Determination of leakage from antibody adsorbent: composition analysis and pH effect.

To study the leakage at different solution pH values, IgG Sepharose 6FF®, a commercially available immunoadsorbent, was used as a model. The leaked substance consists of three parts: (1) ligands and its fragments; (2) ligands plus matrix fragments in which ligands are chemically attached to the adsorbent matrix; and (3) matrix fragments. Buffer solution pH values had a great effect on both the kinetics and the amount of ligand leakage. Cross-linking of the adsorbent matrix could reduce both matrix leakage and antibody leakage at pH 3.0, but its effect was limited at pH 11.0 for ligand leakage.

PEGylation of proteins in organic solution: a case study for interferon beta-1b.

Conventional protein PEGylation is carried out in aqueous solution. However, some hydrophobic proteins seem to be stable in organic solution. In this study, a novel approach of PEGylating IFN-ß-1b in an organic solution of 2-butanol (2-BuOH) was investigated. Compared with protein PEGylation in aqueous solution, the overall modification yields increased more than 37%, while the yield of mono-PEGylated products could be increased by 36%. Furthermore, the PEGylated IFN-ß-1b, which was obtained in organic solution, demonstrated 18% more antiviral potency than those derived from aqueous solution. The PEGylation step could be directly connected to the previous protein separation step for process integration. Dynamic light scattering (DLS) and atomic force microscope (AFM) analysis revealed that IFN-ß-1b formed aggregates both in water and in 2-BuOH solutions. However, the aggregates were much smaller and more homogeneous in 2-BuOH than those in aqueous solution, thereby providing larger solvent accessible protein surfaces, which resulted in a more productive PEGylation process. In addition, the results of circular dichroism (CD), fluorescence spectra, and peptide mapping suggested that the increased bioactivity came from the difference in PEGylation site distribution due to solution environment that induced conformational discrepancy. The results of this study show that PEGylation of IFN-ß-1b in organic solution is a facile and efficient process, which might find applications for other hydrophobic proteins.

Deliberate manipulation of the surface hydrophobicity of an adsorbent for an efficient purification of a giant molecule with multiple subunits.

Hydrophobic interaction chromatography (HIC) is often an inevitable step for a satisfying purification in giant vaccine molecules production. But great mass and activity loss associated with poor purity often occur simultaneously. In this paper, high purity and high bioactivity recovery for the HIC process of hepatitis B surface antigen (rHBsAg) purification were achieved through manipulation of surface hydrophobicity of the adsorbent. Spacer arm length and ligand density were regulated, respectively, through which the interaction between the vaccine and the adsorbent was manipulated deliberately. It was found even in a narrow scope, varying spacer arm length and ligand density resulted in purification factor changing from 1 to 96.5, and rHBsAg recovery from 3 to 91%. The optimal purification performance was achieved when the spacer arm was C8 and the ligand density was 9.2 µmol/g suction-dried wet gel with an average distance of ligands of 3.6 nm. This deliberate regulation strategy represents a new approach of improving purification of giant multi-subunit proteins.

Analysis of gelatin plasma substitutes in blood based on detection of hydroxyproline derivatives.

The gelatin plasma substitute is often polydisperse and heterogenous, making it difficult to determine the elimination rate and half-life in the body. In this study, one method was developed based on quantitative determination of hydroxyproline derivatives. Two plasma substitutes were prepared by succinylation and genipin-crosslinking, respectively. After transfusion, the blood samples were hydrolyzed and derivatized, and then analyzed by HPLC. A two-phase exponential association equation was used for fitting the time-concentration curves. The results indicated that this method could be used for quantitative determination of gelatin in blood, and the pharmacokinetic parameters such as elimination rate and half-life.

PEGylation markedly enhances the in vivo potency of recombinant human non-glycosylated erythropoietin: a comparison with glycosylated erythropoietin.

