A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.

To ensure the optimal safety of plasma derived and new generation recombinant proteins, heat treatment is customarily applied in the manufacturing of such biopharmaceuticals as a means of viral inactivation. In subjecting proteins to anti-viral heat-treatment it is necessary to use high concentrations of thermostabilizing excipients to prevent protein damage, and it is therefore imperative that the correct balance between bioprocessing conditions, maintenance of protein integrity and virus kill is found. In this study we have utilized model proteins (lysozyme, fetuin, and human serum albumin) and plasma-derived therapeutic proteins (factor VIII and factor IX) to investigate the protein modifications that occur during anti-viral heat treatment. Specifically, we investigated the relationship between bioprocessing conditions and the type and extent of protein modification under a variety of industrially relevant wet and lyophilized heat treatments using sucrose as a thermostabilizing agent. Heat treatment led to the formation of disulfide crosslinks and aggregates in proteins containing free cysteine residues. Terminal oligosaccharide sialic acid residues were hydrolyzed from the glycan moieties of glycoproteins during anti-viral heat treatment. Heat treatment promoted sucrose hydrolysis to yield glucose and fructose, leading, in turn, to the glycation of lysine amino groups in those proteins containing di-lysine motifs. During extended hear treatments, 1,2-dicarbonyl type advanced glycation end-products were also formed. Glycation-type modifications were more prevalent in wet heat-treated protein formulations.

During the preparation of therapeutic plasma and recombinant protein biopharmaceuticals heat-treatment is routinely applied as a means of viral inactivation. However, as most proteins denature and aggregate under heat stress, it is necessary to add thermostabilizing excipients to protein formulations destined for anti-viral heat-treatment in order to prevent protein damage. Anti-viral heat-treatment bioprocessing therefore requires that a balance be found between the bioprocessing conditions, virus kill and protein integrity. In this study we have utilized a simple model protein, beta -lactoglobulin, to investigate the relationship between virucidal heat-treatment conditions (protein formulation and temperature) and the type and extent of protein modification in the liquid state. A variety of industrially relevant heat-treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) we show here that protein mo difications do occur with increasingly harsh heat-treatment. The predominant modification under these conditions was protein glycation by either glucose or fructose derived from hydrolyzed sucrose. Advanced glycation end products and additional unidentified products were also present in beta -lactoglobulin protein samples subjected to extended heat-treatment. These findings have implications for the improvement of anti-viral heat-treatment bioprocesses to ensure the safety and efficacy of protein biopharmaceuticals. Copyright (C) 2001 John Wiley & Sons, Ltd.

Heat treatment is routinely used in the preparation of therapeutic protein biopharmaceuticals as a means of viral inactivation. However, in undertaking virucidal heat treatments, a balance must be found between the bioprocessing conditions, virus kill, and the maintenance of protein integrity. In this study, we utilize a simple model protein, hen egg-white lysozyme, to investigate the relationship between antiviral bioprocess conditions (protein formulation and temperature) and the extent and type of protein modification. A variety of industrially relevant wet- and dry-heat treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Although there was no evidence of lysozyme aggregation or crosslinking during any of the heat treatments, using liquid chromatography-electrospray ionization-mass spectroscopy (LC-ESI-MS) and peptide mapping we show that protein modifications do occur with increasingly harsh heat treatment. Modifications were predominantly found after wet-heat treatment, the major covalent modification of lysozyme under these conditions being glycation of Lys(97), by either glucose or fructose derived from hydrolyzed sucrose. The extent of sucrose hydrolysis was itself dependent on both the duration of heat treatment and formulation composition. Advanced glycation end products (AGEs) and additional unidentified products were also present in protein samples subjected to extended heat treatment. AGEs were derived primarily from initial glycation by fructose and not glucose. These findings have implications for the improvement of bioprocesses to ensure protein product quality.

