Rapid chromatographic purification of apolipoproteins A-I and A-II from human plasma

Rapid, large-scale isolation of human apolipoproteins A-I and A-II has been accomplished using two chromatographic procedures. The apolipoproteins adsorbed from plasma onto a column of phenyl-Sepharose are eluted with increasing propylene glycol concentrations. Apolipoproteins A-I and A-II can be resolved by elution with a linear 0 to 80% propylene glycol gradient. Homogeneous preparations of apo A-I and A-II are obtained following gel filtration in 3M guanidinium chloride.     

Rapid and efficient separation and identification of the two molecular forms of human adenosine deaminase by thin-layer gel filtration chromatography

Two molecular forms of adenosine deaminase have been found in human tissues. The column gel filtration method has been used for the separation of the two enzyme forms. Routine separation and analysis of the enzyme forms based on the molecular size difference can be achieved by thin-layer gel filtration on Sephadex G-200 superfine gel. The thin-layer method has been found to be more rapid and efficient than the column method. Enzymes in crude preparations can be studied effectively with the thin-layer method.     

A biologically active fructan from the roots of Arctium lappa L., var. Herkules

From the roots of Arctium lappa L., var. Herkules a low-molecular-weight fructofuranan of the inulin-type has been isolated by water extraction and ethanol precipitation, followed by ion-exchange chromatography and gel filtration of the crude precipitate. The methods employed in structural determination were methylation analysis and 1H and 13C NMR spectral measurements. In tests for antitussive activity in cats the fructan was found to be equally active as some non-narcotic, synthetic preparations used in clinical practice to treat coughing, and in mitogenic and comitogenic tests its biological response was comparable to that of the commercial Zymosan immunomodulator.     

Preparative Procedure for the Purification of Toxoids by Gel Filtration

Sephadex gel filtration can be employed as a preparative procedure for the purification of both tetanus and diphtheria toxoids. A toxoid purification sequence is described in the text. By utilizing the described methods and columns, up to 100,000 human doses of diphtheria toxoid could be processed in a single operation. The method has given an 80% yield of diphtheria toxoid with a purity of 1,900 Lf per mg of N. The analysis of the material by immunodiffusion tests showed that a marked increase in purity was achieved. Antigenicity tests demonstrated that there was no significant difference in antigenic potency between the parent toxoid and its purified fraction. Factors limiting the effective separation of tetanus toxoid by gel filtration are discussed. The construction of the columns used is described in detail, as well as packing procedures and column characteristics such as bed volume, void volume, sample size, and flow rate.     

Molecular composition of the fractions of immunoglobulin preparations studied by gel filtration

The method of gel filtration in columns permitted the separation of aggregated fractions into polymers whose content did not exceed 10% and dimers, their content ranging from 3.6% to 22.11%. The preparations were also found to contain fractions of monomers and fragments, Fab-fragments being detected in 10 out of 20 batches under study (4.04-27.36%). The shelf life of all preparations did not exceed 8-12 months. The use of spectrophotometric techniques ensured obtaining the most objective results in the calculation of the percentage of fractions contained in immunoglobulin preparations. The evaluation of the molecular composition of immunoglobulin preparations by the method of gel filtration is conducive to the improvement of their quality.     

Some characteristics of aggregates of IgG and plasma proteins in heat-treated factor VIII concentrates

Eight batches of commercial heat-treated and one untreated factor VIII concentrate from 6 producers were analyzed for their content of IgG, IgG subclasses, IgG aggregates and the presence of other plasma proteins combined with the IgG as well as for anticomplement activity. Methods used were thin-layer gel filtration, immuno-gel filtration, spot immuno-precipitate assay in a double antibody version and an agarose plate haemolysis inhibition assay of complement fixation. The IgG content varied from 0.1-6.90 g/l. In all preparations IgG existed as monomers and aggregates. Associated with the IgG were also found, at significantly increased amounts compared to normal serum and intravenous immunoglobulin, one to four of the following plasma proteins; fibronectin, fibrinogen, von Willebrand factor antigen, Clq, albumin and IgA. Three batches from two producers had high anticomplementary activity, presumably caused by the IgG aggregates. Two of these deviated strikingly from normal human serum pools in percent distribution of IgG subclasses. It is hypothesized that these aggregates can induce side effects or cause immunological aberrations.     

