Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF.

Recombinant human IFNα2b (rhIFNα2b), as an important immune-related protein, has been widely used in clinic for decades. It is also at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Although with the same amino acid sequence, recombinant proteins are generally heterogeneous due to post-translational modification and chemical reactions during expression, purification, and long-term storage, which could have significant impact on the final product quality. So therapeutic rhIFNα2b must be closely monitored to ensure consistency, safety, and efficacy. In this study, we compared seven rhIFNα2b preparations from six manufacturers in China and one in America, as well as four batches of rhIFNα2b preparations from the same manufacturer, measuring IFNα2b variants and site-specific modifications using a developed LC/Q-TOF approach. Three main forms of N-terminus, cysteine, methionine, and acetylated cysteine were detected in five rhIFNα2b preparations produced in E. coli (1E~5E) and one in Pseudomonas (6P), but only the native form with N-terminal cysteine was found in rhIFNα2b preparation produced in Saccharomyces cerevisiae (7Y). Two samples with the lowest purity (4E and 6P), showed the highest level of acetylation at N-terminal cysteine and oxidation at methionine. The level of oxidation and deamidation varied not only between samples from different manufacturers but also between different batches of the same manufacturer. Although variable between samples from different manufacturers, the constitution of N-terminus and disulfide bonds was relatively stable between different batches, which may be a potential indicator for batch consistency. These findings provide a valid reference for the stability evaluation of the production process and final products.

Development and Validation of a Reporter-Cell-Line-Based Bioassay for Therapeutic Soluble gp130-Fc.

Soluble glycoprotein 130 kDa (sgp130)-Fc fusion protein, an innovative therapeutic bio-macromolecular drug specifically targeting IL-6 trans-signaling, proved to have good potential for application in the treatment of chronic inflammatory diseases. A simple and quick bioassay for sgp130-Fc was developed in this study. First, a stable reporter cell line was obtained by transfecting CHO-K1 cells with a sis-inducible element (SIE)-driving luciferase reporter gene (CHO/SIE-Luc). Sgp130-Fc could inhibit the expression of luciferase induced by IL-6/sIL-6Rα complex, and the dose-response curve fitted the four-parameter logistic model, with 50% inhibitive concentration (IC50) being about 500 ng/mL and detection range between 40 and 5000 ng/mL. Both the intra-assay and inter-assay coefficient of variation (CV) were below 10.0%, and the accuracy estimates ranged from 94.1% to 106.2%. The assay indicated a good linearity (R² = 0.99) in the range of 50% to 150% of optimized initial concentration. No significant difference was found between the test results of new assay and BAF3/gp130 proliferation assay (unpaired t test, p = 0.4960, n = 6). The dose-response effect and copy number of the luciferase gene was basically unchanged after long-term culture (up to passage 60), demonstrating the stability of CHO/SIE-Luc cells. These results suggested that the new reporter assay was suited to routine potency determination of therapeutic sgp130-Fc.

Anti-Tumor Activity and Pharmacokinetics of AP25-Fc Fusion Protein.

AP25 is an anti-tumor peptide with a high affinity for integrins. It exerts its anti-tumor activity by inhibiting angiogenesis and by directly inhibiting the growth of tumor cells. Its half-life time in vivo is only about 50 minutes, which limits its clinical application. In order to prolong the half-life time of AP25 while preserving its anti-tumor activity, several fusion proteins of AP25 and IgG4 Fc were designed and expressed; their anti-tumor activity and pharmacokinetics properties were evaluated. Firstly, four AP25-Fc fusion protein sequences were designed, and the corresponding proteins were expressed and purified. Based on the results of HUVEC migration inhibition assay, HUVEC and tumor cell proliferation inhibition assay and yields of expression by HEK293 cells, the fusion protein designated PSG4R was selected for further evaluation. The anti-tumor effect of PSG4R was then evaluated in vivo on HCT-116 nude mice xenograft model. And the pharmacokinetics properties of PSG4R were investigated in rats. The results showed that PSG4R could inhibit the growth of xenografts of human colon cancer cell line HCT-116 in nude mice by intravenous administration of 40 mg/kg once every two days. The half-life time of PSG4R was 56.270 ± 15.398 h. This study showed that the construction of AP25-Fc fusion protein could significantly prolong the half-life of AP25 while retaining its anti-tumor activity, which provides a new direction for new drug development of AP25.

Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE.

