Recombinant Human Albumin Research Articles

Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis.

Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable and therefore represent the majority of the total protein in the media samples. There are no data in the literature on what non-declared proteins are present in unconditioned (fresh media in which no embryos have been cultured) commercial embryo media. The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analyzed from each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analyzed. For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. Using advanced mass spectrometry and high confidence criteria for accepting proteins (P < 0.01), a total of 110 proteins other than HSA were identified. The average HSA content was found to be 94% (92-97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins. For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions. The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analyzing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in unconditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring. The study was supported by the Danish Agency for Science, Technology and Innovation. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from Merck Sharp & Dohme Corp and Ferring. The authors declare no conflicts of interest.

Miscibility as a Factor for Component Crystallization in Multisolute Frozen Solutions

The relationship between the miscibility of formulation ingredients and their crystallization during the freezing segment of the lyophilization process was studied. The thermal properties of frozen solutions containing myo-inositol and cosolutes were obtained by performing heating scans from -70 °C before and after heat treatment at -20 °C to -5 °C. Addition of dextran 40,000 reduced and prevented crystallization of myo-inositol. In the first scan, some frozen solutions containing an inositol-rich mixture with dextran showed single broad transitions (Tg's: transition temperatures of maximally freeze-concentrated solutes) that indicated incomplete mixing of the concentrated amorphous solutes. Heat treatment of these frozen solutions induced separation of the solutes into inositol-dominant and solute mixture phases (Tg' splitting) following crystallization of myo-inositol (Tg' shifting). The crystal growth involved myo-inositol molecules in the solute mixture phase. The amorphous-amorphous phase separation and resulting loss of the heteromolecular interaction in the freeze-concentrated inositol-dominant phase should allow ordered assembly of the solute molecules required for nucleation. Some dextran-rich and intermediate concentration ratio frozen solutions retained single Tg's of the amorphous solute mixture, both before and after heat treatments. The relevance of solute miscibility on the crystallization of myo-inositol was also indicated in the systems containing glucose or recombinant human albumin.

Chemically defined generation of human cardiomyocytes

Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy, employing a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Along with small molecule-based differentiation induction, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell, and was effective in 11 hiPSC lines tested. This is the first fully chemically defined platform for cardiac specification of hiPSCs, and allows the elucidation of cardiomyocyte macromolecular and metabolic requirements whilst providing a minimally complex system for the study of maturation and subtype specification.

Studying the morphology of lyophilized protein solids using X-ray micro-CT: effect of post-freeze annealing and controlled nucleation.

The objective of this study was to determine how different techniques used during the freezing step of lyophilization affect morphology of the dried protein solids. Aqueous solutions containing recombinant human albumin, trehalose, and sodium phosphate buffer were dried after their freezing by shelf-ramp cooling, immersion in liquid nitrogen, or controlled ice nucleation. Some shelf-frozen solutions were heat treated (annealed) before the vacuum drying. We used three-dimensional (3D) X-ray micro-computed tomography (micro-CT) and scanning electron microscopy (SEM) to study the morphology of solids. The X-ray micro-CT images of the lyophilized microporous solids showed traces of varied size and structure ice crystals that were comparable to corresponding SEM images. A post-freeze heat treatment and a controlled nucleation both induced larger ice crystal ghosts in the solids. The variations in the structure of walls surrounding ice crystals, formed by the different freezing procedures, should affect the water vapor transition during the primary and secondary drying. Some solids also showed higher-density layer in the upper surface. Overall, the simple sample preparation procedures and the ample morphological information make the X-ray micro-CT appropriate for analyzing lyophilized pharmaceuticals.

