Performance of a recombinant fusion protein linking coagulation factor IX with recombinant albumin in one-stage clotting assays.

Essentials Performance of the one-stage clotting (OSC) assay varies with the clotting activator used. Recombinant FIX-albumin fusion protein (rIX-FP) was reliably monitored with most OSC reagents. rIX-FP shows comparable reagent-dependent variability to other rFIX products in the OSC assay. Actin® FS and kaolin-based reagents underestimated rIX-FP activity by around 50% in the OSC assay. SUMMARY: Background Measuring factor IX activity (FIX:C) with one-stage clotting (OSC) assays, based on the activated partial thromboplastin time (APTT), is the current mainstay of diagnostic techniques for hemophilia B. Assessing the performance of new recombinant FIX (rFIX) products in OSC assays is essential, as APTT reagents from different manufacturers yield different potency estimates for rFIX. Objectives To evaluate the extent to which choice of reagent composition influences rFIX potency measurements of recombinant FIX-albumin fusion protein (rIX-FP, IDELVION) activity in OSC assays. Methods rIX-FP was added to FIX-deficient plasma, and FIX:C was assessed centrally and locally in a multicenter international field study with a variety of commercial OSC APTT reagents. Paired sample analysis of clinical samples was performed to compare values of FIX:C from local and central laboratories. In-house bioanalytical investigations with spiked samples were conducted to compare the APTT-reagent dependent variability of rIX-FP with unmodified rFIX and rFIX Fc fusion protein (rFIXFc). Results Central and local assessments of FIX:C from 10 countries and 21 participating centers showed comparable results to those from the central laboratory across the majority of 18 different APTT reagents from both clinical and spiked samples. There was a consistent underestimation of rIX-FP activity of ≈ 50% with OSC assays using Actin FS or kaolin-based APTT reagents. In the bioanalytical study, rIX-FP showed comparable variability in OSC assays to unmodified rFIX and rFIXFc. Conclusions rIX-FP activity can be accurately measured by the use of OSC assays with the majority of commercial reagents. Actin FS or kaolin-based reagents will probably lead to a 50% underestimation of activity.

Guidelines to reach high-quality purified recombinant proteins.

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization-denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS) and circular dichroism (CD)-are revisited with focus on the protein and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.

Scribed transparency microplates mounted on a modified standard microplate.

The immense cost effectiveness of using transparencies as analyte handling implements in microplate instrumentation offers the possibility of application even in resource-limited laboratories. In this work, a standard microplate was adapted to serve as the permanent base for disposable scribed transparencies. The approach is shown to ameliorate evaporation, which can affect assay accuracy when analytes need to be incubated for some time. It also offers assurance against fluorescence measurement errors due to the cross-talk of samples from adjacent wells.

A novel approach to improve the function of FGF21.

BACKGROUND AND OBJECTIVE: Fibroblast growth factor 21 (FGF21) has potent effects on normalizing glucose, lipid, and energy homeostasis, and represents an attractive novel therapy for type 2 diabetes mellitus and obesity. Approaches to improve the pharmacokinetic properties of FGF21, such as conjugation with polyethylene glycol, have been explored for therapeutic development. However, not only is there room for further pharmacokinetic improvements, additional re-engineering approaches to improve the potency and stability of FGF21 have not been reported. Here, we describe a novel approach to modify and improve the function of FGF21 by altering its C-terminal ßKlotho interaction domain. METHODS: We first identified Avimer proteins that are capable of binding ßKlotho. Then we explored replacing the C-terminal ßKlotho interaction domain of FGF21 with a ßKlotho-binding Avimer protein. RESULTS: Such a ßKlotho-binding Avimer protein was able to fully complement the C-terminal domain function of FGF21. The resulting FGF21-Avimer fusion is functionally indistinguishable from wild type FGF21, and more tolerant of C-terminal modification. CONCLUSION: These results demonstrate a viable strategy to modulate the affinity, potency, and engineering of FGF21, paving the way for further improvements of FGF21 as a therapeutic.

Production and characterization of chimeric anti-HLA monoclonal antibodies targeting public epitopes as tools for standardizations of the anti-HLA antibody detection.

