[Purification of biomolecules by the method of the expanded bed adsorption chromatography. III. Method optimization. Aplications].

This review introduces the principles of the expanded bed adsorption (EBA) and serves as a practical guide to the use STREAMLINE adsorbent and columns available on the market. Critical operational parameters will be discussed as well as the principles for the method design and optimization that will ensure maximum operation of this unique unit. The review is illustrated with the examples of different types of biological molecules which have been purified when using the expanded bed adsorption.

[Purification of biomolecules by the method of adsorptional chromatography in the expanded bed. I. Principles of the method]. / Ochistka biomolekul metodom adsorbtsionnoi khromatografii v rasshirennom cloe. I. Printsipy metoda.

Principles the Expanded Bed Adsorption (EBA) have been described in the survey. The paper also deals with critical operation parameters, principles of the method design and optimization, which will guarantee maximum operation of this unique operation stage. All these problems have been discussed. The survey is illustrated by the examples of various types of biological molecules which have been purified using EBA.

[Biomoleculs purification by the method of the expanded bed adsorption chromatography. II. Design of the experiment]. / Ochistka biomolekul metodom adsorbtsionnoi khromatografii vo vzveshennom sloe. II. Proektirovanie éksperimenta.

This review introduces the principles of the Expanded Bed Adsorption (EBA) and serves as a practical guide to the use of STREAMLINE adsorbent and columns available on the market. Critical operating parameters will be discussed as well as principles for the method design and optimization which will ensure maximum exploitation of this unique operation stage. The review is illustrated with examples of different types of biological molecules which have been purified using Expanded Bed Adsorption.

[Blood protein technological industrial developments as a mirror of fundamental studies bgy the Institute of Biochemistry of the Ukrainian National Academy of Sciences]. / Tekhnolohichni rozrobky vyrobnytstva bilkiv krovi iak viddzerkalennia fundamental'nyk doslidzhen' systemy zsidannia krovi v instytuti biokhimiï nan Ukraïny.

We have examined the technology for an industrial chromatographic production highly purified factor VIII concentrate intended for therapy of the hemophilia A and characterized this factor VIII. The final product has been prepared from cryoprecipitate of pooled human plasma using a large-scale procedure combining three conventional chromatographic steps based on AEM and CEM ion exchange and SPG or SHR gel filtration chromatography. The specific activity of the product was 459 +/- 19 IU factor VIII/mg protein (n = 10), corresponding to a purification factor of about 15,000. The concentrate was free of the fibrinogen, alpha-2-macroglobulin, alpha-1-acidglycoprotein, haptoglobin. Only three contaminants could be detected: fibronectin, immunoglobulins A and G (about 0.020, 0.004 and 0.034 microgram/IU factor VIII, respectively). The purity of the final product was confirmed by SDS polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Another examination was concern to the technology for an industrial chromatographic production highly purified factor IX concentrate intended for therapy of the hemophilia B and characterized this factor IX. The final product has been prepared from pooled human plasma using a large-scale procedure combining four conventional chromatographic steps based on AEM ion exchange, AFM affinity and SGS gel filtration chromatography. The specific activity of the product was 149 +/- 10 IU factor IX/mg protein (n = 10), corresponding to a purification factor of about 9000. The concentrate was free of the vitamin K-dependent clotting factors II, VII and X and of proteins C and S. Most of possible contaminants were absent in this new product. High-molecular-weight kininogen, factor VIII, XI, XII or prekallikrein were not detected. There were no activated factors, such as factors IXa and Xa, no thrombin and no phospholipids. Only two contaminants could be detected: C4 and inter-alpha-trypsin inhibitor (about 0.8 and 1.2 mg/IU factor IX, respectively). The purity of the final product was confirmed by SDS polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Thrombogenicity tests in rabbits revealed that the high purified factor IX by Institute of Biochemistry technology tested had a lower thrombogenic power than the commercial factors IX tested. The concentrate has been subjected to a special solvent--detergent treatment for definite time and temperature during its production to virus inactivation (it will be describe in following special examination). These data demonstrate that a highly purified therapeutic clotting factor VIII and IX concentrates can be prepared from human plasma by conventional chromatographic methods developed by Institute of Biochemistry of NAS of Ukraine and Combio Ltd.