Comparative studies on the heterogeneity of plasma-derived and recombinant human albumins in laboratory use

We investigated the thiol-redox state, and the relationship between structural characteristics, such as thermal stability, and functional properties, such as cell growth activity, of commercial plasma-derived (pHSA) and recombinant human serum albumin (rHSA) products. In this study, 5 pHSA products (A1653, A9511, A1887, A8763, and A3782) and 2 rHSA products (A9731 and A9986) were obtained from Sigma-Aldrich. Among them, three kinds of HSA products [A1653 (an initial fractionation product), A3782 (a final purified product), and A9731 (recombinant albumin expressed in rice)] were selected for experiments on the thermal stabilities, analyzed by thermal denaturation curves, and cell growth activities of U937 and THP-1 cell lines using the WST-1 reagent. The secondary and tertiary structures of HSA products were similar, whereas a marked difference was observed in their thermal stabilities. The degree of thermal stability of the three representative products was in the order of A9731 (rHSA)>A1653 (pHSA)>A3782 (pHSA), as was the degree of cell growth activity of these products. One possible explanation for the present results is that albumin-bound fatty acids may have influenced the thermal stabilities and cell growth activities of U937 and THP-1 cells.

Biodistribution of the recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in rats

The recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) is undergoing clinical trials for prophylaxis and on-demand treatment of haemophilia B patients. The aim of this study was to investigate the pharmacokinetics, whole-body and knee joint distribution of rIX-FP following intravenous administration to rats, compared with a marketed, non-fused rFIX and recombinant human albumin. [(3)H]-rIX-FP, [(3)H]-rFIX or [(3)H]-albumin were administered to rats followed by quantitative whole-body autoradiography over 24 or 240 hours, and the tissue distribution as well as elimination of radioactivity were measured. Elimination of all radioactivity derived from the three proteins was shown to occur primarily via the urine. The tissue distribution of [(3)H]-rIX-FP and [(3)H]-rFIX (but not of [(3)H]-albumin) was comparable, both penetrating predominantly into bone, and well-perfused tissues, suggesting that the rIX moiety determines the distribution pattern of rIX-FP, while the albumin moity is responsible for the prolonged plasma and tissue retention. Detailed knee-joint analysis indicated rapid presence of [(3)H]-rIX-FP and [(3)H]-rFIX in synovial and mineralised bone tissue, mostly localised to the zone of calcified cartilage. Longest retention times were observed in the bone marrow and the endosteum of long bones. Intriguingly, [(3)H]-rIX-FP- and [(3)H]-albumin-derived radioactive signals were detectable up to 240 hours, while [(3)H]-rFIX-derived radioactivity rapidly declined after 1hour post-dosing correlating to the extended plasma half-life of [(3)H]-rIX-FP. The prolonged plasma and tissue retention of rIX-FP achieved by albumin fusion may allow a reduction in dosing frequency leading to increased therapeutic compliance and convenience.

Factor XIIa inhibition by Infestin-4: in vitro mode of action and in vivo antithrombotic benefit

Coagulation factor XII (FXII) plays a central role in initiating the intrinsic cascade of blood coagulation. Purified recombinant Human Albumin-tagged Infestin-4 (rHA-Infestin-4) is a recently described FXIIa inhibitor that displayed strong anticoagulant activity without compromising haemostasis in several animal models. We pursued detailed in vitro characterisation of rHA-Infestin-4 and demonstrated that it is a competitive inhibitor of FXIIa with slow on and off rate constants for binding (kon=5x10⁵ M⁻¹s⁻¹, koff=6x10⁻⁴ s⁻¹), it can block FXIIa activation of its physiological substrates (plasma prekallikrein and FXI), and it can inhibit ellagic acid-triggered thrombin generation in plasma. Potency and selectivity profiling in enzyme assays suggest that rHA-Infestin-4 is indeed highly potent on FXIIa (IC50=0.3 ± 0.06, 1.5 ± 0.06, 1.2 ± 0.09 nM, for human, rat, and rabbit FXIIa, respectively) with at least >100-fold selectivity against factors IIa, Xa, IXa, XIa, VIIa, and plasma kallikrein in all three species. rHA-Infestin-4 dose-dependently and markedly reduced clot weight in the arteriovenous shunt thrombosis model in rats and rabbits, accompanied with minimal increase in cuticle bleeding times in either species. rHA-Infestin-4 treatment at 5 mg/kg in rabbit resulted in a 13% reduction in ex vivo FXa activity, demonstrating a modest off-target effect. In summary, our findings confirmed and extended previous reports that inhibition of FXIIa by rHA-Infestin-4 can produce strong antithrombotic efficacy while preserving haemostasis. Our comprehensive selectivity profiling, mode of action, and kinetic studies of rHA-Infestin-4 reveal limitations of this molecule and offer new perspectives on any potential effort of discovering novel FXIIa inhibitors.