Recombinant human non-glycosylated erythropoietin (rh-ngEpo) expressed in E. coli was attached to polyethylene glycol (PEG) chains with different sizes and structures. The pharmacokinetic properties and in vivo potency of the PEGylated protein were investigated and comparisons were drawn between the conjugates and glycosylated recombinant Epo (rhEpo). The rh-ngEpo was modified with linear PEG-aldehyde (PEG-ALD, 20 kDa, 30 kDa, and 40 kDa) and a branched N-hydroxysuccinimide activated PEG (PEG(2)-NHS, 40 kDa). The monoPEGylated proteins were isolated by ion-exchange chromatography. The purified monoPEGylated conjugates suffered 6.5-86.1% loss of in vitro bioactivity compared to the unmodified rh-ngEpo. In addition, PEGylation remarkably increased the resistance of rh-ngEpo against plasma degradation. Pharmacokinetic studies showed that the plasma half-life of rh-ngEpo was increased 9.7-17.4 times by PEGylation, with the two 40k-PEG-rh-ngEpos-treated groups exhibiting better pharmacokinetic performances than rhEpo. Moreover, all the conjugates resulted in markedly enhanced Ret% (the percentage of reticulocyte count in red blood cells) compared with rh-ngEpo after subcutaneous injection. The two 40k-PEG conjugates demonstrated comparable in vivo efficacies compared with rhEpo. Overall, this research provides opportunities for the development of more cost-effective erythropoiesis-stimulating protein drugs.

[Purification and characterization of recombinant human lactoferrin expressed in a cattle mammary bioreactor].

Novel ion exchange adsorbents were synthesized by immobilizing sulfopropyl derivative onto homemade highly cross-linked agarose beads. The effects of different ligand densities (from 0.05 to 0.24 mol/L) on static and dynamic adsorption of the adsorbents were investigated using lysozyme as a model protein. Based on these results, rHLF was purified from the transgenic milk by our SP media. 1 mL high density (0.24 mol/L) adsorbent could handle 50 mL rHLF-containing milk. The mass recovery of rHLF was 86.5% and the purity was 98.5%. CD spectra demonstrated that the native structure of rHLF was not affected in the purification process. The biological functions of the purified rHLF, including iron binding, releasing and antimicrobial activities were then investigated. The results showed that rHLF had comparable iron binding and releasing activity to that of native HLF. 5 g/L concentration of rHLF significantly inhibited the growth of Escherichia coli. These studies lay a solid foundation for the wide application of our self-prepared ion exchange adsorbents in protein purification.

Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli.

Recombinant human erythropoietin produced by mammalian cells contains about 40% carbohydrates which maintain its stability and long residence in body. However, mammalian derived Epo has low yields and high costs of production. In this article, a cost-effective strategy of producing non-glycosylated Epo from Escherichia coli and then PEGylating it to replace the role of sugar chains was investigated. Recombinant human non-glycosylated erythropoietin (rh-ngEpo) was overexpressed as inclusion body in E. coli. As the routine inclusion body washing step resulted in poor protein recovery and purity, a new process scheme of using strong ion-exchange chromatography to purify denatured rh-ngEpo from inclusion body before refolding was developed. The purity of the denatured rh-ngEpo was increased from 59% to over 90%. Rh-ngEpo was then refolded and subsequently purified by one step of weak cation-exchange chromatography to 98% pure. Final protein yield was 129 mg/l, a significant improvement from 49 mg/l obtained via the conventional practice. The in vitro bioactivity of purified rh-ngEpo was comparable with the CHO-expressed Epo and the formation of native secondary structure was also confirmed by CD spectra. Rh-ngEpo was then modified by a 20 kDa methoxy polyethylene glycol (PEG) succinimidyl carbonate. The monoPEGylated protein, which retained 68% bioactivity, had enhanced thermal stability and a remarkably prolonged circulating half-life in rats as compared with that of the unmodified protein. These studies demonstrated the feasibility of PEGylating rh-ngEpo as a promising way for the development of new Epo drugs.

Cooperative effects of urea and L-arginine on protein refolding.

