Use Of Human Serum Albumin Research Articles

Fusion partners can increase the expression of recombinant interleukins via transient transfection in 2936E cells

The expression levels of five secreted target interleukins (IL-11, 15, 17B, 32, and IL23 p19 subunit) were tested with three different fusion partners in 2936E cells. When fused to the N-terminus, human serum albumin (HSA) was found to enhance the expression of both IL-17B and IL-15, cytokines which did not express at measurable levels on their own. Although the crystallizable fragment of an antibody (Fc) was also an effective fusion partner for IL-17B, Fc did not increase expression of IL-15. Fc was superior to HSA for the expression of the p19 subunit of IL-23, but no partner led to measurable levels of IL-32gamma secretion. Glutathione S-transferase (GST) did not enhance the expression of any target and suppressed the production of IL-11, a cytokine which expressed robustly both on its own and when fused to HSA or Fc. Cleavage of the fusion partner was not always possible. The use of HSA or Fc as N-terminal fusions can be an effective technique to express difficult proteins, especially for applications in which the fusion partner need not be removed.

Risk of immunologic reactions to human serum albumin solutions

MADAM We write to highlight the potential risk of immunologic reactions to human serum albumin (HSA) solutions. Recent advances in veterinary emergency and critical care and the greater availability of HSA solutions has led to increased use of these products in small animals. We feel that the potential for adverse reactions to HSA solutions is not widely recognised however. Recent research has identified a dichotomous response to the use of HSA solutions in critically ill versus healthy dogs. In critically ill dogs, HSA was reportedly associated with a low number of mild adverse effects although several serious adverse drug reactions were also potentially attributable to HSA administration (Mathews and Barry 2005, Trow and others 2008). In contrast, administration of HSA to healthy animals may be fatal (Francis and others 2007). A study by Martin and others (2008) suggested this may be due to different patterns of anti-HSA antibody production by these two populations. Importantly, of the 68 healthy dogs with no known prior HSA exposure sampled by Martin and others, 7 per cent had pre-existing anti-HSA antibodies. HSA is antigenic in both healthy and in critically ill dogs and repeat administration of HSA to previously exposed dogs can lead to severe anaphylactic reactions and should not be performed (Cohn and others 2007, Martin and others 2008). Based on these studies the potential risks of using HSA must be carefully considered before the product is administered. Published reports suggest that HSA solutions do increase serum albumin concentrations and colloid osmotic pressure although this may not be associated with a survival benefit. As such, we suggest that other colloids may offer safer alternatives for provision of oncotic support and HSA solutions should only be administered if other forms of treatment have failed. At present, no veterinary licensed preparations containing human serum albumin are available. Use of these products is therefore subject to the cascade as described by the Veterinary Medicines Regulations 2008 thus requiring veterinary surgeons to ensure clients are made aware of the risks of using such a product. At the Queen Mother Hospital for Animals, we record the use of HSA prominently within patients’ medical records. In addition, in our written communications to referring practitioners, we report the use of these solutions in order to prevent inadvertent repeat HSA administration. If the decision to administer HSA is made, we would strongly encourage readers to diligently and prominently record HSA use in the medical records of their patients. In addition, we also feel it is essential that this information be conveyed to any specialist referral centre to which the patient is transferred. Provision of this information may help prevent potentially fatal acute hypersensitivity reactions.

Presence of Soluble CD26 Peptidase Activity in Human Serum Albumin (FlexBumin): Implications for Cell Freezing and Thawing Protocols during Transplantation

