Characterization of a PEGylated protein therapeutic by ion exchange chromatography with on-line detection by native ESI MS and MS/MS.

Detailed profiling of both enzymatic (e.g., glycosylation) and non-enzymatic (e.g., oxidation and deamidation) post-translational modifications (PTMs) is frequently required for the quality assessment of protein-based drugs. Challenging as it is, this task is further complicated for the so-called second-generation biopharmaceuticals, which also contain "designer PTMs" introduced to either enhance their pharmacokinetic profiles (e.g., PEGylated proteins) or endow them with therapeutic activity (e.g., protein-drug conjugates). Such modifications of protein covalent structure can dramatically increase structural heterogeneity, making the very notion of "molecular mass" meaningless, as ions representing different glycoforms of a PEGylated protein may have nearly identical distributions of ionic current as a function of m/z, making their contributions to the mass spectrum impossible to distinguish. In this work we demonstrate that a combination of ion exchange chromatography (IXC) with on-line detection by electrospray ionization mass spectrometry (ESI MS) and methods of ion manipulation in the gas phase (limited charge reduction and collision-induced dissociation) allows meaningful structural information to be obtained on a structurally heterogeneous sample of PEGylated interferon ß-1a. IXC profiling of the protein sample gives rise to a convoluted chromatogram with several partially resolved peaks which can represent both deamidation and different glycosylation patterns within the protein, as well as varying extent of PEGylation. Thus, profiling the protein with on-line IXC/ESI/MS/MS allows it to be characterized by providing information on three different types of PTMs (designer, enzymatic and non-enzymatic) within a single protein therapeutic.

Screening for social phobia in medical in- and outpatients with the German version of the Social Phobia Inventory (SPIN).

OBJECTIVE: To evaluate the German version of the Social Phobia Inventory (SPIN) as a screening device and to report corresponding cut-off scores for different populations. METHOD: In Study 1, 2043 subjects from a representative sample completed the SPIN. Cut-off values were established on the basis of means and standard deviations. In Study 2, different aspects of validity were examined in a clinical sample comprising 164 subjects, including social phobic individuals, individuals with other anxiety disorders and depression, and non-clinical control subjects. Internal consistency was evaluated. Convergent and divergent validity were explored using several established measures. Finally, the sensitivity and specificity of the German SPIN with regard to social anxiety classification were investigated by means of receiver operating characteristics (ROC) analyses. RESULTS: In Study 1, mean scores and standard deviations were used to determine cut-off scores for the German SPIN. In Study 2, excellent internal consistency and good convergent and divergent validity were obtained. ROC analyses revealed that the German SPIN performed well in discriminating between social phobic individuals on the one hand and psychiatric and non-psychiatric controls on the other. A cut-off score of 25 represented the best balance between sensitivity and specificity. CONCLUSION: Comparable to the original version, the German SPIN demonstrates solid psychometric properties and shows promise as an economic, reliable, and valid screening device.

Prevalence and significance of minor anomalies in children with impaired development.

AIM: To compare the prevalence of Waldrop's minor physical anomalies in children with developmental disorders (mental retardation, hearing and visual impairment) and healthy schoolchildren. METHODS: The study was carried out on a sample of 469 children (223 children with developmental disorders and 246 healthy schoolchildren). RESULTS: Significant differences were found between the children with developmental disorders and the healthy children with regard to the number of minor anomalies and their weighted scores according to Waldrop. Multivariate discriminant analysis with two discriminative functions explained as much as 96.51% of the total variability and significantly distinguished the healthy children from the children with developmental disorders. However, no clear distinction was found between the mentally retarded children and those with visual impairment. Interrelation of the number and sum of the weighted scores of minor anomalies showed similar minor anomalies in the mentally retarded children (mean per person 3.65 and 3.82, respectively), the children with visual impairment (3.24 and 3.50), the children with hearing impairment (3.84 and 3.67) and the control group (1.70 and 1.46), although at different levels. CONCLUSIONS: The high prevalence of multiple minor anomalies in children with developmental disorders suggests that during early development, factors which cause the specific developmental disorder and the occurrence of a minor anomaly have a joint effect.

The prevalence of minor physical anomalies in mentally retarded children.