To ensure the safety of plasma and recombinant therapeutic proteins, heat treatment is routinely applied to these biopharmaceuticals as a means of virus inactivation. However, to maintain protein integrity during heat treatment it is necessary to use high concentrations of thermostabilizing excipients, such as sucrose, in order to prevent protein damage. In this study we describe the covalent modifications inferred to a model protein, β‐lactoglobulin A, that occur during typical and extended anti‐viral heat treatments. The chemical derivation and mechanisms by which these modifications arise are addressed. Heat treatment initiated hydrolysis of sucrose to glucose and fructose, which in turn were degraded to glyoxal. Glyoxal and the free reducing sugars reacted with free amino groups in β‐lactoglobulin A to yield Maillard glycation adducts and advanced glycation end products (AGEs). The major mechanism for AGE formation was via degradation of glucose‐derived Schiff‐base adducts. Heat treatment and glycation of β‐lactoglobulin A resulted in thiol‐disulphide interchange reactions leading to protein oligomerization. A small population of β‐lactoglobulin A non‐disulphide‐linked dimers were also observed with increasingly harsh heat treatments. These findings have implications for (i) improvements in the safety and efficacy of heat‐treated protein biopharmaceuticals and (ii) our understanding of the mechanisms of protein glycation and AGE adduct formation.

Prion diseases are neurodegenerative disorders where infectious prion proteins (PrP) accumulate in brain leading to aggregation of amyloid fibrils and neuronal cell death. The amino acid sequence 106–126 from prion proteins, PrP(106–126), is highly amyloidogenic and implicated in prion-induced pathologies. As PrP is known to be expressed in blood following leakage from brain tissue, we sought to investigate its biological effects on human platelets, which have been widely employed as ‘peripheral’ model for neurons. Our findings suggested that, PrP(106–126) (20 μM) induced dramatic 30-fold rise in intracellular calcium (from 105 ± 30 to 3425 ± 525 nM) in platelets, which was attributable to influx from extracellular fluid with comparatively less contribution from intracellular stores. Calcium mobilization was associated with 8–10-fold stimulation in the activity of thiol protease calpain that led to partial cleavage of cytoskeleton-associated protein talin and extensive shedding of microparticles from platelets, thus transforming platelets to ‘activated’ phenotype. Both proteolysis of talin and microparticle release were precluded by calpeptin, a specific inhibitor of calpain. As microparticles are endowed with phosphatidylserine-enriched surface and hence are pro-coagulant in nature, exposure to prion favored a thrombogenic state in the organism.

Background and Objectives: To quantify the cellular isoform of prion protein (PrP c ) in human blood using a new time‐resolved dissociation‐enhanced fluoroimmunoas‐say (DELFIA®). Materials and Methods: The DELFIA was optimised for human blood samples and applied to isolated cell and plasma fractions from blood donations. The physicochemical properties of PrP c were analysed. Results: 26.5% of blood PrP c was associated with the platelet fraction, 0.8% with polymorphonuclear leucocytes, 2.4% with mononuclear leucocytes, 1.8% with red cells and 68.5% with plasma (mean values from 4 processed donations). Conclusion: The majority of blood PrP c is found in the platelet and plasma compartments.

Gas sparged ultrafiltration has been applied to a flat sheet membrane module and the enhancing effect from the injected bubbles is examined experimentally. Two membranes, polysulphone (PS) and polyethersulfone (PES), were used in the experiments, and four proteins, human serum albumin (HSA), human immunoglobulin G (IgG), bovine serum albumin (BSA) and lysozyme (Lys), were chosen as the test media. The effects of gas sparging on permeate flux, single protein transmission and protein fractionation have been investigated. Experimental results show that gas sparging can increase permeate flux and improve the efficiency of protein fractionation.

Ultrafiltration is an attractive process for virusremoval from bioproducts owing to its high throughputas well as the fact that the operation is carried outunder ambient conditions (damage to proteins is highlylimited). The principal concern regarding the adoptionof conventional ultrafiltration membranes for virusremoval is the possibility of the virus passingthrough abnormally large pores or surfaceimperfections on the membrane surface. The chiefprinciple behind the present work is to pretreat themembrane by blocking the abnormally large pores usinglatex particles. Experimental work was conducted tovalidate this pretreatment using the bacteriophageφx174 as a model virus.The results attained were highly encouraging.Different sizes of latex particles were tested bytreating a 100 KD molecular weight cut-off membrane,and the transmission of phage (suspended in buffer)through this membrane assessed. In the absence of anyparticle pretreatment, a virus clearance of 4.78 logreduction value was observed for this membrane. Thetransmission of phage through the membrane could bereduced by an order of magnitude using 0.11 μmlatex particles, or two orders of magnitude using acombination of 0.11 and 0.50 μm particles.The application of latex particles did nothinder the transport of protein through the 100 KDmembrane. Protein sieving coefficients obtained usingthis membrane were 91%, 16% and 2%, for lysozyme,HSA and IgG, respectively.