Protein disulphide-isomerase of chick-embryo tendon.

Protein disulphide-isomerase can be partially purified from the high-speed-supernatant fraction of extensively disrupted chick-embryo tendon tissue. The catalytic properties of the preparation resemble those of the enzyme from mammalian liver. Gel electrophoresis and isoelectric focusing show the enzyme to be very acidic, with pI 4.4 +/- 0.3. Gel filtration indicates an Mr for the active enzyme of 140 000. The enzyme can be partially purified by preparative gel filtration or isoelectric focusing, but its limited stability has prevented purification to homogeneity; active fractions from both gel filtration and isoelectric focusing show two major polypeptide components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The major polypeptides present in partially purified preparations have Mr 45 000 and 55 000; the latter band co-distributes with the enzyme activity in fractionations by both gel filtration and isoelectric focusing. The subcellular location of the enzyme cannot be established from work on homogenates of whole tissue, which are extensively disrupted. In homogenates from isolated tendon cells, the enzyme is located in a vesicle fraction that is excluded from Sepharose 2B but is of low density and can only be sedimented at very high speeds. This fraction is identified as deriving from the endoplasmic reticulum on the grounds of marker-enzyme studies and electron microscopy.     

Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin

Urokinase-activated human plasma was studied by gel electrophoresis, gel filtration, crossed immunoelectrophoresis and electroimmunoassay with specific antibodies and by assay of esterase and protease activity of isolated fractions. Urokinase induced the formation of different components with plasminogen+plasmin antigenicity. At low concentrations of urokinase, a component with a K(D) value of 0.18 by gel filtration and post beta(1) mobility by gel electrophoresis was detected. The isolated component had no enzyme or plasminogen activity. In this plasma sample fibrinogen was not degraded for 10h, but when fibrin was formed, by addition of thrombin, fibrin was quickly lysed, and simultaneously a component with a K(D) value of 0 and alpha(2) mobility appeared, which was probably plasmin in a complex with alpha(2) macroglobulin. This complex showed both esterase and protease activity. After gel filtration with lysine buffer of the clotted and lysed plasma another two components were observed with about the same K(D) value by gel filtration as plasminogen (0.35), but beta(1) and gamma mobilities by gel electrophoresis. They appeared to be modified plasminogen molecules, and possibly plasmin with gamma mobility. Similar processes occurred without fibrin at higher urokinase concentrations. Here a relatively slow degradation of fibrinogen was correlated to the appearance of the plasmin-alpha(2) macroglobulin complex. The fibrin surface appeared to catalyse the ultimate production of active plasmin with a subsequent preferential degradation of fibrin and the formation of a plasmin-alpha(2) macroglobulin complex. The gel filtration and electrophoresis of the plasma protease inhibitors, alpha(1) antitrypsin, inter-alpha-inhibitor, antithrombin III, and C(1)-esterase inhibitor indicated that any complex between plasmin and these inhibitors was completely dissociated. The beta(1) and post beta(1) components appear to lack correlates among components occurring in purified preparations of plasminogen and plasmin.     

Impact of virus preparation quality on parvovirus filter performance

Virus‐removal filtration technology is commonly used in the manufacturing process for biologics to remove potential viral contaminants. Virus‐removal filters designed for retaining parvovirus, one of the smallest mammalian viruses, are considered an industry standard as they can effectively remove broad ranges of viruses. It has long been observed that the performance of virus filters can be influenced by virus preparations used in the laboratory scale studies (PDA, 2010 ). However, it remains unclear exactly what quality attributes of virus preparations are critical or indicative of virus filter performance as measured by effectiveness of virus removal and filter capacity consistency. In an attempt to better understand the relationship between virus preparation and virus filter performance, we have systematically prepared and analyzed different grades of parvovirus with different purity levels and compared their performance profiles on Viresolve® Pro parvovirus filters using four different molecules. Virus preparations used in the studies were characterized using various methods to measure DNA and protein content as well as the hydrodynamic diameter of virus particles. Our results indicate that the performance of Viresolve® Pro filters can be significantly impacted depending on the purity of the virus preparations used in the spike and recovery studies. More importantly, we have demonstrated that the purity of virus preparations is directly correlated to the measurable biochemical and biophysical properties of the virus preparations such as DNA and protein content and monodispersal status, thus making it possible to significantly improve the consistency and predictability of the virus filter performance during process step validations. Biotechnol. Bioeng. 2013; 110: 229–239. © 2012 Wiley Periodicals, Inc.     

