuration, and flow imaging. In addition, novel methods such as flow cytometry and fluorescence microscopy were applied. mAbs were selected based on their hydrophobicity and isoelectric point. All mAbs studied were inherently less stable in human serum as compared to PBS. Particle size and particle counts increased in serum over time. Interestingly, certain mAbs showed significant levels of fragmentation in serum but not in PBS. We conclude that PBS cannot replicate the physical stability measured in serum. The stability of labeled mAbs in human serum did not correlate with their hydrophobicity and isoelectric point . Serum stability significantly differed amongst the tested mAbs.

In recent years, an increasing number of studies assessed the stability of biotherapeutics in biological fluids. Such studies aim to simulate the conditions encountered in the human body and investigate the in vivo stability under in vitro conditions. However, on account of complexity of biological fluids, standard pharmaceutical methods are poorly suited to assess the stability of biotherapeutics. In this study, a fluorescent-labeled therapeutic immunoglobulin G (IgG) was analyzed for proteinaceous particles after mixing with human serum and after incubation at 37°C for 5 days. Samples were analyzed using standard pharmaceutical methods (light obscuration and dynamic imaging). Moreover, we developed a fluorescence microscopy method allowing to semiquantitatively detect IgG particles in serum. Several hundred IgG particles were detected after exposure to serum. Moreover, particle counts and particle size increased in serum over time. The results showed that an IgG may form particles on mixing with serum and novel methods such as fluorescence microscopy are required to gain insight on the stability of biotherapeutics in biological fluids. Furthermore, we showed distinct advantages of machine learning over traditional threshold-based methods by analyzing microscopy images. Machine learning allowed simplifying particles in regards to count, size, and shape.

In-depth characterization of the three capsid viral proteins (VPs 1, 2, and 3) of adeno-associated viruses (AAVs) is immediately needed to ensure the consistency in gene therapy products and processes. These proteins are typically present at very low concentrations in matrices containing high concentrations of excipients and salts. Thus, there is a need for convenient methods for sample preparation before proteomic analysis. The aim of this study was to meet this need by developing a fast, reliable approach for isolating VPs in a manner enabling their efficient digestion and in-depth characterization using liquid chromatography-mass spectrometry (LC-MS).VPs from Anc80 were precipitated with different organic solvents, and the resulting precipitates were dissolved in either sodium deoxycholate (SDC) and N-dodecyl-beta-D-maltoside (DDM) or guanidine hydrochloride (Gu-HCl). The peptides obtained by the following enzymatic digestion by either trypsin or Asp-N were analyzed using LC-MS/MS.We found that precipitation with chloroform/methanol/water results in fast, efficient preparation of VP samples, allowing 100% and 99.2% amino acid sequence coverage of VP1 for trypsin and Asp-N digestion, respectively. This also allowed complete sequence confirmation of VP1, VP2, and VP3 of Anc80, as well as characterization of the amino acid sequences of the N- and C-terminal regions of each VP, together with their post-translational modifications (PTMs).The presented method enables fast, reliable, and relatively cheap sample preparation for identifying AAV serotypes and characterizing the heterogeneity of capsid viral proteins, including their PTMs.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a poly-disperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

The main goal of this work is to study the role that Bulgaria’s small towns play in the development of its rural regions. It posits that small towns should serve as administrative and socio-economic transmissions in their interrelations with the rural regions, acting as focal points of population and functions with the goal of closer territorial integration and creation of active, self-sustainable rural regions. Critical historic, geographic, demographic, and comparative analyses are used to test the above hypothesis, assess and forecast the changes in the characteristics of settlements and regions, and provide recommendations, in support of science-based geospatial policy-making in Bulgaria. In addition to stressing some methodological and data deficiencies, the results point to the conclusion that the majority of the small towns in Bulgaria do not have the necessary capacity to fulfil their role as development ‘transmissions’ for the country’s rural areas. Policy changes have been offered.