The poor selectivity of membranes has been regarded as one of the critical factors limiting the application of membrane systems to protein fractionation. This study demonstrates that ultrafiltration enhanced by gas sparging, together with proper adjustment of solution conditions, can dramatically improve the selectivity of a commercially available tubular PVDF membrane (MWCO 100 kD) for the fractionation of the HSA/IgG mixture, as well as significantly increase permeate flux. For the system studied, the optimal solution condition was found to be at pH 8 and salt concentration of 40 mM. In this system the transmission of IgG was much higher than that of HSA, i.e. a. reversed selectivity was observed. The results were explained on the basis of the balance of the hydrodynamic and electrostatic forces acting on the molecules when they approach and enter the membrane pores. The effect of other operating parameters, including TMP, air and liquid flowrates and protein feed concentration, were also investigated and the optimal conditions were identified. With gas sparged ultrafiltration under those conditions, almost complete separation of the two proteins was achieved. Finally, the possible damage to proteins induced by air bubbles was studied and it was concluded that the damage was negligible under the selected operation conditions.

Over the last four years, a number of different studies have been conducted examining the effect of injecting gas bubbles into the liquid crossflow stream when ultrafiltering/microfiltering a solution of macromolecules/particles, with the intention of gaining an understanding of the performance characteristics of this flux enhancement technique as well as an understanding of mechanism of flux enhancement. In this report, possible mechanisms are discussed. Example applications of this technique and their associated benefits are presented, including protein concentration, protein fractionation and virus removal from bioproducts.

Gas sparged ultrafiltration experiments are performed using a tubular membrane module with solutions of dextran and human serum albumin (HSA) as the test media. Air is injected, in a controlled manner with the ability to adjust bubble size and frequency independently, into the membrane module to create a gas–liquid two-phase crossflow operation. The effects of bubble size and frequency on the permeate flux of the sparged ultrafiltration are studied experimentally. It is found that the permeate flux increases with the bubbling frequency in the examined range. The effect of bubble size on flux can be divided into two regions, an increasing region for smaller bubbles and a plateau region for larger slugs. The results are discussed on the basis of bubble wake hydrodynamics.

Ultrafiltration experiments were performed on mixtures of BSA and lysozyme with the aim of fractionating the two proteins. Pilot-plant scale polysulphone hollow fiber membrane modules with molecular weight cutoffs of 150 000 and 200 000 were used. The membrane process was enhanced via the injection of gas slugs into the hollow fiber membrane modules. The gas slugs served to disrupt concentration polarization near the membrane surface with the subsequent aim of improving membrane selectivity. It was found that hollow fiber membranes were effective at fractionating BSA and lysozyme, with the membrane selectivity being greater than 20 across a range of operating conditions. The introduction of gas slugs further improved membrane selectivity by a factor of 3–5. The sieving mechanism of the membrane and electrostatic interactions between proteins and the membrane were discussed and used to explain experimental observations.

This study focuses on the use of gas-liquid two-phase crossflow to overcome concentration polarisation in the ultrafiltration of macromolecular solutions as applied to hollow fibre membrane systems. The experimental work was conducted on a purpose built pilot-plant scale rig with albumin and dextran as the test media, The effect of gas injection on the permeate flux and membrane sieving coefficient was examined experimentally at different transmembrane pressures, feed concentrations and gas to liquid flow ratios.The results were encouraging, with flux enhancements of 20-50% obtained for dextran and 10-60% for albumin, when air was injected into the system over the range of process variables examined, The sieving coefficient of albumin was considerably reduced when gas-liquid two-phase cross-flow was used. These results were compared to those obtained with tubular membrane systems, and an additional mechanism, based on physical displacement of the concentration polarisation boundary layer is proposed, The operational difficulty related to protein foaming is also discussed.