Hematopoietic cells are commonly exposed to human serum albumin (HSA) in the clinical setting and fetal bovine serum (FBS) in the research setting. Serum is utilized as a standard reagent across multiple research disciplines for tissue culture (10–20% FBS). HSA is a common reagent in the clinical setting and is routinely added to hematopoietic cells (0.5–4% HSA) in preparation for cryopreservation. Many labs also wash cryopreserved hematopoietic stem cells with HSA (1–5%) prior to autologous or allogenic transplantation to rid the cells of the DMSO used during cryopreservation. Lastly, HSA is sometimes infused directly into patients in the clinic for such indications as the short-term treatment of nephrotic syndrome. We have generated data suggesting that the peptidase CD26 (dipeptidylpeptidase IV/DPPIV) regulates HSC/HPC function, release of HPC out of the bone marrow (BM) during G-CSF induced mobilization, and engraftment of HSC/HPC into the BM during transplantation. It is within this context that we evaluated the presence of CD26/DPPIV activity in HSA. HSA of 20%, 10%, 2.5%, and 1% were prepared by diluting Flexbumin 25% (Baxter Healthcare) with 0.9% NaCl. FBS (Hyclone) concentrations were prepared likewise. Bovine Serum Albumin Fraction V (BSA) concentrations were prepared weight by volume. CD26/DPPIV activity was assayed utilizing the Gly-Pro-p-nitroanilide substrate in a 96-well format. Standard p-nitroanilide (pNA) curves were created by serially diluting pNA with 5mM Tris-HCl pH 7.5. 4mM Gly-Pro-pNA substrate was then combined with each test solution in 5mM Tris-HCl. Absorbance readings (A405) were then taken at 37° C every 5 minutes for 60 minutes. CD26 inhibition was achieved by treating with Diprotin-A (Ile-Pro-Ile). Prior to measurement of CD26 activity, HSA, BSA, or FBS were combined with Diprotin-A incubated at 37° C for 15 minutes. Final Diprotin-A concentrations during treatment were 5000μM, 500μM, 50μM, 5μM, 0.5μM, 0.05μM, 0.005μM, and 0 μM. Evaluation of CD26 activity in 1%, 2.5%, 10%, and 20% HSA solutions revealed a statistically significant and dose dependent level of CD26 activity within the serum (p<0.05). CD26 activity of 10% HSA was quantitated at 49.62±1.5 pmoles/min and activity of 10% FBS was quantitated at 53.42±1.44 pmoles/min. Evaluation of CD26 activity in 2.5%, 10%, or 20% FBS also showed a quantifiable and dose dependent CD26 activity (p< 0.05). 1%, 2.5%, 10%, and 20% BSA Fraction V had no detectable levels of CD26 activity as compared to saline alone. Furthermore, treatment with the CD26 inhibitor, Diprotin A, actively inhibits CD26 activity ex vivo in a dose dependent manner (p< 0.05). These results suggest that significant levels of CD26 activity are present in HSA (Flexbumin) that is utilized routinely for clinical purposes. Additional evaluations are indicated to test whether this is an industry wide phenomenon across multiple preparation procedures and manufactures. Additionally, these results suggest that the CD26 activity that is present is specific and can be inhibited utilizing CD26 inhibitors. No clinical protocol methodology currently exists for the treatment of HSA in the clinical or research setting. Lastly, these results suggest that the use of HSA that may contain CD26 activity during clinical freezing and thawing procedures of transplant units should be reconsidered, especially if the HSA remains with the unit during infusion. Future studies will be needed to evaluate if the results these pre-clinical experiments necessitate changes to clinical methodologies.

Transmissible spongiform encephalopathy agent clearance by the immunoaffinity and anion‐exchange chromatography steps of the ReFacto® manufacturing process

ReFacto (moroctocog alfa), a recombinant factor VIII approved for the treatment of haemophilia A, is produced by a mammalian cell-culture process that includes therapeutic-grade human serum albumin (HSA) in the cell-culture medium. While to date there have been no cases of transmissible spongiform encephalopathy (TSE) resulting from the clinical use of HSA, Wyeth conducted a study to demonstrate that the ReFacto manufacturing process has significant capacity to remove a TSE agent if it were present as a contaminant in the HSA. The immunoaffinity (8A4 Sepharose) and anion-exchange (Q Sepharose) chromatography steps were evaluated for the clearance of the hamster TSE agent, strain 263K. This Good Laboratory Practice study was performed using appropriately qualified, laboratory-scale chromatography systems. Filtered brain homogenate from TSE-infected hamsters was added to loads of both chromatographic columns, and the concentration of TSE agent in the loads and product pools were determined using a validated western blot quantitation method. Replicate chromatography runs were consistent, as demonstrated by the < or =0.7 log(10) difference observed in TSE agent reduction between each pair of runs. The immunoaffinity and anion-exchanges steps demonstrated 3.8 log reduction and >5.2 log reduction respectively. These data provide a high degree of assurance that in the unlikely event of a TSE contamination of the HSA used in the ReFacto cell-culture process, the purification steps have the potential to remove the infectious agent to extremely low levels, thereby significantly reducing the risk to patients receiving ReFacto.