The sterility of drug products intended for parenteral administration is a critical quality attribute (CQA) because it serves to ensure patient safety, and is thus a key requirement by health authorities. Whilst sterility testing is a probabilistic test, the assurance of sterility is a holistic concept including adequate design of manufacturing facilities, process performance, and product design. Container closure integrity testing (CCIT) is necessary to confirm the integrity of a container closure system (CCS), until the end of a products’s shelf life. The new and revised United States Pharmacopeia (USP) informational chapter is a comprehensive guidance on CCI. Nevertheless, practical considerations including the choice of CCIT methods, the acceptance criteria, or the positive control samples (artificial leaks) must be addressed by the pharmaceutical manufacturer. This study is the first to provide a systematic comparison of four commonly used physical CCIT (pCCIT) methods [Helium (He) leak, vacuum decay, laser-based headspace analysis (HSA), and dye ingress] and four commonly used modes of creating artificial leaks (laser-drilled micro holes, copper wire introduced leaks, and two types of capillary leaks). The results from these experiments provide comprehensive data to allow a direct comparison of the capabilities of the individual methods. The results confirmed that the He leak detection method, which is considered the “gold-standard” for pCCIT regarding method sensitivity, indeed demonstrates the highest detection sensitivity (lowest detection limit). In comparison to the dye ingress method, HSA and vacuum decay, also demonstrated better detection sensitivity in our study. Capillary leaks with orifice diameter (capillary leak with flow according to an ideal orifice) and micro holes yielded similar leak rates, whereas capillaries with nominal diameters yielded significantly lower leak rates. In conclusion, method sensitivity cannot be compared by means of a leak diameter, but requires the consideration of multiple impacting factors (e.g. path length, uniformity).

Introduction Polysorbates (PS) are used in biopharmaceuticals to stabilize therapeutic proteins. Oxidative degradation of (poly)unsaturated fatty acids (PUFAs) contained in PS was shown to lead to α,β-unsaturated carbonyls. Aim The n-6-PUFA linoleic acid accounts for up to 18% of all FAs contained in multi-compendial grade PS80. 4-Hydroxynonenal (HNE) is highly reactive towards nucleophilic amino acids, potentially leading to covalent protein modifications. This study tests whether HNE may be a pharmaceutically relevant PS80 peroxidation product. Methods Since HNE was not directly detectable in the PS80 matrix by UV and MS, a new quantification method was established. After derivatization with 2,4-dinitrophenyl hydrazine (DNPH) and extraction of the formed hydrazone with a salting-out assisted liquid-liquid extraction, the HNE-DNPH adduct was analyzed by multiple reaction monitoring. Kinetic oxidation studies were conducted incubating PS80 in presence and absence of the antioxidant butylhydroxytoluene (BHT). Results HNE was confirmed as PS80 degradant in oxidatively stressed samples. BHT was shown to prevent its formation. Conclusion HNE is a detectable PS80 degradation product raising questions about the potential impact on critical quality attributes of biopharmaceuticals formulated with PS80. Addition of BHT prevented HNE formation under oxidative stress. Consequently, BHT might be a valuable additive in PS used in biopharmaceuticals.

A manuscript version without peer-review revisions was mistakenly processed and published.

Intravitreal (IVT) injection is currently the state of the art for drug delivery to the back of the eye. Drug Products (DP) intended for IVT injections usually pose challenges such as a very low injection volume (e.g. 50 μL) and high injection forces. DPs in vials are typically transferred and injected using disposable polymer syringes, which can feature a silicone oil (SO) coating. In our syringe in-use study, we compared dead volume, total SO content and SO layer distributions of three IVT transfer injection syringes. We assessed multiple potential impact factors such as protein concentration, needle gauge, injection speed, surfactant type and the impact of the in-use hold time on sub-visible particle (SvP) formation and injection forces. Pronounced differences were observed between the syringes regarding SvP generation. Siliconized syringes showed higher SvP counts as compared to non-siliconized syringes. In some cases injection forces exceeded 20 N, which caused needles to burst off during injection. The syringes also showed relevant differences in total SO content and dead volume. In conclusion, specific consideration in the selection of an adequate transfer injection syringe are required. This includes extensive testing and characterization under intended and potential in-use conditions and the development of in-use handling procedures.