During the past two decades, recombinant human interleukin-12 (rhIL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies. Purity is a critical quality attribute (CQA) in the quality control system of rhIL-12. In our study, rhIL-12 bulks from manufacturer B showed a different pattern in non-reduced SDS-PAGE compared with size-exclusion chromatography (SEC)-HPLC. A small fragment was only detected in non-reduced SDS-PAGE but not in SEC-HPLC. The results of UPLC/MS and N-terminal sequencing confirmed that the small fragment was a 261⁻306 amino acid sequence of a p40 subunit of IL-12. The cleavage occurs between Lys260 and Arg261, a basic rich region. With the presence of 0.2% SDS, the small fragment appeared in both native PAGE and in SEC-HPLC, suggesting that it is bound to the remaining part of the IL-12 non-covalently, and is dissociated in a denatured environment. The results of a bioassay showed that the fractured rhIL-12 proteins had deficient biological activity. These findings provide an important reference for the quality control of the production process and the final products of rhIL-12.

Bioactivity Determination of a Therapeutic Recombinant Human Keratinocyte Growth Factor by a Validated Cell-based Bioassay.

The therapeutic recombinant human keratinocyte growth factor 1 (rhKGF-1) was approved by the FDA for oral mucositis resulting from hematopoietic stem cell transplantation for hematological malignancies in 2004. However, no recommended bioassay for rhKGF-1 bioactivity has been recorded in the U.S. Pharmacopoeia. In this study, we developed an rhKGF-1-dependent bioassay for determining rhKGF-1 bioactivity based on HEK293 and HaCat cell lines that stably expressed the luciferase reporter driven by the serum response element (SRE) and human fibroblast growth factor receptor (FGFR2) IIIb. A good responsiveness to rhKGF-1 and rhKGF-2 shared by target HEK293/HaCat cell lines was demonstrated. Our stringent validation was completely focused on specificity, linearity, accuracy, precision, and robustness according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, AAPS/FDA Bioanalytical Workshop and the Chinese Pharmacopoeia. We confirmed the reliability of the method in determining rhKGF bioactivity. The validated method is highly timesaving, sensitive, and simple, and is especially valuable for providing information for quality control during the manufacture, research, and development of therapeutic rhKGF.

Nuclear-translocated endostatin downregulates hypoxia inducible factor-1α activation through interfering with Zn(II) homeostasis.

Hypoxia­inducible factor­1α (HIF­1α) is key in tumor progression and aggressiveness as it regulates a series of genes involved in angiogenesis and anaerobic metabolism. Previous studies have shown that the transcriptional levels of HIF­1α may be downregulated by endostatin. However, the molecular mechanism by which endostatin represses HIF­1α expression remains unknown. The current study investigated the mechanism by which nuclear­translocated endostatin suppresses HIF­1α activation by disrupting Zn(II) homeostasis. Endostatin was observed to downregulate HIF­1α expression at mRNA and protein levels. Blockage of endostatin nuclear translocation by RNA interference of importin α1/ß1 or ectopic expression of NLS­deficient mutant nucleolin in human umbilical vein endothelial cells co­transfected with small interfering (si)­nucleolin siRNA compromises endostatin­reduced HIF­1α expression. Nuclear­translocated apo­endostatin, but not holo­endostatin, significantly disrupts the interaction between CBP/p300 and HIF­1α by disturbing Zn(II) homeostasis, which leads to the transcriptional inactivation of HIF­1α. The results reveal mechanistic insights into the method by which nuclear­translocated endostatin downregulates HIF­1α activation and provides a novel way to investigate the function of endostatin in endothelial cells.

A novel reporter gene assay for recombinant human erythropoietin (rHuEPO) pharmaceutical products.

Accurate determination of in vitro biological activity of therapeutic erythropoietin is essential in quality control of recombinant human erythropoietin (rHuEPO) pharmaceutical products. However, most of currently-used methods leave much to be desired so that a simpler, quicker and more accurate method is urgently needed. The bioassay described here utilizes a sub clone of UT-7/epo cell line stably transfected with luciferase gene under the control of sis inducible element and interferon γ-activated sequence element promoter. Active erythropoietin could induce the expression of luciferase by signaling through the erythropoietin receptor and the dose-response curve showed good linearity, yielding a coefficient of determination of 0.99 or higher. The optimized assay was simpler with the operation completed within 24h and more sensitive with EC50 being 0.077IU/mL. The accuracy estimates ranged from 81.7% to 102.4%, and both intra-assay and inter-assay precision was below 15.0%. The robustness of the assay was demonstrated by no effect of passage levels of the cells on the performance of the assay (p values: 0.772 for sample 1 and 0.943 for sample 2). Besides, Bland-Altman analysis showed a high consistency of the new assay with in vivo reticulocyte assay in results. These results suggested that the new reporter gene assay can be a viable supplement to the traditional reticulocyte assay and employed in potency determination of rHuEPO pharmaceutical products.

Novaferon, a novel recombinant protein produced by DNA-shuffling of IFN-α, shows antitumor effect in vitro and in vivo.