Synthesis of Pyrophosphate-Containing Compounds that Stimulate V&amp;#947;V&amp;#948;2 T Cells: Application to Cancer Immunotherapy

Human Vgamma2Vdelta2 T cells recognize nonpeptide antigens, such as isoprenoid pyrophosphomonoester intermediates, alkylamine compounds, and bisphosphonate drugs, as well as some tumor cells. Although attempts have been made to derive novel cancer immunotherapies based on the discovery of these unconventional antigens, effective therapies remain to be developed. Here, we synthesized a series of pyrophosphate-containing compounds and examined the chemical requirements for the recognition of pyrophosphomonoester antigens by gammadelta T cells. The structural analysis clearly demonstrated that a proximal methylene moiety plays a crucial role in the stimulatory activity of the antigens. For optimal gammadelta T cell proliferation, we find that the use of human serum albumin was preferred and that pyrophosphomonoesters were superior to nitrogen-containing bisphosphonate compounds. Using these techniques, we have successfully expanded gammadelta T cells from healthy donors as well as from cancer patients using one of the most active compounds, 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP). The resulting expanded gammadelta T cells exhibited potent, cytotoxic activity against a wide variety of tumor cell lines. Even gammadelta T cells from a patient with advanced liver carcinoma efficiently responded to 2M3B1PP and exhibited strong cytotoxic activity against tumor cells. The pretreatment of tumor cells with nonpeptide antigens was essential for efficient cytotoxicity via TCR-gammadelta. The present study suggests a novel strategy for cancer immunotherapy using synthetic small pyrophosphate-containing compounds and nitrogen-containing bisphosphonates.

A Use of Modified Cyclodextrins as a Transporter for a Radiolabeled Tracer NMR Investigation

Cyclodextrins (D. Duchene (ed.): New Trends in Cyclodextrins and Derivatives (1991)) have long been shown to be capable of modifying the water solubility of a number of hydrophobic guests through the formation of inclusion complexes. Among the three natural cyclodextrins (α, β and γ-cyclodextrins containing 6, 7 and 8 d-glucopyranose units, respectively), β-cyclodextrin is by far the most commonly used although it exhibits a weaker solubility in water (and therefore a weaker solubilization power). This specific feature has encouraged the synthesis of derivatives exhibiting an increased solubility in water. Methylated cyclodextrins are amongst the simplest derivatives, and their properties regarding the solubility and the solubilization power for hydrophobic guests are well documented especially concerning Heptakis (2,6-di-Omethyl)-cyclodextrin (DIMEB) and Heptakis (2,3,6-tri-Omethyl)-cyclodextrin (TRIMEB) K. Koizumi et al.:J. Chromatogr. 368, 329–337 (1986). In order to avoid the use of human serum albumin (HSA), this property has been applied here to the solubilization of a very sparingly water-soluble fatty acid derivative (16-iodo-3-methylhexadecanoic acid), which is known to localise in viable myocardial cells, allowing the generation of functional images reflecting the viability of the cardiac tissue through the use of radiolabeled analog (Demaison et al.: J. Nucl-Med. 29, 1230–1236 (1998)). Nuclear magnetic resonance (NMR) was used throughout this study to evidence that the observed solubilization and stabilisation (under conditions required for sterilisation) induced by cyclodextrins are due to the formation of a true inclusion complex and not to non-specific interactions; This technique further allows to derive thermodynamic as well as structural informations for this complex. On one hand, the inclusion complex prevents thermal degradation during sterilisation process compared to HSA. On the other hand, NMR displacement experiments against HSA showed that the complex likely dissociates in vivo.