Polysorbates are critical stabilizers in biopharmaceutical protein formulations. However, they may degrade in drug substance (DS) or drug product (DP) during storage. Degradation catalyzed by lipases present in host cell proteins (HCPs) is one suspected root cause. The purpose of this study was to develop an assay to detect lipolytic activity in biopharmaceutical DS and DP formulations. The assay is based on the hydrolysis of the lipase substrate 4-methylumbelliferyl oleate to yield the fluorescent product 4-methylumbelliferone. First, the assay components and their concentrations (buffer salts and pH, solvent and inhibitor Orlistat) were established and optimized using a model lipase (Porcine pancreatic lipase) and cell culture harvest fluid that exhibited lipolytic activity. The assay was then successfully applied and thereby qualified in protein formulations and at lipase concentrations possibly encountered in actual biopharmaceutical DS and DP formulations. The lipase assay can be used to detect lipolytic activity in intermediate and final DS, for example during process optimization in downstream purification, to better and specifically reduce the level, or deplete, lipases from HCPs. The assay is also suitable to be applied during root cause investigations related to polysorbate degradation in biopharmaceutical DP.

Significant efforts are made to characterize molecular liabilities and degradation of the drug substance (DS) and drug product (DP) during various product life-cycle stages. The in vivo fate of a therapeutic protein is usually only considered in terms of pharmacokinetics (PKs) and pharmacodynamics (PDs). However, the environment in the human body differs substantially from that of the matrix (formulation) of the DP and may impact on the stability of an injected therapeutic protein. Stabilizing excipients used in protein formulations are expected to undergo more rapid distribution and dissociation in vivo, compared to a protein as a highly charged macromolecule. Thus, in vivo stability may significantly differ from shelf-life stability. In vivo degradation of the therapeutic protein may alter efficacy and/or safety characteristics such as immunogenicity. Studying the stability of a therapeutic protein in the intended body compartment can de-risk drug development in early stages of development by improving the selection of better clinical lead molecules. This review assesses the considerations when aiming to evaluate the in vivo fate of a therapeutic protein by comparing the physiology of relevant human body compartments and assessing their potential implications on the stability of a therapeutic protein. Moreover, we discuss the limitations of current experimental approaches mimicking physiologic conditions, depending on the desired route of administration, such as intravenous (IV), subcutaneous (SC), intravitreal (IVT), or intrathecal (IT) administration(s). New models more closely mimicking the relevant physiologic environment and updated analytical methods are required to understand the in vivo fate of therapeutic proteins.

Prefilled syringes (PFSs) are increasingly preferred over vials as container closure systems (CCSs) for injectable drug products when facilitated or self-administration is required. However, PFSs are more complex compared to CCSs consisting of vial, rubber stopper, and crimp cap. Container closure integrity (CCI) assurance and verification has been a specific challenge for PFSs as they feature several sealing areas. A comprehensive understanding of the CCS is necessary for an appropriate CCI assessment as well as for packaging development and qualification. A comprehensive CCI assessment of 6 different PFSs from 3 different manufacturers (including 1 polymeric PFS) was conducted using helium leak testing. PFS components were manipulated to systematically assess the contribution of the different sealing areas to CCI, namely rigid needle shield (RNS)/needle, RNS/tip cone, and the individual ribs of a syringe plunger. The polymeric PFS required an equilibrium measurement for accurate container closure integrity testing. The different sealing areas and a single plunger rib were shown to provide adequate CCI. Acceptable tip cap movement until the point of CCI failure was estimated. The assessment of acceptable tip cap movement demonstrated the importance of considering the RNS/tip cone seal design to ensure CCI of the PFS upon post assembly possesses and shipment.

Asymmetric flow field-flow fractionation is a valuable tool for the characterization of protein aggregates in biotechnology owing to its broad size range and unique separation principle. However, in practice asymmetric flow field-flow fractionation is non-trivial to use due to the major deviations from theory and the influence on separation by various factors that are not fully understood. Here, we report methods to assess the non-ideal effects that influence asymmetric flow field-flow fractionation separation and for the first time identify experimentally the main factors that impact it. Furthermore, we propose new approaches to minimize such non-ideal behavior, showing that by adjusting the mobile phase composition (pH and ionic strength) the resolution of asymmetric flow field-flow fractionation separation can be drastically improved. Additionally, we propose a best practice method for new proteins.