Two chimeric monoclonal antibodies (MoAbs) were designed with variable parts of the mouse antibodies W6/32 and F3.3, and the human constant parts C-gamma1 and C-kappa. These chimeric MoAbs are specific for HLA-class I and HLA-class II public epitopes, respectively. The anti-class I MoAb recognizes all HLA-class I tested so far, and the anti-class II MoAb recognize all HLA-DR, DP, and only DQ antigens of the DQ2 subgroup. These Moabs were used as positive controls in routine tests for the detection of IgG anti-HLA antibodies in the sera of patients (Luminex, flow cytometry, and complement-dependent lymphocytotoxicity assay: CD-LCT). In tests with the LABScreen MIX assay, serial dilutions of the two MoAbs have allowed to determine the thresholds of detection by the Boltzmann sigmoidal regression. The thresholds of detection in mean of fluorescence intensity (MFI) were 926 and 866 for the anti-class I MoAb and the anti-class II MoAb, respectively. The thresholds defined as mean+3SD of MFI values obtained with 30 negative control sera were 411 and 251 for the anti-class I beads and the anti-class II beads, respectively. For the daily validation of the anti-HLA IgG antibodies screening by LABScreen MIX we decided to use the positive control MoAbs at three concentrations (5, 50, and 500ng/ml) and we measured the repeatability (<10%) and reproducibility (<16%) of the method. Used as positive controls in tests with the LABScreen Single Antigen kit, the two MoAbs allowed to estimate daily the quality of beads coated with HLA antigens. The two anti HLA MoAbs were also used as positive control in the cross-match assay by flow cytometry or CD-LCT. In total these two chimeric anti-HLA-MoAbs, which have no equivalent so far, are valuable positive controls for the daily validations of most techniques used for the detection of anti-HLA antibodies according to the good practice guidelines for laboratories.

A sandwich ELISA for assessment of pharmacokinetics of HSA-(BNP)2 fusion protein in mouse plasma.

Brain natriuretic peptide (BNP) is a circulating hormone of cardiac origin that plays an important role in the regulation of intravascular blood volume and vascular tone. HSA-(BNP)(2), derived from the joining of human BNP to the C-terminus of human serum albumin (HSA), has been developed to prolong the BNP pharmacodynamic action. For the analysis of pharmacokinetics of the new drug, a novel sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated to quantify HSA-(BNP)(2) fusion protein in mouse plasma. The ELISA method was calibrated with 1:10 and 1:100 dilutions of blank mouse plasma spiked with HSA-(BNP)(2) standard and validated with respect to parallelism, precision (intra- and inter-assay variation), accuracy (recovery), specificity and stability. The practical working range was estimated to be 31.2-2000ng/ml with the limit of detection was 7.8ng/ml. Recoveries ranged from 80.5 to 108.4%, while the intra- and inter-assay precisions were <2.73% and <4.32%, respectively. The terminal half-life of HSA-(BNP)(2) was 2.14h, which had extended more than 40 times compared to 3.1min half-life of BNP monomer in mouse.

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.

[Development and application of quality control materials for human papillomavirus types 16 and 18 nucleic acid test].

OBJECTIVE: To develop quality control materials for human papillomavirus (HPV) types 16 and 18 molecular detection and to evaluate the applicability of the materials in external quality assessment (EQA) of HPV-16, 18 clinical detection. METHODS: The target gene from HPV16 was cloned into the pEGFP-C1 plasmid, then the recombinant plasmid pEGFP-C1-HPV16 was transfected into the HPV18 carrying HeLa cells. The cultured epithelial cells carrying both HPV16 target gene and HPV18 were collected and fixed using methanol. EQA samples for HPV types 16 and 18 test diluted to the concentrations 1:10, 1:50, 1:100, 1:500 were prepared from the above prepared cells and distributed to 73 EQA participants nationwide to undergo RT-PCR. The reports from the participants were summarized and evaluated. Three samples were blindly mailed to Urumqi, Hohhot, and Xiamen respectively to observe the influence of post. RESULTS: Forty-four of the 73 PCR laboratories sent feedback. For the quality control materials of the concentrations 1:10, 1:50, 1:100, and 1:500 the corresponding Ct values were 29.10, 31.19, 32.15, and 32.73 respectively for HPV-16, and 30.32, 32.13, 32.22, and 35.55 respectively for HPV-18. Stability test indicated that the quality control materials were stable at least for 40 days when stored at 4 degrees C. The EQA data from 44 participants showed, that the average fit rate was 95.1% for the high concentration positive samples and was 57.4% for the low concentration samples, and was 98.3% for the negative samples. No significant changes were detected by real-time PCR in the returned EQA samples that were blindly mailed to Urumqi, Hohhot, and Xiamen. CONCLUSION: A quality control materials for HPV types 16 and 18 molecular detection has been developed, and quality assessment verifies its applicability in EQA.

Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant products.