Recombinant Human Serum Albumin and Hyaluronan Can Replace Blood-Derived Albumin in Embryo Culture Media

Objective: Human preimplantation embryos are routinely cultured in the presence of a macromolecule, typically in the form of patient’s serum or human serum albumin (HSA). Although embryo development can be achieved in the absence of a macromolecule, both embryo development and handling are improved by the presence of a macromolecule. Exposure of an embryo to serum is not an appropriate procedure, whereas the use of HSA is associated with both biological variation and the possibility of disease transmission. Albumin is the most abundant macromolecule in the oviduct, while significant levels of hyaluronan (HA) are present in the uterus. We have therefore investigated the efficacy of recombinant HSA (rec-HSA) and HA (rec-HA) in a mouse embryo culture system.

Human serum albumin enhances DNA transfection by lipoplexes and confers resistance to inhibition by serum

Cationic liposome–DNA complexes (‘lipoplexes’) are used as gene delivery vehicles and may overcome some of the limitations of viral vectors for gene therapy applications. The interaction of highly positively charged lipoplexes with biological macromolecules in blood and tissues is one of the drawbacks of this system. We examined whether coating cationic liposomes with human serum albumin (HSA) could generate complexes that maintained transfection activity. The association of HSA with liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane and dioleoylphosphatidylethanolamine, and subsequent complexation with the plasmid pCMVluc greatly increased luciferase expression in epithelial and lymphocytic cell lines above that obtained with plain lipoplexes. The percentage of cells transfected also increased by an order of magnitude. The zeta potential of the ternary complexes was lower than that of the lipoplexes. Transfection activity by HSA-lipoplexes was not inhibited by up to 30% serum. The combined use of HSA and a pH-sensitive peptide resulted in significant gene expression in human primary macrophages. HSA-lipoplexes mediated significantly higher gene expression than plain lipoplexes or naked DNA in the lungs and spleen of mice. Our results indicate that negatively charged HSA-lipoplexes can facilitate efficient transfection of cultured cells, and that they may overcome some of the problems associated with the use of highly positively charged complexes for gene delivery in vivo.

Preparation and characterization of protein C-loaded PLA nanoparticles

This paper deals with the preparation and the characterization of poly(lactic acid) (PLA) nanoparticles containing protein C, a plasma inhibitor. Nanoparticles were prepared by the double emulsion method (w/o/w), using methylene chloride as an organic solvent and polyvinyl alcohol (PVA) or human serum albumin (HSA) as a surfactant. The influence of experimental constraints such as sonication and organic solvent on protein C activity was evaluated. It appears that a short time of sonication as well as the addition of acetone to methylene chloride (1/1) limited the lost of protein C activity. The study of protein C adsorption on blank PLA nanoparticles gave evidence to hydrophobic interactions between these two entities. The increase in PLA molecular weight on the characteristics of the protein C-loaded nanoparticles led to both a slightly decreased particle size and a lower polydispersity index, whereas the entrapment efficiency of protein C was not affected. The use of HSA as a surfactant allowed the increase in the entrapment efficiency of protein C but prevented its release. Finally, the evaluation of the activity of released protein C clearly illustrates that it was disturbed during the nanoparticle preparation. Thus, the obtained results emphasize the potential of protein C-loaded biodegradable nanoparticles for protein progressive delivery in plasma.