Efficacy, PK and Safety Results of Clinical Study Of Recombinant Fusion Protein Linking Coagulation Factor IX With Albumin (rIX-FP) In Previously Treated Patients With Hemophilia B

BackgroundThe standard of care for patients with severe hemophilia B is replacement treatment with Factor IX (FIX) 2-3 times a week. A fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin (rIX-FP) was developed with the aim to extend the half-life of FIX. In the completed Phase I pharmacokinetic study, the mean half-life of rIX-FP was found to be over 5 times longer than the subjects' previous FIX. Thus, rIX-FP has potential to prevent bleedings for longer periods, allowing reduction in the frequency of injections compared to standard FIX and to reduce the number of injections required to treat a single bleed. AimsThis was a Phase I/II open-label, multicenter study of rIX-FP in previously treated patients 12-65 years of age with severe hemophilia B (FIX ≤ 2%). The study evaluated the safety and efficacy of rIX-FP, including prevention of bleeding episodes during weekly prophylaxis of rIX-FP. MethodsAfter completion of a 14-day rIX-FP pharmacokinetic assessment, 13 subjects in the prophylaxis arm received weekly prophylaxis of rIX-FP for approximately 11 months, and 4 subjects in the on-demand arm received rIX-FP upon occurrence of bleeding events. The treatment doses were initially selected based upon the pharmacokinetic profile of rIX-FP and subject's bleeding phenotype, and doses could be adjusted at the Investigator's discretion. ResultsSeventeen subjects were enrolled from hemophilia treatment centers in Israel and Bulgaria; the mean age was 26 years (range 13 to 46 years). Following a single injection of 25 IU/kg rIX-FP (n=13), the mean FIX activity level was 3.75% and 2.67% above baseline at Day 7 and Day 14, respectively, and the mean half-life of rIX-FP was 95 hours (comparable to the previously reported Phase I data).Over the 11 month treatment period, rIX-FP demonstrated a good safety profile with a total of over 700 EDs. The treatment was well tolerated and no FIX inhibitor formation was observed. There was no AE considered to be related to treatment with rIX-FP. No subject was withdrawn from the study due to safety concerns or lack of hemostatic efficacy.All 13 prophylaxis subjects were successfully maintained on a weekly routine regimen of rIX-FP for the entire duration of the study, with annualized spontaneous bleeding rates of 1.255 and 1.134 (mean and median respectively). Furthermore, three prophylaxis subjects who received only on-demand treatment prior to study entry had greater than 80% reduction in the annualized bleeding rate compared to their annualized bleeding rate prior to study entry. All bleeding events were treated successfully with ≤ 2 injections of rIX-FP, with approximately 90% of bleeds treated with a single injection of rIX-FP. The mean weekly consumption of rIX-FP was reduced markedly compared to the subjects' weekly consumption of the previous FIX product. ConclusionThis Phase I/II study demonstrated the clinical efficacy of rIX-FP for once weekly routine prophylaxis to prevent spontaneous bleeding episodes and for the treatment of bleeding episodes. In addition, rIX-FP showed an excellent safety and an improved PK profile over currently marketed factor IX products. Disclosures:Lubetsky:CSL Behring: Investigator for CSL clinical trial of rIX-FP Other. Lissitchkov:CSL Behring: Investigator for CSL Behring clinical trial of rIX-FP Other. Santagostino:CSL Behring: Honoraria, Investigator for CSL Behring clinical trial of rIX-FP Other, Research Funding, Speakers Bureau. Jotov:CSL Behring: sub-investigator for CSL Begring trial of rIX-FP Other. Barazani-Brutman:CSL Behring: study coordinator for CSL Behring rIX-FP trials Other. Voigt:CSL Behring: Employment. Moises:CSL Behring: Employment. Jacobs:CSL Behring: Employment. Martinowitz:CSL Behring: Honoraria, Investigator for CSL rIX-FP trials Other, Speakers Bureau.