Altered N----F transition in bovine serum albumin dimer and tetramer

The monomer, dimer and tetramer of bovine serum albumin were separated by gel filtration on a Sephacryl S-300 column. Their purity was established by gel filtration, polyacrylamide gel electrophoresis and SDS polyacrylamide gel electrophoresis. These preparations were identical in bilirubin binding, cross-reactivity against anti-bovine serum albumin antiserum and UV spectral features. However, the three preparations differed in their N----F transition. The mid points of the N----F transitions for monomer, dimer and tetramer were found to be 3.75, 3.60 and 3.40 respectively. We presume that intermolecular interactions and/or alterations in the pK values of carboxyl groups are responsible for increased stability in the case of dimer and tetramer.     

The effect of heparin on the affinity chromatography of plasminogen. Demonstration of heparin-plasminogen interaction.

Evidence is presented that heparin binds rabbit plasminogen types I and II under affinity chromatographic conditions using the single stage technique earlier described (Hatton, M.W.C. and Regoeczi, E. (1974) Biochim. Biophys. Acta 359, 55-65). Thus, the affinity of types I and II for Sepharose-lysine is markedly increased in the presence of heparin and elution by epsilon-aminohexanoic acid requires a steeper gradient to recover the plasminogen types. Furthermore by adding sufficient epsilon-aminohexanoic acid to non-heparinised plasma to suppress plasminogen affinity, the presence of heparin is shown to encourage binding of plasminogen (type II more so than type I) to the gel. However, the heparin effect is quickly reversed by washing the column with 0.5 M NaCl prior to elution by epsilon-aminohexanoic acid. No evidence of a stable plasminogen-heparin complex has been found from gel filtration studies and any interaction between plasminogen and heparin probably only takes place when heparin is bound to an affinity site. Studies with 35-S-labelled heparin have shown the mucopolysaccharide to bind to the free amino group of Sepharose-lysine and Sepharose-cadaverine and to be displaced by 0.5 M NaCl elution but not by 0.1 M epsilon-aminohexanoic acid. The plasminogen types produced from heparinised plasma are free from heparin and closely resemble preparations from non-heparinised plasma when compared by polyacrylamide gel electrophoresis, Sephadex gel filtration and arginine esterase activity after urokinase activation.     

Isolation from a pregnant woman of a serum protein responsible for inhibiting an in vitro cytotoxicity reaction

A non specific immunosupressive factor able to block an in vitro cytotoxicity reaction is demonstrated in the serum of pregnant women. A solution containing this blocking factor is obtained by gel filtration and precipitation of plasma by polyethylene glycol 4 000. Then after immunisation of rabbits the immune serum can be used for affinity chromatography. An alpha 2 glycoprotein has been separated which inhibits the in vitro cytotoxicity reaction and whose molecular weight determined by gel filtration and polyacrylamide gel is about 200 000 daltons.     

Analysis of the mitogenic effect of fetuin preparations on arterial smooth muscle cells: the role of contaminant platelet-derived growth factor

Fetuin, a major protein of fetal calf serum, partially purified by the method of Pedersen, stimulated growth of aortic smooth muscle cells. More highly purified fetuin preparations stimulated growth less than Pedersen fetuin, as previously described for other cell types, suggesting that this activity is due to a contaminant. Recently bovine alpha 2-macroglobulin or "Embryonin" has been proposed as the mitogenic component of crude fetuin preparations. We found that active fetuin preparations did contain alpha 2-macroglobulin that stimulated smooth muscle cell growth. However, alpha 2-macroglobulin purified directly from platelet-poor bovine plasma or fetuin purified from Pedersen fetuin by gel filtration lacked appreciable mitogenic effect on smooth muscle cells. Since alpha 2-macroglobulin can bind platelet-derived growth factor (PDGF), and since highly acidic fetuin might bind the very basic PDGF molecule non-specifically, we measured the PDGF content of various fetuin preparations and found a good correlation between the PDGF content and mitogenic activity. Gel filtration experiments demonstrated that in Pedersen fetuin PDGF occurred both free, and in association with alpha 2-macroglobulin. We conclude that the principal mitogenic component for smooth muscle cells in crude fetuin preparations is PDGF, since purified bovine alpha 2-macroglobulin or fetuin do not appreciably affect growth of these cells. These results help to resolve a long-standing controversy regarding the nutrition of cultured cells. In addition, we suggest that before alpha 2-macroglobulin or "Embryonin" is accepted as a bona fide growth factor for a given cell type, the role of contamination with PDGF should be assessed.     