R-061. The use of human serum albumin in sperm preparation for intracytoplasmic sperm injection

R-061. The use of human serum albumin in sperm preparation for intracytoplasmic sperm injection

Prospective randomized study comparing human serum albumin with fetal cord serum as protein supplement in culture medium for in-vitro fertilization

The use of human serum albumin (HSA) instead of fetal cord serum (FCoS) as protein supplement highly simplifies the preparation of culture medium for human in-vitro fertilization (IVF) but whether they are equivalent in sustaining embryo development is still controversial. We performed a prospective randomized study of patients undergoing IVF or intracytoplasmic sperm injection (ICSI) where embryos were cultured in Earle's balanced salt solution containing either 8% (v/v) FCoS or 0.4% (w/v) HSA as protein source. Fertilization rates, morphological embryonic quality and pregnancy rates were compared. A total of 2189 oocytes from 210 cycles were cultured in medium supplemented with HSA in patient group 1 and 2109 oocytes from 203 cycles in medium supplemented with FCoS in patient group 2. The fertilization rate, defined as the presence of two nuclei, for microinjected oocytes was similar in both patient groups (77.4 and 76.7%, respectively). The fertilization rate for inseminated oocyte-cumulus complexes was significantly higher in the HSA group than in the FCoS group (62.9 versus 53.8%, P < 0.025). The embryonic quality was significantly better after culture in medium supplemented with HSA than with FCoS (13.7 versus 9.9% morphologically excellent embryos, P < 0.001). Implantation rates per transferred embryo were not significantly different (22.5 versus 18.2%), but there was a significantly higher pregnancy rate per embryo transfer in the HSA group (45.7 versus 35.9%, P < 0.05, respectively). Non-evolutive pregnancy rates were significantly different (27.4 and 16.7%). Our data demonstrate that the use of human serum albumin as a protein supplement for culture medium in human IVF programmes is associated with improved embryonic quality and significantly higher pregnancy rates. For this reason as well as the additional benefits of being virus-free and being purified, HSA is preferable to FCoS for the preparation of culture media in human IVF.

Potency measurement of factor VIII in recombinant concentrates.

In preliminary studies on the stability of recombinant factor VIII (rFVIII) concentrates post reconstitution, a rise in potency to 200% of labelled values was observed in concentrates stored at 22 °C over 24 h. This was observed in potency estimates by the one-stage clotting, but not the two-stage clotting or chromogenic assays, and was not observed for intermediate-purity product derived from plasma (IPVIII). Use of human serum albumin (HSA), rather than the usual bovine material (BSA), to dilute product for the stability study abolished the rise in potency. Incorporating purified von Willebrand Factor (VWF) in the diluent buffer abolished the rise observed in BSA. A similar rise was observed upon incubating rFVIII in the presence of 10(-4) u of thrombin per mL in HSA buffer. Potency estimates using the HSA in the dilution buffer resulted in severe underpotency in relation to the label claim when using the two-stage clotting and the chromogenic assays, but not the one-stage clotting assay. Predilution in severe haemophilic plasma restored potency levels to those claimed. We conclude that (i) commercial preparations of BSA may be unsuitable for inclusion in buffers for rFVIII studies; (ii) FVIII in rFVIII concentrates is exquisitely sensitive to activation by thrombin, presumably as a result of the lack of VWF; (iii) accurate potency estimation in the two-stage assay systems requires VWF in the assay system.

A case of severe ovarian hyperstimulation in a healthy oocyte donor

To report an unexpected case of severe ovarian hyperstimulation syndrome (OHSS) and to compare E2 levels and number of follicles to other oocyte donors. Private assisted reproduction technology center. Healthy oocyte donors with normal menstrual cyclicity. Prophylactic and therapeutic use of human serum albumin infusions. The clinical development of signs and symptoms of severe OHSS. More than 60% of other oocyte donors had higher E2 levels and 12% had higher number of follicles without associated OHSS. The risk of developing severe OHSS cannot be predicted accurately to be low even in the absence of "risk factors."

Factors affecting the fine specificity and sensitivity of serum antiganglioside antibodies in ELISA