Elective single cryopreserved embryo transfer of blastocysts cultured in defined media with recombinant human albumin: a randomized study

Human serum albumin (HSA), a commonly used protein source for ART, may cause biological variation and disease transmission. Recombinant human albumin (recHA) could reduce these risks; however, existing literature on the use of recHA is limited. Here, we evaluated the efficacy of chemically defined media containing recHA for blastocyst culture of human embryos. Prospective randomized study. This study included patients from our center undergoing elective cryopreservation of all embryos. Ninety-five patients received IVF/ICSI treatment between July 2012 and October 2012, which resulted in ≥6 2PN embryos 18 h after insemination. They were randomized using sealed envelopes into 2 groups for embryo culture in G1/G2 media (Vitrolife) containing either 5 mg/mL HSA (standard commercial media, group A) or 0.5 mg/mL recHA (group B). We used the same recHA concentration used in our previous study with bovine embryos (Murakami et al., 2012). After culture, only good-quality embryos were vitrified by day 6. We evaluated the clinical data for patients with vitrified blastocysts in groups A (n = 35) and B (n = 33). Patient age (years) was similar for groups A and B (35.5 [3.3] vs. 35.7 [3.7], P = 0.857). A total of 120 and 70 blastocysts were vitrified in groups A and B, respectively. By the end of March 2013, groups A and B underwent a total of 51 and 36 cycles of single ET with the vitrified-warmed blastocysts, respectively. All vitrified blastocysts survived after warming. Clinical pregnancy rate/ET was 24/51 (47.1%) for group A and 25/36 (69.4%) for group B (P = 0.049). Ongoing pregnancy rate/ET was 22/51 (43.1%) for group A and 19/36 (52.8%) for group B (P = 0.632). Culture of human embryos in the defined media with recHA can yield good-quality blastocysts, resulting in a high pregnancy rate after single cryopreserved ET. These results could help develop serum-free defined ART systems. We are currently evaluating the perinatal outcomes after ET with more participants.

Impact of heat treatment on miscibility of proteins and disaccharides in frozen solutions

The purpose of this study was to elucidate the effect of heat treatment (annealing) on the miscibility of concentrated protein and disaccharide mixtures in the freezing segment of lyophilization. Frozen solutions containing a protein (e.g., recombinant human albumin, chicken egg lysozyme, bovine plasma immunoglobulin G, or a humanized IgG1k monoclonal antibody) and a non-reducing disaccharide (e.g., sucrose or trehalose) showed single thermal transitions of the solute mixtures (glass transition temperature of maximally freeze-concentrated solutes: Tg′) in their first heating scans. Heat treatment (e.g., −5°C, 30min) of some disaccharide-rich mixture frozen solutions at temperatures far above their Tg′ induced two-step Tg′ transitions in the subsequent scans, suggesting the separation of the solutes into concentrated protein–disaccharide mixture phase and disaccharide phase. Other frozen solutions showed a single transition of the concentrated solute mixture both before and after heat treatment. The apparent effects of the heat treatment temperature and time on the changes in thermal properties suggest molecular reordering of the concentrated solutes from a kinetically fixed mixture state to a more thermodynamically favorable state as a result of increased mobility. The implications of these phenomena on the quality of protein formulations are discussed.