Isolation of the factor VIII–von Willebrand factor complex directly from plasma by gel filtration

A high capacity gel filtration system was developed with the purpose of isolating factor VIII (FVIII) and von Willebrand factor (vWF) directly from plasma in significantly higher yields than obtained by cryoprecipitation, the technique most commonly used to recover FVIII–vWF from human plasma. After laboratory-scale gel filtration of plasma, a FVIII-containing fraction was collected containing about 90% of FVIII in the applied plasma and with almost tenfold higher purity than that obtained by cryoprecipitation. The gel filtration step has been scaled up for use as the initial step in the manufacturing process for a FVIII preparation (Nordiate).     

A study of the single polypeptide nature of rhodanese. A comparison of different preparations.

The enzyme rhodanese (EC 2.8.1.1) appears as a single polypeptide chain protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this species is approx. 33 000. This contrasts with previous reports that rhodanese behaves on gel filtration chromatography as a rapidly equilibrating monomer-dimer system composed of identical subunits with a molecular weight of 18 500. We have investigated this apparent discrepancy by isolating the enzyme by the two different preparative procedures used in the above investigations. The two crystalline samples were subjected to gel filtration chromatography under a wide variety of conditions and to sodium dodecyl sulfate disc gel electrophoresis. The two preparations yielded rhodanese which behaved identically and no evidence for the monomeric species was obtained under any experimental condition tested. Thin-layer gel chromatography of clarified liver homogenates gave no evidence of rhodanese species other than that present in the purified samples. The variation in molecular weights observed in gel filtration chromatography may be a reflection of the conformational mobility of the enzyme leading to solvent-dependent changes in Stokes radius. If rhodanese is dimeric, special interactions must stabilize it under the conditions tested here.     

A reexamination of the properties of spinach nitrite reductase: protein and siroheme content heterogeneity in purified preparations

Recent preparations of nitrite reductase do not display the heterodimeric quaternary structure obtained previously (total molecular weight 85,000; subunit molecular weights 24,000 and 61,000), but rather yield only the 61,000 molecular weight subunit, even when buffers containing the protease inhibitor phenylmethylsulfonyl fluoride are used. Nevertheless, such preparations retain the high ratio of ferredoxin-linked to methyl viologen-linked enzyme activity which has been previously taken as a characteristic of only the heterodimeric form. These preparations display a siroheme prosthetic group to protein ratio of 1.1. When nitrite reductase samples are frozen during the purification scheme, even though the ferredoxin-linked specific activity does not significantly decrease, enzyme activity-stained native gel electrophoresis of the subsequently purified protein reveals that gels with several bands of activity can be obtained. Further evidence of protein heterogeneity in these preparations comes from N-terminal amino acid analysis which reveals that even nonfrozen preparations contain two major peptides with valine and cysteine as the N-termini. Formation of complexes of purified nitrite reductase with ferredoxin resulted in siroheme difference electronic spectra which resembled those observed previously for monomeric preparations. However, the siroheme midpoint potential of recent preparations of nitrite reductase (-287 mV) is close to that of the heterodimeric preparations. Ultrafiltration studies of crude extracts of the enzyme indicate that, at least at certain stages of the preparation, higher molecular weight forms of the enzyme may exist. We conclude that the 24,000 molecular weight polypeptide is a contaminant and that the heterodimeric quaternary structure model for spinach nitrite reductase is incorrect. Furthermore, the monomeric preparations we do obtain display both significant protein heterogeneity and facile loss of siroheme upon gel filtration.     

