Development of a diphtheria toxin based antiporcine CD3 recombinant immunotoxin

Anti-CD3 immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.     

GMP production and characterization of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1) for phase I/II clinical trials

The bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(UCHT1), was developed for treatment of T-cell leukemia, autoimmune diseases and tolerance induction for transplantation. To obtain clinical grade bivalent anti-T cell immunotoxin for phase I/II clinical trials, a single batch of 120 L bioreactor culture was performed using the Pichia pastoris mutEF2JC307-8(2) strain expressing the bivalent anti-T cell immunotoxin. After 162 h induction of the culture by methanol, the culture medium was harvested by a 0.1 microm hollow-fiber microfiltration step. The recombinant protein was purified by a 3-step purification procedure (Butyl 650 M capturing step, borate anion exchange step and final Poros anion exchange step). The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C. Expression level was 207 mg/L of culture supernatant and the final production yield was 69.6% or 144.2mg/L of culture supernatant. The final product was characterized by multiple assays. Vialed product was sterile. The drug concentration was 0.8 mg/mL in 150 mM NaCl, 5% glycerol, 1mM EDTA, and 5mM Tris (pH 8.0). Purity by SDS-PAGE was 98%. Aggregates by Superdex 200 HPLC were <1%. Potency revealed a 20 h IC(50) of 17f M on Jurkat cells. Endotoxin level was 0.02 U/mg. Chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule. The drug did not react with tested frozen human tissue sections except for T cells. LD(10) in mice was between 500 and 75 0microg/kg. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 1.5 year at -80 degrees C. The scalable synthesis of this protein drug should be useful for production for phase I/II clinical trials and can be applicable for other diphtheria toxin fusion drugs for clinical development.     

A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa

A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 μM. The protease did not have antifungal or ribonuclease activity.     

Molecular cloning, expression, and characterization of a Ca2+-dependent nuclease of Arabidopsis thaliana

Yeast Pichia pastoris has been widely utilized to express heterologous recombinant proteins. P. pastoris expressed recombinant porcine interleukin 3 (IL3) has been used for porcine stem cell mobilization in allo-hematopoietic cell transplantation models and pig-to-primate xeno-hematopoietic cell transplantation models in our lab for many years. Since the yeast glycosylation mechanism is not exactly the same as those of other mammalian cells, P. pastoris expressed high-mannose glycoprotein porcine IL3 has been shown to result in a decreased serum half-life. Previously this was avoided by separation of the non-glycosylated porcine IL3 from the mixture of expressed glycosylated and non-glycosylated porcine IL3. However, this process was very inefficient and lead to a poor yield following purification. To overcome this problem, we engineered a non-N-glycosylated version of porcine IL3 by replacing the four potential N-glycosylation sites with four alanines. The codon-optimized non-N-glycosylated porcine IL3 gene was synthesized and expressed in P. pastoris. The expressed non-N-glycosylated porcine IL3 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50 HQ. In vivo mobilization studies performed in our research facility demonstrated that the non-N-glycosylated porcine IL3 still keeps the original stem cell mobilization function.     

Arabinoxylan fractionation on DEAE-cellulose chromatography influenced by protease pre-treatment

An arabinoxylan mixture was extracted with saturated barium hydroxide from a water unextractable residue of rye bran. The mixture was fractionated on an anion exchange column which was eluted with water, 0.0025 M sodium borate, 0.025 M sodium borate and 0.4 M sodium hydroxide. It was possible to fractionate the arabinoxylan mixture into fractions with different structural features. The fractionation was repeated with prior protease treatment of the arabinoxylan mixture, but most of the arabinoxylan did not bind to the column by any mechanism that the protease treatment affected, As the largest fraction was still eluted with 0.4 M sodium hydroxide. Protease treatment affected the proportion of disubstituted xylose residues (dXyl) in the water, 0.0025 M sodium borate and 0.025 M sodium borate fractions, indicating that protein associated with arabinoxylans with a high dXyl content is more liable to the protease treatment or that protein is mainly associated with these structures. This study gives indications that protein is involved in the separation mechanism of rye arabinoxylan on a DEAE–cellulose column.     

Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15

The porcine CD3 specific monoclonal antibody 898H2-6-15 has been used in allo- and xeno-transplantation studies as a porcine CD3 marker and as an effective T cell depletion reagent when conjugated to the diphtheria toxin mutant, CRM9. A recombinant anti-porcine CD3 immuntoxin was recently developed using single-chain variable fragments (scFv) derived from 898H2-6-15. In this study, using published sequence data, we have expressed the porcine CD3 ectodomain molecules in E. coli through inclusion body isolation and in vitro refolding approach. The expressed and refolded porcine CD3 ectodomain molecules include CD3ε, CD3γ, CD3δ, CD3εγ heterodimer, CD3εδ heterodimer, CD3εγ single-chain fusion protein and CD3εδ single-chain fusion protein. These refolded porcine CD3 ectodomain molecules were purified with a strong anion exchange resin Poros 50HQ. ELISA analysis demonstrated that only the porcine CD3εγ ectodomain single-chain fusion protein can bind to the porcine CD3 specific monoclonal antibody 898H2-6-15. The availability of this porcine CD3εγ ectodomain single-chain fusion protein will allow screening for affinity matured variants of scFv derived from 898H2-6-15 to improve the recombinant anti-porcine CD3 immunotoxin. Porcine CD3εγ ectodomain single-chain fusion protein will also be a very useful reagent to study the soluble phase interaction between porcine CD3εγ and porcine CD3 antibodies such as 898H2-6-15.     

Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography

Weak partitioning chromatography (WPC) has been proposed for the purification of monoclonal antibodies using an anion exchange (AEX) resin to simultaneously remove both acidic and basic protein impurities. Despite potential advantages, the relationship between resin structure and WPC performance has not been evaluated systematically. In this work, we determine the structure of representative AEX resins (Fractogel® EMD TMAE HiCap, Q Sepharose FF, and POROS 50 HQ) using transmission electron microscopy and inverse size exclusion chromatography and characterize protein interactions while operating these resins under WPC conditions using two mAb monomers, a mAb dimer, mAb multimers, and BSA as model products and impurities. We determine the isocratic elution behavior of the weakly bound monomer and dimer species and the adsorptive and mass transfer properties of the strongly bound multimers and BSA by confocal laser scanning microscopy. The results show that for each resin, using the product K_p value as guidance, salt, and pH conditions can be found where mAb multimers and BSA are simultaneously removed. Isocratic elution and adsorption mechanisms are, however, different for each resin and for the different components. Under WPC conditions, the Fractogel resin exhibited very slow diffusion of both mAb monomer and dimer species but fast adsorption for both mAb multimers and BSA with high capacity for BSA, while the Sepharose resin, because of its small pore size, was unable to effectively remove mAb multimers. The POROS resin was instead able to bind both multimers and BSA effectively, while exhibiting a greater resolution of mAb monomer and dimer species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:425–434, 2017     

Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins

Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.     

Densonucleosis virus purification by ion exchange membranes

Preparative chromatography is widely used in the downstream purification of biopharmaceutical products. Replacement of resins by membranes as chromatographic supports, overcomes many of the limitations associated with resin-based chromatography such as high-pressure drops, slow processing rates due to pore diffusion and channeling of the feed through the bed. In particular, adsorptive membranes may be ideally suited for virus capture. Virus capture is critical in a number of applications. In gene therapy and vaccine production, large-scale purification of virus vectors is often essential. In the manufacture of biopharmaceuticals, validation of virus clearance is critical.Here results for purification of Aedes aegypti densonucleosis virus (AeDNV) using anion and cation exchange membranes are presented. AeDNV is a non-enveloped, single-stranded mosquito-specific parvovirus. Virus particles are around 20 nm in size. AeDNV could find potential applications in integrated vector-borne disease control programs. In addition, capture of parvovirus for validation of virus clearance in the manufacture of biopharmaceuticals is of commercial importance.By adjusting the pH of the feed stream, AeDNV particles may be adsorbed by both anion and cation exchange membranes. However, strongly basic anion exchange membranes were the most effective in adsorbing AeDNV particles. Adsorption and subsequent elution of AeDNV by anion exchange membranes leads to significant virus concentration. Dynamic and static capacities for anion exchange membranes were similar. Further, a sharp elution curve was obtained suggesting that pore diffusional resistances are insignificant. The adsorption of AeDNV particles by anion exchange membranes may be described by a linear isotherm.     