Endocytosis of Albumin by Podocytes Elicits an Inflammatory Response and Induces Apoptotic Cell Death

The presence of albuminuria is strongly associated with progression of chronic kidney disease. While albuminuria has been shown to injure renal proximal tubular cells, the effects of albumin on podocytes have been less well studied. We have addressed the hypothesis that exposure of podocytes to albumin initiates an injury response. We studied transformed human-urine derived podocytes-like epithelial cells (HUPECS, or podocytes). Upon differentiation, these cells retain certain characteristics of differentiated podocytes, including expression of synaptopodin, CD2AP, and nestin. We exposed podocytes to recombinant human albumin, which lacks lipids and proteins that bind serum albumin; this reagent allowed a direct examination of the effects of albumin. Podocytes endocytosed fluoresceinated albumin and this process was inhibited at 4°C, suggesting an energy-dependent process. Exposure to albumin at concentrations of 5 and 10 mg/ml was associated with increased cell death in a dose-dependent manner. The mechanism of cell death may involve apoptosis, as caspase 3/7 were activated and the pan-caspase inhibitor z-VAD reduced cell death. Albumin exposure also increased nuclear factor (NF)-κB activation and increased transcription and release of interleukin (IL-) 1β, tumor necrosis factor (TNF), and IL-6. We extended these findings to an in vivo model. Glomeruli isolated from mice with nephrotic syndrome also had increased expression of IL-1β and TNF RNA. These data suggest that while podocyte injury begets albuminuria, albumin in the glomerular ultrafiltrate may also beget podocyte injury. Thus, an additional mechanism by which anti-proteinuric therapies are beneficial in the treatment of glomerular diseases may be a reduction in injury to the podocyte by albumin.

Safety of a 2-dose Regimen of a Combined Measles, Mumps, Rubella and Varicella Live Vaccine Manufactured With Recombinant Human Albumin

ProQuad, a vaccine containing antigens from M-M-RVAXPRO (measles, mumps and rubella vaccine) and VARIVAX (varicella vaccine), is indicated for simultaneous vaccination against measles, mumps, rubella and varicella (MMRV) in individuals from 12 months of age. To eliminate blood-derived products of human origin from the manufacturing process of the MMRV vaccine, recombinant human albumin was selected as a replacement for human serum albumin. This open-label, multicenter clinical trial (clinicaltrials.gov identifier NCT00560755) was designed to describe the safety profile of a 2-dose schedule of the MMRV vaccine at a 1-month interval in healthy children aged 12-22 months. In total, 3388 children received at least 1 dose of the MMRV vaccine. Overall, 3376 (99.65%) children were included in the post-dose 1 safety analysis and 3342 (98.64%) in the post-dose 2 safety analysis. After doses 1 and 2, the frequencies of children experiencing solicited injection-site reactions (post-dose 1: erythema 14.31%; swelling 5.57% and pain 10.31%; post-dose 2: erythema 30.46%; swelling 13.23% and pain 11.49%), rashes of interest (post-dose 1: 11.4%; post-dose 2: 2.78%), vaccine-related nonserious systemic adverse events (post-dose 1: 34.86%; post-dose 2: 13.4%) and temperature ≥39.4 °C (post-dose 1: 25.24%; post-dose 2: 12.06%) were consistent with those observed in previous studies of the MMRV vaccine manufactured with human serum albumin. Neither serious allergic-type adverse events nor anaphylactic reactions were reported. The results confirm the good safety profiles of MMRV and of measles, mumps and rubella vaccines manufactured with recombinant human albumin.

Quality enhancement of bovine blastocysts using defined culture media supplemented with recombinant human albumin, with or without myo-inositol

Human serum albumin (HSA), a commonly used protein source for ART, may cause biological variation and disease transmission. Using recombinant human albumin (recHA) can reduce these risks, but the extant literature is limited. Here, using bovine embryo models, we evaluated the efficacy of adding myo-inositol (MI) shown to have embryotrophic properties to defined embryo culture media containing recHA.

Alanine and Glycine During the Cleavage Stages Are Detrimental to IVM/IVF Mouse Embryo Development.