Publisher Summary This chapter focuses on hemoglobin polymerization. A reagent to cross-link hemoglobin (Hb) is needed to stabilize the tetramer (64 kDa) by intramolecular cross-links and to produce oligomers ( 500 kDa) by intermolecular cross-links. Intramolecular cross-links between tetramer subunits prevent dissociation into excretable dimers (32 kDa). Intermolecular cross-links are necessary to reduce colloid osmotic pressure as well as to improve the circulatory half-life of the molecule as a cell-free solution. The chapter introduces a reliable two-step procedure in which the crosslinking is performed by a simple molecule and the oxygen affinity is modified by a separate simple molecule. Glycolaldehyde is a very useful, flexible, reliable, and cost-effective option in the spectrum of chemical reagents available for the modification of Hb solutions. It is remarkably compatible with a variety of additional chemical derivatives (2-nor-2-formylpyridoxal 5'-phosphate (NFPLP) and bis(3,5-dibromosalicyl) fumarate (DBBF)) and the oxy and deoxy forms of Hb. The use of glycolaldehyde in the scaleup of Hb modification and processing on a large scale for the production of therapeutic, clinical grade material maybe realistically contemplated.

Three different coupling chemistries that have been tried and tested for use in affinity chromatography are described. These methods are particularly recommended for use by workers who do not have access to, or do not wish to use, complex organic chemical synthetic procedures. They have been demonstrated repeatedly to be reliable, efficient, low cost, and easily scaleable up or down in size. The periodate oxidation method works best with Sephacryl type gels and uses only low toxicity reagents and couples well to proteins with both high efficiency and high capacity. The vinyl sulfone method is more reactive and couples both carbohydrates and proteins. The bis-epoxide method, although less reactive, can be used under more extreme conditions of pH to couple otherwise unreactive molecules, such as synthetic polymers, drugs, and so forth.

The profile of proteins bound to immobilised heparins in hirudin-anticoagulated human plasma was analysed. In molar terms, antithrombin III was the most abundant protein bound to therapeutic doses of unfractionated heparin (M(r) = 12,000), whereas heparin cofactor II constituted <1% of the protein bound. Histidine-rich glycoprotein was the only plasma protein likely to influence anticoagulant activity by direct competition with antithrombin III, though significant quantities of complement Factor H, fibrinogen, fibronectin, vitronectin and apolipoprotein B were also detected. Only traces of von Willebrand factor, complement factor I, inter-alpha-trypsin inhibitor, alpha(2)-macroglobulin, serum amyloid P and transferrin were identified, and neither thrombospondin nor platelet factor 4 were measurable. Binding of both antithrombin III and histidine-rich glycoprotein varied with the ratio of heparin to plasma. Clexane (M(r) = 4,500) also bound antithrombin III, but both histidine-rich glycoprotein and vitronectin were quantitatively significant neutralising proteins. Neutralising proteins dominated the binding profile for Oligo H (M(r) = 2,200).

SUMMARY. An enzyme linked immunosorbent assay (ELISA) has been developed to measure VIII:Ag in plasma and concentrates. The assay utilizes two commercially available monoclonal antibodies to VIII:Ag and provides an alternative to the established immunoradiometric assay (IRMA). It has the advantage of not requiring the use of radioactive material and human antibodies. The assay sensitivity is 0.006 u/ml and the interassay coefficient of variation is 6.3%. Forty‐eight samples with VIII:Ag levels ranging from 0.006 to 1.5 u/ml were assayed by both ELISA and IRMA. The coefficient of correlation between the two assays was 0.89. In addition to measuring human VIII:Ag, it is also possible to detect antigen in several animal plasma and sera.

A direct dye-binding procedure was established for the quantification of protein after its immobilization on a solid phase, using IgG and BSA as model proteins. The assay, which in the range 0-5 mg protein/ml gel correlates well with indirect protein determination by A280 as well as determination of protein hydrolyzed from the gel, is based on a modified Bradford dye-binding assay. As the protein coupled to the gel binds the dye, a decrease in A465 of the supernatant is measured. Three solid supports commonly used for protein immobilization (Sepharose, Sephadex, Sephacryl) were found to be compatible with the dye-binding assay while nonspecific dye binding was found to HEMA gels. Protein was coupled to Sephacryl S-1000 using three different activation methods (aldehyde, hydrazine, and adipic acid dihydrazide). Artifactual dye-binding was not observed using any of the three different "linkers." The assay is easily carried out and represents a useful tool, e.g., when optimizing procedures for protein immobilization.