[Thrombolytic effect of plasmin, miniplasmin and fibrinolysin in simulated intraocular hemorrhage]. / Tromboliticheskoe deistvie plazmina, miniplazmina i fibrinolizina v usloviiakh modelirovaniia vnutriglaznogo krovoizliianiia.

The fibrinolytic properties of the preparations of plasmin (Pm), mixture of plasminogen and streptokinase (Pg+Sk), miniplasmin (m-Pm) and fibrinolysin have been investigated on the models of rabbit's hyphema. The action of preparations of Pm and Pg+Sk had approximately equal therapeutic index--the total destruction of clots proceeded for 12.0 days, that was 6.6 days less than in the control group. The therapeutic effect for m-Pm was 3.6 days. The dose of preparation of m-Pm, necessary for total destruction of clots, was 15-20% greater than for the same dose of Pg+Sk. The preparation of fibrinolysin, used in the same quantities of proteolytic activity, as Pg and m-Pg, (16 caseinolytic units per 1 injection) had no therapeutic effect.

[Hydrolysis of polymeric fibrin by plasmin, miniplasmin, microplasmin and trypsin]. / Osobennosti gidroliza polimernogo fibrina plazminom, miniplazminom, mikroplazminom i tripsinom.

125I-labeled polymeric fibrin hydrolyzed with plasmin, Val442-plasmin (miniplasmin, Lys530-plasmin (microplasmin) and trypsin has been studied for radioactivity of its separate electrophoretic bands. The reaction of hydrolysis was stopped at a moment of a two-fold decrease of the fibrin clot turbidity (t1/2) at the wave length 350 nm. For plasmin, miniplasmin, microplasmin and trypsin taken in the same caseinolytic activities t1/2 was 12.4, 40.0 164.1 and 76.8 min, respectively. Differences in composition of fibrin digests taken at t1/2, are demonstrated: the content of high-molecular components of digests decreases in the order of plasmin greater than miniplasmin greater than microplasmin greater than trypsin, thus showing differences in the processes of fibrin clot structure disruption by the enzymes.

[Role of the K4 and K5 plasmin heavy chain kringles in the fibrin clot structure destruction]. / Rol' kringlov K4 i K5 tiazheloi tsepi plazmina v razrushenii struktury fibrinovogo sgustka.

A comparative analysis of the rates of polymeric fibrin structure destruction by plasmin (Pm) and its proteolytic derivatives such as Val354-plasmin (c-Pm), Val442-plasmin (m-Pm) and Lys530-plasmin (mu-Pm) has been undertaken. It was shown, that Pm, c-Pm, m-Pm and mu-Pm at equal proteolytic activity, have dissolved fibrin clots with relative rates 40.3:38.0:4.6:1.0 correspondingly. The Pm, m-Pm and mu-Pm relative rates were changed by epsilon-aminocaproic acid to 4.6:1.5:1.0 correspondingly. In this case fibrin clot destruction time was increased for Pm and m-Pm and was not changed for mu-Pm. The rates of fibrinogen hydrolysis were nearly equal for these forms of enzyme. It was suggested, that the specific interactions between plasmin K4 and K5 kringles and solid phase fibrin substrate determine the polymer fibrin structure destruction rate.

[Binding of lys-plasminogen to E-fragment of fibrinogen]. / Sviazyvanie liz-plazminogena s E-fragmentom fibrinogena.

The interaction of Lys-plasminogen and its fragments with fibrinogen fragment E was studied by equilibrium affinity binding. A quantitative analysis of binding parameters revealed two types of binding sites responsible for Lys-plasminogen interaction with the immobilized fragment E, i.e., with a high (Kd = 1.5 x 10(-6) M) and low (Kd = 82 x 10(-6) M) affinity ones. Among plasminogen fragments, only miniplasminogen and KI-3 bound immobilized fragment E and were eluted by epsilon-aminocaproic acid. Hence, two lysine binding sites may be involved in the binding of Lys-plasminogen to fragment E; they are localized in the KI-3 and K5 kringle structures.