Solubilization, Characterization, and Partial Purification of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-, Quisqualate-, Kainate-Sensitive l-Glutamate Binding Sites from Porcine Brain Synaptic Junctions

L-[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X-114 in the presence of KCl. The solubilized binding sites bound L-[3H]glutamate reversibly with KD and Bmax values of 1.48 +/- 0.18 microM and 178.2 +/- 15.9 pmol/mg of protein, respectively. These binding sites appeared to be integral membrane glycoproteins, with sugar moieties recognized by wheat germ agglutinin. A 49.3-fold purification of these binding sites was achieved by Triton X-114 solubilization, anion-exchange chromatography, and affinity chromatography using wheat germ agglutinin-Sepharose. The apparent molecular mass of the partially purified binding sites was 620 +/- 50 kDa. L-[3H]Glutamate bound to the solubilized preparation could be effectively displaced by agonists of non-N-methyl-D-aspartate (NMDA) L-glutamate receptors but not by NMDA or alpha-amino-4-phosphonobutyrate. The rank order for the competitive ligands in displacing L-[3H]glutamate was: quisqualate greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid greater than L-glutamate greater than kainate.     

Purification and characterization of human hemoglobin: effect of the hemolysis conditions

Human hemoglobin was isolated and purified by anion exchange chromatography. To isolate hemoglobin, outdated red blood cells (RBC) were transformed into carbonylhemoglobin, by reaction with carbon monoxide, and submitted to washing/centrifugation procedures, to eliminate other plasma proteins. Albumin was quantified in each supernatant, by the bromcresol green method. Hemolysis was performed in three different hypotonic media (water, 0.01 M NaCl and 5 mM Tris/HCl buffer at pH 7.4), at 8 degrees C for 24 h. Sonication for 5 min was also used to lyse RBC. After isolation of hemoglobin, additional purification was carried out by anion exchange chromatography on AG MP-1, Q-SFF and both exchangers. Hemoglobin concentration of hemolysates and of purified solutions were determined by the hemiglobincyanide method. Residual phospholipids were extracted from the four different hemolysates, as well as from the purified hemoglobin solutions, and were analyzed by high performance liquid chromatography. Native and SDS-polyacrylamide gel electrophoresis experiments were performed on purified hemoglobin samples to verify the presence of proteins other than hemoglobin. According to the results, the hemolysis conditions have influence on the purification of hemoglobin.     

High performance liquid chromatography of integral glycoproteins of peripheral nerve myelin

Peripheral nerve myelin contains a large quantity of integral glycoproteins, such as PO and PASII protein. The present paper reports a fast and sensitive method for separation of these glycoproteins. High performance liquid chromatography (HPLC) with TSK-GEL 3000 SW column in the presence of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate (LDS) was used. Whereas the separation of PO and PASII was inadequate with low concentrations of the detergent, better separation profiles were obtained with high concentrations (1–2%) of the detergent in 0.1 M phosphate buffer. The two glycoproteins were able to be purified by rechromatography. High concentration of the detergent presumably diminished hydrophobic interaction between these glycoproteins. LSD-phosphate, SDS-lithium citrate or SDS-Tris buffer as an eluent was also compared with SDS-phosphate system. This method will be applicable to the detection and purification of proteins from myelin or other organelles.     

Use of borate buffers for purification of placental β-glucuronidase on DEAE-cellulose

The compiexing effect of borate ions with glycoproteins has been used to obtain a high degree of purification of placental β-glucuronidase by chromatography on DEAE-cellulose columns.     

抗HIV融合多肽CP32M的制备工艺研究

目的:研究抗HIV融合多肽CP32M的制备工艺,为其临床前试验奠定基础。方法:采用固-液相混合策略规模合成目标肽,用离子交换色谱、反相高效液相色谱对粗肽进行纯化。结果:获得了利于提高合成及片段缩合效率的3条优选片段,CP32M粗品经DEAE离子交换色谱及C18反相纯化后纯度高于98%。结论:CP32M按3条片段(Ac-1-9-OH、Fmoc-10-21-OH、H-22-32-NH2)划分,可显著提高片段合成及缩合效率,DEAE阴离子交换色谱能除去大部分杂质,提高了第二步C18反相色谱纯化效率。     

Systematic Separation of Human Urinary Peptides

Urinary peptides were roughly fractionated by combined columns of cation and anion exchange resins, and the peptides eluted from each column were further fractionated by a combination of various ion exchange resins and DEAE-cellulose column chromatography, paper chromatography and other methods. From the fractions adsorbed on cation exchange resin, 13 homogeneous peptides could be isolated, and from the ones adsorbed on anion exchange resin, 8 glycopeptides could be found. Their amino acid compositions were analyzed.

Although some fractions remain univestigated, an outline of the whole aspect of main urinary peptides has been clarified by this study.     