The fractional composition of immunoglobulin preparations studied by gel filtration on different carriers and by high-pressure liquid chromatography]

The fractional composition of immunoglobulin preparations produced by different manufacturing enterprises of this country has been studied by gel chromatography in columns packed with different carriers (Sephadex G-200 and ultragel AcA-34) and by high-performance liquid chromatography (HPLC). This study has revealed the nonstandard character of immunoglobulin preparations produced according to the same technological procedure (modified Cohn's method). The fractionation of immunoglobulins on different carriers with the use of different methods has yielded similar results confirmed by the statistical processing of the data. The results obtained in the study of the fractional composition of immunoglobulin preparations evidence that gel filtration with the use of ultragel and HPLC have greater resolving capacity in comparison with the method of gel filtration on traditionally used Sephadex G-200.     

Chromatographic analysis and immunobiologic properties of tuberculins]

Immunobiologic activities of tuberculin preparations and their components have been comparatively studied using gel filtration and high-performance liquid chromatography (HPLC) techniques. It is shown that high-molecular weight fraction of protein-purified derivate tuberculin (PPD) had higher activity as compared to nonfractionated preparation in skin tests on guinea pigs sensitized with Mycobacterium bovis BCG as well as in enzyme-linked immunosorbent assay with affinity purified rabbit antibodies against PPD. Using the preparative HPLC-technique we failed to isolate a component of PPD having greater tuberculin test potency than nonfractionated preparation.     

Are cardiovascular and sympathoadrenal effects of human "new pressor protein" preparations attributable to human coagulation beta-FXIIa?

"New pressor protein" (NPP) derived from normal human plasma is an extra renal enzyme that shares strong sequence homology with human coagulation beta-FXIIa. Under our bioassay conditions, human NPP (10-20 microl plasma equivalent/ approximately 300 g rat iv) can raise the systolic blood pressure (SBP) by 40-50 mmHg, the diastolic blood pressure (DBP) by 15-20 mmHg, and the heart rate (HR) by 70-90 beats/min. Plasma epinephrine (of adrenal medullary origin) and norepinephrine rise by about 50- and 10-fold, respectively. Because beta-FXIIa is not normally associated with pressor properties, we endeavored to substantiate that the hypertensive effects of impure NPP preparations used in our experiments are attributable to their content of beta-FXIIa. We carried out comparisons with highly purified (>90%) commercial human beta-FXIIa and found that by gel filtration (Sephadex G-100 and G-75), NPP bioactivity appeared in the approximately 30-kDa elution zone, consistent with the molecular mass of beta-FXIIa. Retention time using fast-protein liquid chromatography anion exchange chromatography was identical. Molecular mass and comigration were confirmed by SDS-PAGE gel electrophoresis, and the recovered approximately 30-kDa protein bands yielded beta-FXIIa fragments identified by mass spectrometry. Matched doses of the NPP preparations produced dose-response curves very similar to those elicited by beta-FXIIa with respect to increments of SBP, DBP, and HR, whereas plasma catecholamine increments were generally comparable. We propose that beta-FXIIa is substantially, if not exclusively, responsible for the observed effects of our NPP preparations and that this points to a novel axis connecting the FXII coagulation cascade and the sympathoadrenal gland to other cardiovascular regulatory mechanisms.     

一种新型助滤剂在血液制品生产中的应用研究

目的:探讨新型硅藻土用于血液制品组份分离过滤的适用性。方法:对比新型硅藻土与传统硅藻土的电镜扫描物理结构、金属离子含量、比表面积、离心湿密度等物理表征数据,并考察其在血浆蛋白分离阶段各组份过滤效果及最终产品的质量。结果:新型硅藻土与传统硅藻土比较,具有颗粒孔隙致密、金属离子含量低、比表面积大和离心湿密度小的特点;在血浆蛋白分离阶段的过滤过程中,按照传统硅藻土用量的70%左右使用新型硅藻土时,过滤时可得到理想的滤液浊度、较快的过滤速率、更小的过滤压力和均匀的滤饼分布;所得最终产品的质量符合《中国药典》及公司内控标准。结论:新型硅藻土可替代传统硅藻土用于血液制品的规模化生产,同时降低使用量。该应用研究对于降低血液制品的生产成本和提高产品质量具有一定的实际应用价值和参考意义。