The prevalence of minor physical anomalies was examined in a sample of 109 children with idiopathic mental retardation (65 boys and 44 girls). Control group consisted of 246 healthy schoolchildren (123 boys and 123 girls) aged 8 to 12 years. A comparison was made between number of found minor anomalies per child (W1) and their Waldrop weight scores (W2) in healthy and mentally retarded (MR) children. The MR children were found to have a higher number of minor anomalies per child. In their group predominated those with four or more anomalies (56.9%), whereas among healthy children only 7.7% had four anomalies or more. In contrast to the high weighted score value (W2) of five or greater in 36.7% of MR children, it was absent in all control group subjects. There were highly significant differences between the MR and healthy children in the average value of the number of minor anomalies per child (W1) and in the average weighted score (W2). The average number of minor anomalies per child (W1) in MR and well children was 3.65 and 1.7, respectively. In MR children the average weighted score (W2) was 3.82, being 1.46 in healthy children. Our results suggest that common etiological factors, which had led to a physical and mental disorder, were active early in the development of MR children. The finding of high incidence of multiple minor anomalies in MR children indicates that genetic factors may play an important role in the etiology of the underlying disorder in the child group studied.

DNA sequencing up to 1300 bases in two hours by capillary electrophoresis with mixed replaceable linear polyacrylamide solutions.

This paper presents results on ultralong read DNA sequencing with relatively short separation times using capillary electrophoresis with replaceable polymer matrixes. In previous work, the effectiveness of mixed replaceable solutions of linear polyacrylamide (LPA) was demonstrated, and 1000 bases were routinely obtained in less than 1 h. Substantially longer read lengths have now been achieved by a combination of improved formulation of LPA mixtures, optimization of temperature and electric field, adjustment of the sequencing reaction, and refinement of the base-caller. The average molar masses of LPA used as DNA separation matrixes were measured by gel permeation chromatography and multiangle laser light scattering. Newly formulated matrixes comprising 0.5% (w/w) 270 kDa and 2% (w/w) 10 or 17 MDa LPA raised the optimum column temperature from 60 to 70 degrees C, increasing the selectivity for large DNA fragments, while maintaining high selectivity for small fragments as well. This improved resolution was further enhanced by reducing the electric field strength from 200 to 125 V/cm. In addition, because sequencing accuracy beyond 1000 bases was diminished by the low signal from G-terminated fragments when the standard reaction protocol for a commercial dye primer kit was used, the amount of these fragments was doubled. Augmenting the base-calling expert system with rules specific for low peak resolution also had a significant effect, contributing slightly less than half of the total increase in read length. With full optimization, this read length reached up to 1300 bases (average 1250) with 98.5% accuracy in 2 h for a single-stranded M13 template.

Routine DNA sequencing of 1000 bases in less than one hour by capillary electrophoresis with replaceable linear polyacrylamide solutions.

Long, accurate reads are an important factor for high-throughput de novo DNA sequencing. In previous work from this laboratory, a separation matrix of high-weight-average molecular mass (HMM) linear polyacrylamide (LPA) at a concentration of 2% (w/w) was used to separate 1000 bases of DNA sequence in 80 min with an accuracy close to 97% (Carrilho, E.; et al. Anal. Chem. 1996, 68, 3305-3313). In the present work, significantly improved speed and sequencing accuracy have been achieved by further optimization of factors affecting electrophoretic separation and data processing. A replaceable matrix containing a mixture of 2.0% (w/w) HMM (9 MDa) and 0.5% (w/w) low-weight-average molecular mass (50 kDa) LPA was employed to enhance the separation of DNA sequencing fragments in CE. Experimental conditions, such as electric field strength and column temperature, as well as internal diameter of the capillary column, have been optimized for this mixed separation matrix. Under these conditions, in combination with energy-transfer (BigDye) dye-labeled primers for high signal-to-noise ratio and a newly developed expert system for base calling, the electrophoretic separation of 1000 DNA sequencing fragments of both standard (M13mp18) and cloned single-stranded templates from human chromosome 17 could be routinely achieved in less than 55 min, with a base-calling accuracy between 98 and 99%. Identical read length, accuracy, and migration time were achieved in more than 300 consecutive runs in a single column.