Traditionally, amino acids are added to embryo culture media in a premixed formulation developed for somatic cells; MEM essential (EAA) and non-essential (NEAA) amino acids. Previous work has suggested that essential amino acids are inhibitory when present in the first step of embryo culture, and thus are generally absent. However, both EAA and NEAA are present in oviductal fluid. Our objective was to evaluate the effect of lower concentrations of EAA, as well as higher, physiological concentrations of alanine (0.5 mM, ALA) and glycine (2.0 mM, GLY), two of the most abundant NEAA in oviductal fluid, on mouse embryo development in vitro. Zygotes were produced following 18 hours in vitro maturation in a defined, in house maturation medium and 6 hours in vitro fertilization in HTF, using B6D2F1 gametes. All defined media contained 2.5 mg/mL recombinant human albumin (G-MM Recombumin, Vitrolife, Göteborg, Sweden). Embryo cleavage was recorded 22 hours post insemination, and blastocyst development examined on day 4 (96 hours) and day 5 (114 hours) of culture. In Experiment 1 (n=597), 4 treatments were examined: 1) Control (G1 48 hours, G2 66 hours; Vitrolife); 2) G1 with 0.25X EAA, ALA and GLY followed by G2; 3) G1 followed by G2 with ALA and GLY; and 4) G1 with 0.25X EAA, ALA and GLY followed by G2 with ALA and GLY. Neither embryo cleavage nor day 4 blastocyst development (control, 68.4 ± 3.8; G1+EAA+ALA+GLY/G2, 62.9 ± 4.1; G1/G2+ALA+GLY, 72.5 ± 3.6; G1+EAA+ALA+GLY/G2+ALA+GLY, 61.1 ± 4.0) was significantly affected by treatment. Inclusion of ALA, GLY and EAA in G1 decreased day 4 hatching blastocysts (control, 20.4 ± 3.3; G1+EAA+ALA+GLY/G2, 4.9 ± 1.8; G1/G2+ALA+GLY, 22.9 ± 3.4; G1+EAA+ALA+GLY/G2+ALA+GLY, 34.2 ± 3.9; p<0.01), and tended to decrease day 5 hatching blastocysts (control, 52.0 ± 4.1; G1+EAA+ALA+GLY/G2, 38.5± 4.1; G1/G2+ALA+GLY, 51.2 ± 4.1; G1+EAA+ALA+GLY/G2+ALA+GLY, 45.6 ± 4.1; p<0.07). In Experiment 2 (n=911), we further examined specific AA in step 1 of culture (0 to 48 hours) using a defined in house culture medium: 1) Control (1X NEAA, 0.5 mM alanyl-glutamine, 0.1 mM taurine); 2) 0.5x EAA; 3) ALA and GLY; 4) Methionine (0.05 mM, METH). All embryos were then cultured in defined second step medium (48 to 114 hours, also prepared in house) with ALA and GLY. When present during the first 48 hours of culture, ALA and GLY decreased day 4 hatching blastocysts (control, 39.4 ± 3.3; EAA, 48.9 ± 3.3; ALA+GLY, 32.2 ± 3.1; METH, 48.9 ± 3.3:p=0.03), although embryonic cleavage, blastocyst development (control, 72.1 ± 3.0; EAA, 76.7 ± 2.8; ALA+GLY, 66.5 ± 3.1; METH, 72.7 ± 2.9), day 5 hatching blastocysts, total blastocyst cell number (control, 94.5 ± 7.3; EAA, 83.6 ± 5.0; ALA+GLY, 79.3 ± 5.5; METH, 82.5 ± 5.4) or percentage ICM of total cells were not affected. Addition of 0.5X EAA or methionine alone to the first step of culture did not significantly improve development; neither was it inhibitory. In summary, exposure to physiological concentrations of alanine (0.5 mM) and glycine (2.0 mM) during the early cleavage stages of in vitro embryo culture slows development and inhibits the onset of hatching in mouse IVM/IVF embryos.

Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media

Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum.