The need for a closer and enhanced connection with nature, as the basis for human wellbeing, reinforces the importance of natural heritage governance, including wider and much improved access to information about nature-based tourism and recreation. This research seeks to reveal how Internet content can support the digital identification of nature sites and their branding as tourist destinations. To this end, it analyzes 20 exemplary websites, each known around the world, and highlights important current trends in the structuring and attractiveness of the information provided. Based on the devised benchmarking criteria, the study develops a benchmarking approach that improves digital “packaging” and marketing of natural heritage for the purposes of tourism, recreation, leisure, and sports, and then tests it in Bulgaria. The procedure includes the selection of 57 natural heritage sites and rating the content of their official websites. The results show an unsatisfactory level of representation of the heritage value of the Bulgarian NH and a severe depreciation of the provision of the necessary tourism and recreation information, in comparison to the selected global benchmark. Besides the outline of the most frequent gaps and shortcomings, the results provide managers with suggestions about the development of a “natural heritage” brand for tourism and recreation, which features amplification of site identity and messaging, plus enhancement of its visibility. The conclusions are applicable to a wide variety of geographical contexts and audiences in nature-based destination governance.

Polysorbates are stabilizers typically required in therapeutic protein formulations. On account of their chemical structure, polysorbates are prone to degradation, which can render a pharmaceutical product instable or incompliant. The purpose of this study was to investigate if the addition of butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) protects polysorbate 20 (PS20) and polysorbate 80 (PS80) against oxidative degradation. PS20 and PS80 solutions containing BHA, BHT—or as control without an antioxidant—were stressed by exposure to air at 40°C for 7 weeks. The following assays were performed: ferrous oxidation-xylenol orange assay, liquid chromatography coupled to UV and mass spectrometry (MS), pH measurement, liquid chromatography fluorescence micelle assay, headspace–gas chromatography coupled with MS. PS20 and PS80 solutions containing an antioxidant were found to be more stable, as demonstrated by lower peroxide levels, lower free fatty acid contents, stable pH, intact polysorbate micelle structure/composition, and less volatile degradants. PS20 and PS80 solutions containing BHT or BHA are more stable against oxidative degradation compared to nonstabilized solutions. It might be beneficial to formulate bulk polysorbate with the antioxidant(s) to ensure stabilization during all process steps.

Geography of the twenty-first century is expected to contribute to the development of human capital and the knowledge society, to offer place-specific solutions for sustainable regional development and use of the planet’s natural and human capital. With this idea, we prepared this book which presents selected contributions from the International Conference “Smart Geography: 100 years Bulgarian Geographical Society”. They are focused on various themes related to smart spatial solution in different geographical disciplines as well as interdisciplinary studies with pronounced spatial aspect. The book illustrates the great variety of themes the contemporary geography is dealing with. Most of them have real potential to contribute by smart spatial solution to the human well-being. The examples presented in the book cover case studies from Bulgaria and many other countries worldwide.

Glass fogging is a phenomenon occurring in lyophilized drug products and can be described as a thin product layer deposited on the inner surface of the glass container, in the area not covered by the lyo cake itself. It is often considered a cosmetic defect; however, the loss of container closure integrity is a potential consequence of the fogging’s expansion to the vial neck region, making this a potential critical defect. Thus, a method for predicting the extent of vial fogging before the actual freeze-drying is of particular interest for the pharmaceutical industry. For that reason, we evaluated a simple method (“simulated fogging”) applicable to drug product formulations in a specific container closure system. Two different vial types with different surface hydrophilicity were tested using 3 model protein formulations, comparing the simulated fogging test and the degree of fogging after actual lyophilization. The simulated fogging method could predict fogging and showed a correlation to fogging in lyophilized drug product glass vials. We observed that all formulations showed fogging in the hydrophilic vials. By contrast, hydrophobic vials prevented fogging, however, interestingly with remaining defects of so-called droplet formation. Other than extent of fogging, no additional differences of lyophilized cake properties or other product quality attributes were observed between products using the different glass vial types tested.