The use of low concentrations of urea, guanidinium chloride or arginine has been reported in the literature to increase protein refolding and yield of active proteins by suppressing aggregate formation. However, no studies have yet examined whether these substances can exert synergistic or cooperative effects when used in combination. In this work, a comparative study was carried out on refolding of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in the presence of different concentrations of urea, guanidinium chloride or arginine. All three folding aids could inhibit the formation of insoluble aggregates of rhG-CSF but with different efficacies. A low concentration of guanidinium chloride was found to denature protein, so that rhG-CSF was not fully or correctly folded even if concentration was reduced to 1M. Low concentration of urea (2M) or arginine (0.5M) did not cause rhG-CSF denaturation, but urea was unable to suppress the formation of soluble oligomers, which persisted at a level of about 30% in refolded soluble rhG-CSF. Arginine, in contrast, could inhibit formation of all soluble oligomers. Based on these phenomena, we tested rhG-CSF folding in a mixture of 2M urea and 0.5M arginine. Kinetic analysis indicated that urea aided in suppressing insoluble precipitates, while arginine prevented formation of soluble oligomers produced by hydrophobic interaction. With this combination system, the refolding yield of rhG-CSF could be increased 2-fold.

Different effects of L-arginine on protein refolding: suppressing aggregates of hydrophobic interaction, not covalent binding.

Arginine is one of the most favorable additives in protein refolding. However, arginine does not work for certain disulfide-bond-containing proteins, which is not yet well explained. In this work, refolding of three proteins in the presence of 0-2 M arginine was investigated and compared. Bovine carbonic anhydrase B (CAB), containing no cysteine, was successfully refolded with the help of arginine. The refolding yield could reach almost 100% in the presence of 0.75 M arginine. However, recombinant human colony stimulating factor (rhG-CSF), containing five cysteines, could only achieve 65% refolding yield. The formation of aggregates was found. Blocking of free SH groups of the denatured rhG-CSF by iodoacetamide and subsequently refolding of the protein could reduce the aggregate formation substantially. Further investigation on recombinant green fluorescence protein (GFP), containing two cysteines, also revealed the accumulation of oligomers. The content of oligomers increased with the concentration of arginine, reaching about 30% at 2 M arginine. Comparison of reduced and nonreduced SDS-PAGE revealed that the oligomers were formed through intermolecular disulfide binding. Analysis of the refolding kinetics indicated that intermolecular disulfide bonds were probably formed in the intermediate stage where arginine slowed down the refolding rate and stabilized the intermediates. The accumulated intermediates with unpaired cysteine possessed more chances to react with each other to form oligomers, whereas arginine failed to inhibit disulfide bond formation.

Identification of an oxidative refolding intermediate of recombinant consensus interferon from inclusion bodies and design of a two-stage strategy to promote correct disulfide-bond formation.

Dilution refolding of recombinant consensus IFN (interferon) from inclusion bodies suffers from low yield. A stable intermediate was found to mix with the correct product and to have an antiviral activity of less than 10% of the latter. This intermediate would form precipitates upon removal of the precipitation inhibitor arginine. Compared with the native protein, the intermediate moved more slowly on non-reducing SDS/PAGE. The CD and fluorescence spectra indicated that it had formed a native-like structure, but had only one disulfide bond: Cys(29)-Cys(139). Further evidence showed that the formation of Cys(29)-Cys(139) is specific and very likely to happen, even in the presence of a high concentration of reducing agent, whereas pairing of the other disulfide (Cys(1)-Cys(99)) needed a stronger oxidative condition. It competed with intermolecular disulfide bonding to form covalent oligomers. On the basis of this discovery, a two-stage refolding step strategy was designed that employed a modified dilution refolding step followed by a dialysis refolding step. The first stage used a high concentration of reducing agent together with the precipitation inhibitor arginine. The purpose was to hinder any reaction through Cys(1) or Cys(99) but allow the intramolecular disulfide bonding of Cys(29)-Cys(139). The second stage was a dialysis step that gradually increased the oxidative agent concentration and simultaneously decreased the arginine concentration. The refolding yield was increased from 35 to 82%, while the mass recovery was increased from 60 to 96%. Moreover, this strategy could suppress precipitation even after arginine was completely removed.