The major problem associated with ELISA of serum antiganglioside antibodies is the high background values (absorbancy of sera added to wells without ganglioside), which interfere with the accurate assessment of the fine specificity and sensitivity of these antibodies. This investigation identifies factors elevating the background values and/or decreasing the fine specificity, and describes strategies to minimize their influence. Using sera of neuropathy and melanoma patients, we found that highest background values were observed with the polystyrene ‘tissue culture’ microtiter plates; of the various ‘non-tissue culture’ microtiter plates tested, the lowest background values (> 0.060) were observed with Costar-3590 (H), Immunolon-3, Immunolon-1, Falcon-3915 (in increasing order). Background artifact of polystyrene microtest plates was significantly reduced by gamma irradiation (at 40 kRad) and/or use of detergent Tween-20 (0.1%) in the washing step. Even after controlling the background values, the fine specificity, namely, the ability of the antibody to distinguish between the target epitope of an antigen and epitopes of related antigens (when moles of antigen/well is constant) varied with different microtiter plates. Using sera with high affinity and specificity for G M2, G D3 or G M3, we observed that Immunolon-1, Immunolon-3 and particularly Falcon-3915 were superior for assessing the abilities of the antibodies to distinguish closely related epitopes found on other gangliosides. The reactivity of antiganglioside antibodies was more consistent after detergent treatment. The reactivity of antibodies to G D3 is significantly enhanced after treatment with Tween-20, but that of antibodies reacting to G M1 and G M2 is reduced. Fine specificity of the antiglycolipid antibodies was resolved better by coating glycolipids in mol/well rather than by weight/well. Based on these results, a protocol for a sensitive and reproducible ELISA for serum antiganglioside antibodies is recommended. The protocol takes into consideration the suitability of polystyrene plates, coating based on the number of molecules, pertinency of the solvent for coating, use of human serum albumin for blocking, dilution and washing steps and use of 0.1% Tween-20 to further minimize the background absorbancy.

Albumin. 1. Background and use.

THE INCREASING use of human serum albumin, which recently has threatened to stress the usual annual supply and to add considerably to health care costs, prompted a cooperative study in early 1975 between the federal government and the private sector. This led to the development of three projects: (1) A workshop was convened in Bethesda, Md, under joint sponsorship of the Division of Blood Diseases and Resources, National Heart and Lung Institute, and the Division of Blood and Blood Products, Bureau of Biologics. This meeting in February 1975 defined the present state of knowledge of the structure, synthesis, and function of albumin and reviewed in depth its current clinical use. The proceedings were subsequently published. 1 (2) Recommendations were formulated on areas needing further basic or applied research. (3) Guidelines for the appropriate clinical use of albumin were proposed and are currently being circularized to concerned persons for comment. Although

Albumin

THE INCREASING use of human serum albumin, which recently has threatened to stress the usual annual supply and to add considerably to health care costs, prompted a cooperative study in early 1975 between the federal government and the private sector. This led to the development of three projects: (1) A workshop was convened in Bethesda, Md, under joint sponsorship of the Division of Blood Diseases and Resources, National Heart and Lung Institute, and the Division of Blood and Blood Products, Bureau of Biologics. This meeting in February 1975 defined the present state of knowledge of the structure, synthesis, and function of albumin and reviewed in depth its current clinical use. The proceedings were subsequently published. 1 (2) Recommendations were formulated on areas needing further basic or applied research. (3) Guidelines for the appropriate clinical use of albumin were proposed and are currently being circularized to concerned persons for comment. Although

99m Tc-labelled serum albumin in cardiac output and blood volume studies.

The use of human serum albumin labelled with iodine-131 (RIHSA) as the radioactive indicator in the measurement of cardiac output by the external counting technique is not ideal since the emission characteristics, physical half-life, and physiological fate restrict the amount that can be administered. A more suitable material, especially because of the short physical half-life of the isotope involved, is albumin labelled with technetium-99m. This communication describes a simple three-stage process for the preparation of such a complex which was then tested in subjects with no impairment of cardiac performance for its suitability in assays of cardiac output by the external counting method. Values were within the same range as those obtained with RIHSA in comparable subjects. Blood volumes estimated with ^99mTc-albumin were within the limits of physiological variation of values derived with RIHSA in the same subjects. Serial blood samplings and urine collection during a period of 24 hours after administration showed that the preparation was lost continuously from the circulation at a more rapid rate than RIHSA. The greatest loss was in the first few hours when most of the urinary excretion of the isotope occurred. Comparison of the present data with published results using an alternative preparation suggests that our product may be more stable.