Throughout the development process of protein therapeutics a broad array of analytical techniques is applied on a routine basis to measure various molecular attributes. These measurements are carried out in the context of the quality control system of the product (routine monitoring of the product quality e.g. regarding stability during long-term storage) or as various characterization studies aimed at broadening the knowledge of the molecule. All stages of product development rely on analytical support: cell line and purification development, formulation development, compatibility studies, comparability exercises and also assessment of stability during production, storage and shipping.

The goal of this study is to diagnose the demographic situation in rural Bulgaria and analyze the impact of the last 25 years of transformations. The main proposition is that, during the post-1989 transition and its first EU membership decade, Bulgaria’s rural area expanded territorially and suffered a severe demographic depletion, which led to large and widening discrepancies between the national and local/regional scales and became one of the main factors to influence the country’s overall development for many decades to come. The study uses GIS-aided comparative analysis and assessment of local-level statistical information, and field monitoring to territorially identify and evaluate the rate and scale of the ongoing rural depopulation, as well as highlight the most vulnerable regions and pinpoint the few areas of exception. The research reveals tendencies of importance to the research and administration of the Bulgarian and EU rural regions. It concludes that Bulgaria’s EU membership has so far not been able to terminate the process of deep and continuing depopulation and the ensuing peripheralization of the vast majority of the local and regional administrative units in the country. The fact that the land state boundary and the mountainous areas are the most vulnerable parts of rural Bulgaria implies significant geostrategic consequences for EU’s sensitive southeastern outer border.

The goal of this study is to produce a comparative analysis of the socio-economic transformations in a typicalmountain administrative district at the NUTS 3 level since the 1989 transition to market economy in Bulgaria. Demographic and economic indicators are utilized to provide a diagnosis of the current state of affairs, but also to highlight tendencies, which are noteworthy for the investigation and administration of districts that belong to both border and mountain peripheries. The main proposition of this paper is that in the post-1989 conditions the Oblast’s “double” periphery position is severely impairing its overall development in relation to the rest of the country. The large and widening discrepancies between national and regional demographic and economic indicators point to a pronounced process of marginalization of certain areas, which necessitates the implementation of a specifically targeted development policy.

Administration into the subcutaneous (SC) tissue is a typical route of delivery for therapeutic proteins, especially for frequent treatments, long-term regimens, or self-administration. It is currently believed that the maximum volume for SC injections is approximately 1.5 mL. Larger SC injection volumes are considered to be associated with injection pain and adverse events at the injection site. However, no controlled clinical studies and actual evidence exist to support this assumption. In this review, we discuss current and publically available data related to SC administration volumes. We conclude that injection volumes higher than 3.5 mL are worth exploring if required for the development of efficacious drug treatments. Studying tissue back pressure, injection site leakage, local tolerability, and injection-related adverse events, such as injection pain, should be considered for the development of higher SC injection volumes.

A number of new techniques for subvisible particle characterization in biotechnological products have emerged in the last decade. Although the pharmaceutical community is actively using them, the current knowledge about the analytical performance of some of these tools is still inadequate to support their routine use in the development of biopharmaceuticals (especially in the case of submicron methods). With the aim of increasing this knowledge and our understanding of the most prominent techniques for subvisible particle characterization, this study reports the results of a systematic evaluation of their accuracy. Our results showed a marked overcounting effect especially for low concentrated samples and particles fragile in nature. Furthermore, we established the relative sample size distribution as the most important contributor to an instrument's performance in accuracy counting. The smaller the representation of a particle size within a solution, the more difficulty the instruments had in providing an accurate count. These findings correlate with a recent study examining the principal factors influencing the precision of the subvisible particle measurements. A more thorough understanding of the capabilities of the different particle characterization methods provided here will help guide the application of these methods and the interpretation of results in subvisible particle characterization studies.

The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.