Purification of bovine hemoglobin via fast performance liquid chromatography

Bovine hemoglobin (bHb) was purified from bovine red blood cells (bRBCs) via anion exchange chromatography preceded by dialysis. This is a fast and effective way to obtain bHb from bRBCs using Q Sepharose XL, a strong anion exchange resin. This resin had double the binding capacity for bHb compared to three other anion exchange resins that were studied in this work. Methemoglobin levels remained below 2% with bHb concentrations between 0.7 and 1.7 mM. The high purity of bHb was confirmed via SDS-PAGE and size exclusion chromatography (SEC).     

Differential release of high mannose structural isoforms by fungal and bacterial endo-β-N-acetylglucosaminidases

Endo-β-N-acetylglucosaminidases (ENGases) are widely used to remove N-linked oligosaccharides from glycoproteins for glycomic and proteomic studies and biopharmaceutical processes. Although several ENGases are widely available and their main oligosaccharide structural preferences are generally known (i.e. high mannose, hybrid or complex), the preferences of ENGases from different kingdoms for individual structural isoforms within the major classes of N-linked oligosaccharides have previously not been compared. In this work, a fungal ENGase (Endo Tv) was purified for the first time from a commercial Trichoderma viride chitinase mixture by sequential anion exchange and size exclusion chromatography, a commonly used strategy for purification of chitinases and endo enzymes. Oligosaccharides released from substrate glycoproteins by Endo Tv were identified and quantified by high pH anion exchange chromatography with pulsed amperometric detection and verified by mass spectrometric analysis. Unlike the widely-used bacterial ENGases, Endo H and Endo F1, Endo Tv released exclusively high mannose N-linked oligosaccharides from RNase B, ovalbumin, and yeast invertase. Endo Tv did not hydrolyze fucosylated, hybrid, complex type or bisecting N-acetylglucosamine-containing structures from bovine fetuin, ovalbumin and IgG. When compared to the bacterial ENGase, Endo H, the relative ratio of high-mannose oligosaccharide structural isoforms released from RNase B by Endo Tv was found to differ, with Endo Tv releasing more Man?GlcNAc and Man?GlcNAc isoform I and less Man(9)GlcNAc from RNase B. Based on these data, it is suggested that use of ENGases from multiple sources may serve to balance an introduced bias in quantitative analysis of released structural isoforms and may further prove valuable in biochemical structure-function studies.     

Smooth muscle myosin light chain kinase: rapid purification by anion exchange high-performance liquid chromatography

A rapid procedure for the purification of myosin light chain kinase present in chicken gizzard smooth muscle using anion exchange high-performance liquid chromatography is described. The procedure allows preparation of microgram amounts of the protein directly from the extract of gizzard myofibrils and then is suitable for the study of myosin light chain kinase in small muscles. The protein was judged to be greater than 95% pure by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme retains its activity since it catalyzes the calcium-calmodulin-dependent phosphorylation of the 20,000-Da myosin light chain.     

Salt tolerant membrane adsorbers for robust impurity clearance

Clearance of impurities such as viruses, host cell protein (HCP), and DNA is a critical purification design consideration for manufacture of monoclonal antibody therapeutics. Anion exchange chromatography has frequently been utilized to accomplish this goal; however, anion exchange adsorbents based on the traditional quaternary amine (Q) ligand are sensitive to salt concentration, leading to reduced clearance levels of impurities at moderate salt concentrations (50–150 mM). In this report, membrane adsorbers incorporating four alternative salt tolerant anion exchange ligands were examined for impurity clearance: agmatine, tris‐2‐aminoethyl amine, polyhexamethylene biguanide (PHMB), and polyethyleneimine. Each of these ligands provided greater than 5 log reduction value (LRV) for viral clearance of phage ?X174 (pI ～ 6.7) at pH 7.5 and phage PR772 (pI ～ 4) at pH 4.2 in the presence of salt. Under these same conditions, the commercial Q membrane adsorber provided no clearance (zero LRV). Clearance of host‐cell protein at pH 7.5 was the most challenging test case; only PHMB maintained 1.5 LRV in 150 mM salt. The salt tolerance of PHMB was attributed to its large positive net charge through the presence of multiple biguanide groups that participated in electrostatic and hydrogen bonding interactions with the impurity molecules. On the basis of the results of this study, membrane adsorbers that incorporate salt tolerant anion exchange ligands provide a robust approach to impurity clearance during the purification of monoclonal antibodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009