Alterations in Recovery from Spinal Cord Injury in Rats Treated with Recombinant Human Bone Morphogenetic Protein-2 for Posterolateral Arthrodesis

Treatment of trauma-related spinal instability with use of recombinant human bone morphogenetic protein-2 (rhBMP-2) may appear as a viable option, but little is known of the direct effects of rhBMP-2 on the injured spinal cord. In the current study, we investigated the acute and long-term effects of using rhBMP-2 in the posterolateral spine at the level of a spinal cord injury in rats. Fifty-two rats underwent a T10 dorsal hemisection and were assigned to one of two groups: the vehicle control group (twenty-four rats) or the rhBMP-2 group (twenty-four rats). Within each group, animals were further subdivided according to the follow-up period: one week and six weeks after the lesion. For the acute phase, an additional group of four rats received recombinant human albumin, to account for the cross-species inflammatory response. Postoperatively, locomotor function was assessed on a weekly basis with use of an open field scale and digital footprint analysis. After the animals were killed, they were perfused and the spinal cords analyzed for inflammatory markers, gliosis, and extracellular matrix proteins with use of immunohistochemistry. At one week, there was a significant increase in reactive astrocyte, macrophage-microglia, and fibroblast immunoreactivity around the lesion in the rhBMP-2-treated rats relative to controls. Additionally, there was increased staining for chondroitin sulfate proteoglycans. Similar intergroup morphologic differences persisted at six weeks. Functionally, in the acute phase, rhBMP-2-treated animals demonstrated more open field and fine motor control deficits relative to the controls. By six weeks, both groups had equivalent functional scores, but those treated with rhBMP-2 retained significantly greater paw angle changes than the control animals. Our findings indicate that in a rat model, rhBMP-2 use in the vicinity of a penetrating spinal cord injury triggers detrimental changes in the morphology of the spinal cord lesion and alters functional recovery.

Issues arising during the treatment of Jehovah's Witnesses by in vitro fertilisation

Four Jehovah's Witness couples were treated between 2000 and 2009 using in vitro fertilization or intracytoplasmic sperm injection. A review of the issues encountered during their treatment, and the options chosen, was made in an attempt to provide effective treatment without compromising the moral views of the individuals concerned. Considerations specific to the Jehovah's Witness faith were identified that required departure from the standard operating procedures used in the clinic, and the development of modified protocols prior to treatment is recommended to minimize the stress and anxiety of patients and staff alike. Issues raised included the collection of semen by masturbation, the use of donor gametes, the number of oocytes inseminated, the discard of poor quality embryos conventionally thought unsuitable for transfer or embryo cryopreservation. A common request was the avoidance of blood products as a culture medium supplement. The use of recombinant human albumin circumvented this, although a recent shift by the Watchtower Bible and Tract Society of Pennsylvania in the interpretation of the Bible opens the possibility of using blood fractions if not whole blood, and so the use of standard culture medium containing human serum albumin is now acceptable to some Jehovah's Witnesses.

Modified Surface Coatings and their Effect on Drug Adsorption within the Extracorporeal Life Support Circuit