Organization of health care in Croatia: needs and priorities.

AIM: Description and analysis of the present situation of health care system in Croatia, and its characteristics in the transitional process of restructuring. METHODS: A descriptive method was used. The data from the regular statistical publications were used for the analysis. RESULTS: Croatia is faced with problems similar to those in other countries of the Central and Eastern Europe (CEE), such as control of health expenditure, balancing the development of different segments of health care services, stabilization of effectiveness and quality of care, transition from one-party system to pluralistic democracy, introduction of a free market economy, war devastations, etc. On the other hand, the Croatian experience in the development of a decentralized and integrated primary health care, decentralized health insurance system, education of general/family practitioners, and a tradition in the implementation and development of public health measures, have facilitated and contributed positively to the whole process of transition. CONCLUSION: In contrast to the economic difficulties, war devastations, and changing the social system, the Croatian health care system proved its stability and sustainability. The highest priority and needs are now related to coping with unhealthy behavior of the population, such as smoking, accidents, physical inactivity, and nutritional problems, which should be solved and controlled by the implementation of preventive programs, organization and management of public health services, and further focusing onto the integrated type of primary health care in the organization of services. Hospital services need more intensive and skilled management, as well as support measures for better quality of work.

Labeling reactions applicable to chromatography and electrophoresis of minute amounts of proteins.

Chromatography and electrophoresis have become extremely valuable and important methods for the separation, purification, detection and analysis of biopolymers and HPLC/HPCE may become the premier, preferable approaches for both qualitative and quantitative analyses of most proteins, especially from recombinant materials. This includes smaller peptides, polypeptides, proteins, antibodies and all types of protein or antibody-conjugates (antibody-enzyme, protein-fluorescent probe, antibody-drug and so forth). This entire Topical Issue of Journal of Chromatography emphasizes the application of chromatography and electrophoresis to protein analysis. This particular review deals with approaches to the selective tagging or labeling of proteins at trace (minute) levels, again using either chromatography or electrophoresis, with the emphasis on modern HPLC/HPCE methods and approaches. We discuss here both pre- and post-column labeling methods and reagents, techniques for realizing selective labeling of proteins or antibodies, applicable approaches to protein preconcentration in both HPLC and HPCE areas and in general, methods for improving (lowering) detection limits for proteins utilizing chemical or physical derivatization and/or preconcentration techniques. There are really two major goals or emphases in that which follows: (1) methods for selective labeling of proteins prior to or after HPLC/HPCE and (2) labeling of proteins at trace levels for improved separation-detection and lowered detection limits. We discuss here a large number of specific references related to both pre- and post-column/capillary derivatizations for proteins, as well as methods for improved detectability in both HPLC and HPCE by, for example, analyte preconcentration on a solid-phase extractor or membrane support, capillary isotachophoresis and other methods. Selective reactions or derivatizations on proteins refers to the ability to tag the protein at specific (e.g. reactive amino sites) in a controlled manner, with the products having the same number of tags all at the very same site or sites. The products are all the same species, having the same number of tags at the same locations on the protein. Selective reactions can also refer to the idea of tagging all of the protein sample at only a single, same site or at all available sites, homogeneously.

Capillary isoelectric focusing as a tool in the examination of antibodies, peptides and proteins of pharmaceutical interest.

This paper describes the recent history and development of capillary isoelectric focusing (cIEF), as it has evolved over the past 10 years forming a distinct mode of high-performance capillary electrophoresis (HPCE). The theory, equations, fundamentals and basics of cIEF are discussed and described, including modes of focusing and mobilization, coated vs uncoated capillaries, different detection schemes, resolutions possible, peak capacity possible and final commercialized approaches now available. Then, the applications of the technique are emphasized, as applied to smaller peptides, larger proteins and still larger antibodies and antibody-protein complexes. The emphasis has been on the application of capillary electromigration techniques in drug analysis. Throughout, attempts have been made to emphasize the potential applications and uses of cIEF methods, and how these might be successfully utilized in drug analysis and assays for larger biopolymers.