We have evaluated the diagnostic utility of six antigenic regions of the Toxoplasma gondii MIC2, MIC3, M2AP, GRA3, GRA7, and SAG1 gene products, assembled in recombinant chimeric antigens by genetic engineering, in order to replace the soluble, whole-cell tachyzoite extract in serological assays. Serum samples from 100 adults with acquired T. gondii infection and from 30 infants born to mothers with primary toxoplasmosis contracted during pregnancy, of whom 20 were congenitally infected, were included. Immunoglobulin G (IgG) and IgM antibodies against epitopes carried by chimeric antigens were measured by performing parallel enzyme immunoassays (recombinant enzyme-linked immunosorbent assays [Rec-ELISAs]), and the results obtained by standard commercial assays with the whole-cell Toxoplasma antigen and assays with the chimeric antigens were compared. Our results demonstrate that IgG and IgM Rec-ELISAs with individual chimeric antigens have performance characteristics comparable to those of the corresponding commercial assays. Furthermore, we show that IgM-capture assays based on chimeric antigens improve the ability to diagnose congenital toxoplasmosis postnatally compared with the ability to diagnose congenital toxoplasmosis by the use of standard assays. The use of recombinant chimeric antigens is effective in distinguishing T. gondii-infected individuals from T. gondii-uninfected individuals and shows that immunoassays based on recombinant products could provide the basis for standardized commercial tests for the serodiagnosis of toxoplasmosis.

Protein production: feeding the crystallographers and NMR spectroscopists.

Protein purification efforts for structural genomics will focus on automation for the readily-expressed proteins, and process development for the more difficult ones, such as membrane proteins. Thousands of proteins are expected to be produced in the next few years. The purified proteins will be valuable reagents for the entire research community.

Capillary electrophoresis for purity estimation and in-process testing of recombinant GB virus-C proteins.

Protein purity estimation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with scanning densitometry is a critical component in the manufacture of recombinant proteins for commercial diagnostic assays. However, the procedure is time consuming and often difficult to reproduce because commercial dyes that are used for visualizing proteins do not bind in a stoichiometric fashion for all proteins. The present report describes the use of a rapid and dye-independent SDS polymer-filled capillary gel electrophoresis (CE-SDS) method to estimate protein purity. The CE-SDS method was used for in-process and final purity testing of GB virus-C (GBV-C) fusion proteins produced in E. coli, and was directly compared with the conventional SDS-PAGE method using purified Coomassie blue dye to reduce protein staining anomalies. The CE-SDS method may serve as an alternative or replacement method to the lengthy and tedious SDS-PAGE method. This study also demonstrates that the observed molecular weight of the fusion protein, determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), provides higher accuracy than values estimated by either CE-SDS or SDS-PAGE methods.

Quantitation of IgG subclass antibody responses after immunization with a group B meningococcal outer membrane vesicle vaccine, using monoclonal mouse-human chimeric antibodies as standards.

An ELISA method was developed to quantitate gravimetrically (microgram/ml) the IgG subclass response against a Norwegian vaccine composed of outer membrane vesicles (OMV) isolated from a Neisseria meningitidis B:15:P1.7,16 epidemic strain. Chimeric mouse-human anti-hapten NIP (5-iodo-4-hydroxy-3-nitrophenacetyl) antibodies of each subclass were used for calibration purposes. Before vaccination, low amounts of IgG1 and IgG2 antibodies against OMV were detectable in all vaccinees, whereas IgG3 was only detectable in one of the 21 vaccinees. After vaccination, IgG1 antibodies dominated the response followed by IgG3 and low to moderate levels of IgG2 antibodies. IgG4 was only detectable at very low levels in a few vaccinees. All sera showed close to parallel dose-response curves to each other for IgG1 and IgG3, whereas the IgG2 curves were not parallel to chimeric IgG2 and could thus not be quantitated gravimetrically. For IgG3, 1/3 of the vaccinee sera showed non-parallel dose-response curves to the rest of the vaccinee sera and to chimeric IgG3 and could not be gravimetrically quantitated. The rest of the sera showed parallel dose-response curves with the chimeric IgG3 and gravimetric quantitation was possible. This study illustrates that chimeric antibodies can be used as calibrators to quantitate IgG subclass antibody responses against OMV in gravimetric units and that the vaccine mainly induces IgG1 and IgG3 antibodies in humans.

Standard calibration proteins for Western blotting obtained by genetically prepared protein A conjugates.

Genetically prepared protein A fusion proteins, having retained antibody binding capacity, were used to design different well-defined standard molecular weight marker proteins for Western blotting. The blotted marker proteins are developed at the same time and with the same reagents as the protein sample of interest.