A recently completed study quantified the percent of fentanyl or morphine sulfate lost to uncoated polyvinylchloride (PVC) tubing or to one of two hollow fiber oxygenators within the extracorporeal life support (ECLS) circuit. The results demonstrated the majority of drug loss was due to adsorption by the PVC tubing. The purpose of this study was to determine if a tubing coating process affects fentanyl or morphine Sulfate adsorption. The goal was to quantify fentanyl or morphine sulfate lost due to adhesion within surface modified tubing. The following surface modifications were studied: 1) Maquet Safeline® (synthetic immobilized albumin); 2) Maquet Softline® (a heparin free biopassive polymer); 3) Maquet Bioline® (recombinant human albumin + heparin) (Maquet Cardiopulmonary AG, Hirrlingen, Germany); 4) Terumo X Coating™ (poly2methoxylacrylate)) (Terumo Cardiovascular Systems Corporation, Ann Arbor, MI); 5) Medtronic Carmeda® (covalently bonded heparin); and 6) Medtronic Trillium® (covalently bonded heparin) (Medtronic, Minneapolis, MN). A total of 36 individual circuits were built from the above six available modified surface coatings, for a total of six individual circuits of each coating type. Blood samples were drawn at 5 minutes, 120 minutes, and 360 minutes followed by High-Performance Liquid Chromatography to determine available circulating levels of either fentanyl or morphine sulfate. Fentanyl concentrations decreased to an average final available concentration of 35% (±5%) within the 18 circuits. Morphine sulfate however, decreased to a final available concentration of 57% (±1%) in all Maquet tubing and the Medtronic Trillium tubing, while it decreased to a final concentration of 35% (±1%) in the Medtronic Carmeda coated tubing and in the Terumo X Coating tubing. Biocompatible ECLS circuit surface coatings affected drug-adsorption and availability. Further evaluation is necessary to understand the adsorptive loss of other drugs administered to our patients while on modified surface coated ECLS circuits.

Albinterferon Alfa-2b Was Not Inferior to Pegylated Interferon-α in a Randomized Trial of Patients With Chronic Hepatitis C Virus Genotype 2 or 3

A phase 3 active-controlled study was conducted to assess the efficacy/safety of albinterferon alfa-2b (albIFN), a novel, long-acting, genetic fusion polypeptide of recombinant human albumin and interferon alfa-2b, in patients with chronic hepatitis C virus (HCV) genotype 2/3. In all, 933 patients were randomized to open-label subcutaneous treatment with pegylated interferon-alfa-2a (Peg-IFNalfa-2a) 180 μg/wk, or albIFN 900 or 1200 μg every 2 weeks for 24 weeks, each administered with oral ribavirin 800 mg/day. The primary end point of the study was sustained virologic response (SVR) (HCV-RNA level, <15 IU/mL at week 48). During the study, the data monitoring committee recommended dose modification for all patients receiving albIFN 1200 μg to 900 μg, impacting 38% of this treatment arm. By intention-to-treat analysis, SVR rates were 84.8% (95% confidence interval, 80.4%-88.6%), 79.8% (95% confidence interval, 74.9%-84.1%), and 80.0% (95% confidence interval, 75.1%-84.3%) with Peg-IFNalfa-2a, and albIFN 900 and 1200 μg, respectively. The primary hypothesis of noninferiority of SVR was established for albIFN 900 μg (P = .009) and 1200 μg (P = .006). Independent positive predictors of SVR by multivariate regression analysis were pretreatment HCV-RNA level less than 400,000 IU/mL, age younger than 45 years, body mass index less than 30 kg/m(2), genotype 2, normal γ-glutamyl transpeptidase and increased alanine aminotransferase levels at baseline, fibrosis stage F0-F2, no steatosis, and Asian geographic region (Peg-IFNalfa-2a only). The 3 treatment groups showed similar rates of serious (7%-8%) and severe (13%-16%) adverse events, and discontinuations owing to adverse events (3.6%-5.5%). Albinterferon alfa-2b 900 μg every 2 weeks provides an alternative efficacious treatment option in patients with chronic HCV genotype 2 or 3.

The production, characterisation and enhanced pharmacokinetics of scFv–albumin fusions expressed in Saccharomyces cerevisiae

An expression system is described for the production of monomeric scFvs and scFv antibody fragments genetically fused to human albumin (at either the N- or C-terminus or both). Based upon strains of Saccharomyces cerevisiae originally developed for the production of a recombinant human albumin (Recombumin®) this system has delivered high levels of secreted product into the supernatant of shake flask and high cell density fed-batch fermentations. Specific binding to the corresponding ligand was demonstrated for each of the scFvs and scFv–albumin fusions and pharmacokinetic studies showed that the fusion products had greatly extended circulatory half-lives. The system described provides an attractive alternative to other microbial systems for the manufacture of this type of product.