[A comprehensive program for prevention of chronic non-infectious diseases in basic population groups--the Belec Study. V. Changes in mortality and causes of death after intervention]. / Cjeloviti program borbe protiv kronicnih nezaraznih bolesti u osnovnim populacijskim skupinama--studija Belec. V. Promjene mortaliteta i strukture uzroka smrti nakon intervencije.

After an earlier analysis of mortality and causes of death in the rural community Belec for the period from 1977 to 1981, analysis was repeated for the period from 1983 to 1987. In the meantime, in 1984, the Health Committee and the Guidance Clinic for Hypertension were founded, as important elements of Comprehensive Programme for Fighting Non-infectious Chronic Diseases in Basic Population Groups of Belec Study, started in 1981. The order of causes of death did not change significantly in the period analyzed. However, 12% decrease of the crude death rate was noted, as well as the decrease of both cardiovascular diseases death rate (33%) and malignant neoplasms death rate (17%). Cerebrovascular diseases death rate increased for 3%. At the same time, data for the Republic of Croatia show an increase of 16% of crude death rate, as well as an increase of cardiovascular diseases death rate (20%), cerebrovascular diseases death rate (24%) and malignant neoplasms death rate (11%). Results suggest positive impact of the Programme.

[A comprehensive program in the fight against chronic non-infectious diseases in a basic population group--the Belec Study. IV. The interventional model]. / Cjeloviti program borbe protiv kronicnih nezaraznih bolesti u osnovnim populacijskim skupinama--studija Belec. IV. model intervencije.

As part of a comprehensive programme aimed at the struggle against chronic non-infectious diseases in basic population groups, an intervention model in the rural community Belec has been set up. The development, application and evaluation of this intervention model with an emphasis on positive experiences and difficulties connected with such a model have been described. The role of important parts of the programme has been emphasized--the work of the Health Committee and Guidance Clinic of Elevated Blood Pressure. The results show that there is necessary and possible to efficaciously involve the population of the community into the priority assessment, planning, application and follow-up of such a programme. However, the close cooperation with the local health service and other community sectors and their support is indispensible. It is also stressed that the economic and social development of the rural community is the prerequisite for the continuous intervention measures to be carried out and health care system promoted.

[Is a military hospital needed in the Republic of Croatia?]. / Treba li Republici Hrvatskoj vojna bolnica?

The paper is an attempt to answer the question whether the military hospital is necessary or not in such a small country. Some arguments (and contra-arguments) are listed and discussed. Between the others are: (1) the necessity of developing "war" medical skills and knowledge (cannot be developed during peace-time neither in the military nor civil hospitals, and civilian health services are capable to adapt and to fulfill specific war tasks as it was shown in war in Croatia in 1991), (2) the possibility to develop specific, even peculiar, specialties as hyperbaric or space medicine (these are necessary at the airports, navy bases etc, not in military hospitals), (3) specific health needs of the population of soldiers, officers, and their families (as a rule this population is younger and positively selected i. e. healthier than the general population), (4) security reasons (the data are more accessible from the military service in one place than from the scattered civilian health services), (5) privileges in health care for population of soldiers, officers, and their families due to their particular merits (military forces themselves deny this reason; also, separate military health services is not really the privilege due to bureaucracy, and rather narrow choice of services, (6) separate services could be less expensive and more efficient than the civilian one (experiences from the other countries are completely opposite). The conclusion is that, for such expensive parts of health services, as hospital care (spending between 40% and 70% of the total health expenditure), there is the growing need for rational and planned development within the comprehensive and integral healthcare system. Inside such comprehensive system the military hospital does not look like a rational solution.

Self-help hypertensive groups and the elderly in Yugoslavia.

This study examines aging in Yugoslavia in the context of rapid demographic and social changes over the last several decades. Since this time, urbanization and the population aging of the society have begun to place a stress on the ability of the family and the formal health care system to deal with the needs of the elderly. Responses to this stress have included new types of residences for the elderly and various types of self-help groups dealing with a variety of health care needs. This paper focuses on the development and functioning of self-help groups to deal with the problem of hypertension among the elderly in the Republic of Croatia. Preliminary results indicate that such groups have been relatively successful in regulating blood pressure and slightly reducing mortality. However, the success of groups requires a long term commitment from public health institutions and a recognition of how such groups must be adapted to different social environments.