Subvisible particles (SVPs) are an obligatory critical quality attribute of the product, and yet, they are found in all biopharmaceutical products intended for infusion or injection. Light obscuration (LO) is the primary pharmacopeial method used to quantify SVPs. However, the method may not be equally sensitive toward all particles that can possibly occur. Calibration of LO instruments is usually performed using polystyrene beads suspended in water. In this study, the dependence of the sizing accuracy of LO analysis was evaluated by using a calibration suspension of lower refractive index beads made of silica suspended in sucrose solution. It was demonstrated that the sizing accuracy was strongly dependent on the reference material's properties used for calibration. It was also demonstrated that flow imaging microscopy suffered from the same artifact, albeit to a smaller extent. We further tested different LO sensors and instruments. Interestingly, our results show that the sizing accuracy varied from instrument to instrument, strongly depending on the properties of the sensor. To summarize, sizing and counting accuracies were dependent on the material used for calibration and its optical properties as well as the calibration curve, the sensor, and the instrument supplier. Closer match of optical properties between calibration system and test system seems to improve the sensitivity of the measurement. The results of this study raise the following major practical implications: (1) LO and flow imaging microscopy are not truly orthogonal analytical methods, (2) while matching optimal properties of material used for calibration and test items increased sensitivity, this is of poor practical applicability given that analytes contain multiple particles, and (3) setting product-specific limits for SVPs require special considerations with regard to the data sets used.

Polysorbates are commonly added to protein formulations and serve an important function as stabilizers. This paper reviews recent literature detailing some of the issues seen with the use of polysorbate 80 and polysorbate 20 in protein formulations. Based on this knowledge, a development strategy is proposed that leads to a control strategy for polysorbates in protein formulations. A consortium of Biopharmaceutical scientists working in the area of protein formulations, shared experiences with polysorbates as stabilizers in their formulations. Based on the authors experiences and recent published literature, a recommendation is put forth for a development strategy which will lead into the appropriate control strategy for these excipients. An appropriate control strategy may comprise one or more elements of raw material, in-process and manufacturing controls. Additionally, understanding the role, if any, polysorbates play during stability will require knowledge of the criticality of the excipient, based upon its impact on CQAs due to variations in concentration and degradation level.

The aim of the EU Biodiversity Strategy to 2020 isto maintain and enhance ecosystem services (ES) in Europe and requires all Member States to map and assess the state of ecosystems and their services in the respective national territories. The EU-funded project ESMERALDA analyses ES mapping and assessment methods and approaches in their biophysical, social and economical perspectives, as well as their application in different case studies. The project also aims at the development of an integrated and consistent assessment framework. In Bulgaria, methodological guides for evaluation and mapping of the services provided by the nine main types of ecosystems have been prepared together with the respective proposals for their implementation in the national assessment. The Bulgarian research team analyses and tests various aspects of ecosystem services mapping and assessment, such as alternative economic assessments, multi-criteria analyses and biophysical assessment approaches, mapping challenges and local population surveys. In this paper paper, we review the ES activities in Bulgaria and present selected mapping and assessment methods tested in the Central Balkan case study area. It provides relevant examples for the implementation of integrated mapping and assessment of ecosystem services at local and regional level, where different mapping approaches and techniques are embedded within diverse policy contexts. The main goal of the study is to investigate how the assessment results can support the integration of the ecological functions of the Central Balkan National Park with the economic opportunities that it creates for the local and regional communities. A tiered approach has been used to organise the mapping and assessment exercises in the study area, in order to meet the needs for integrated ecosystem assessment and overcome the limitations of data availability. At tier 1, the study performs identification and initial ES mapping of the whole area. At tier 2, it applies economic valuation for the Municipality of Karlovo by using statistical data and the contingent valuation method. At tier 3, the investigation applies modelling methods to assess carbon storage and flood regulation on a larger scale. The results are presented in the form of maps at all levels, which use a uniform 0 to 5 assessment scale. The integrated approach presented here ensures a clear communication of the end results to the respective decision-makers.

The article observes the problems of the mountain regions in Southeastern Europe. The other aspect of the publication is related with review of the main researches and activities for the mountain regions in Bulgaria and Southeastern Europe. The two main European conventions for the mountain regions (Alpine and Carpathian) are also presented. Development of the convention for the mountain regions in Southeastern Europe (Balkan convention) is key problem of the study. The experience and results of Alpine and Carpathian conventions can be use for the model in the process of foundation and realization of the new convection for the mountain regions in Southeastern Europe., Problems of Geography, 2018, Vol.1-2, p. 159-164