OBJECTIVE: A recombinant antitumor/antiviral protein (Novaferon, Nova) is a new type of interferon, which is produced by artificial design technology combining DNA-shuffling and High Throughput Screening (HTS). METHODS: The in vitro biological activities, such as anti-tumor activity and antiviral activity of Nova and recombinant human interferon alpha-2b (rhIFN-α2b) was performed; in vivo anti-tumor activity in nude mice was also tested. Flow cytometry, histo-pathological analysis including HE staining and immunohistochemistry, and surface plasmon resonance assay were performed to investigate the underlying mechanisms analysis. RESULTS: Nova exhibited stronger anti-cancer effects compared to rhIFN-α2b in vitro and in vivo. The antitumor mechanisms of Nova may be related to S phase arrest, pro-apoptosis, and inhibition of tumor angiogenesis. Moreover, Nova exhibited a higher binding affinity for IFN receptor 2 (IFNR2) than rhIFN-α2b, which is one of the possible reasons accounting for its stronger actions against tumor cells compared with rhIFN-α2b. CONCLUSION: Nova has strong antitumor activity and could be a potentially effective therapeutic drug for cancer.

Improved detection of variants in recombinant human interferon alpha-2a products by reverse-phase high-performance liquid chromatography on a core-shell stationary phase.

The detection of variants is one of the important aspects in quality control of recombinant DNA drugs. In this study, a gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method with fluorescence detection is described for the separation of interferon alpha-2a (rhIFN α-2a) from several product related variants. The methodology employed a core-shell C18 column with a linear gradient elution of 0.2% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 1.0mL/min, and the temperature of the column was maintained at 60°C. The method was validated in terms of linearity, sensitivity, intra- and inter-day variations. Compared to the European Pharmacopeia RP-HPLC method of rhIFN α-2a analysis, this new method can separate N-methionylated variant in both drug substance and finished product, and analyze the variants in untreated, oxidized sample and slightly degraded samples more efficiently. In conclusion the method has an improved capability to detect variants in rhIFN α-2a products.

[Measurement of the amino acid sequence for the fusion protein FP3 with LC-MS/MS].

The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.

[Quality control of recombinant oncolytic adenovirus/p53].

To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis. The result conformed that all modified regions were in consistent with theoretical ones. Particle number was 2.0 x 10(11) mL(-1) determined by UV (A260). Infectious titer was 5.0 x 10(10) IU mL(-1) analyzed by TCID50. In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A450 ratio of nucleoprotein in virus infection group to control group was 5.2. Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI(IC50) of this gene therapy preparation was 1.0. The tumor cells targeted replication ability of recombinant virus was determined by TCID50 titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI. TCID50 titer ratio of tumor cell infection group to normal cell infection control group was 398. The IE-HPLC purity of virus was 99.5%. There was less than 1 copy of wild type adenovirus within 1 x 10(7) VP recombinant virus. Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume III. The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard. The study also provided reference for quality control of other oncolytic viral vector products.

Synthesis and activity of an octapeptide inhibitor designed for SARS coronavirus main proteinase.

The outbreak of SARS, a life-threatening disease, has spread over many countries around the world. So far there is no effective drug for the treatment of SARS. Stimulated by the binding mechanism of SARS-CoV Mpro with the octapeptide AVLQSGFR reported recently as well as the "Chou's distorted key" theory, we synthesized the octapeptide AVLQSGFR for conducting various biochemical experiments to investigate the antiviral potential of the octapeptide against SARS coronavirus (BJ-01). The results demonstrate that, compared with other compounds reported so far, AVLQSGFR is the most active in inhibiting replication of the SARS coronavirus, and that no detectable toxicity is observed on Vero cells under the condition of experimental concentration.

Expression of human nerve growth factor beta gene in central nervous system mediated by recombinant adeno-associated viruses type-2 vector.

BACKGROUND: Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer's disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor beta (hNGFbeta) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption. METHODS: rAAV-2 containing hNGFbeta gene was constructed. The ability of hNGFbeta gene mediated by rAAV-2 vector (rAAV-2/hNGFbeta) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFbeta in the culture supernatant of BHK-21 cells infected by rAAV-2/hNGFbeta. rAAV-2/hNGFbeta and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFbeta concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope. RESULTS: After 48 hours, hNGFbeta content in supernatant was up to (188.0 +/- 28.6) pg/ml when BHK-21 cells were infected by rAAV-2/hNGFbeta at multiplicity of infection (MOI) 1.0 x 10(6) vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hNGFbeta. Whole brain hNGFbeta content in rAAV-2/hNGFbeta transferred group was up to (636.2 +/- 140.6) pg/ml. hNGFbeta content of BBB disruption in rAAV-2/hNGFbeta infused group increased significantly compared to the control group (P <0.05). GFP expression was clearly observed in brain sections of rAAV-2/GFP transferred group. CONCLUSION: rAAV-2/hNGFbeta successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.

[Study on methods for quality control of recombinant human neu epitope peptide 12].

AIM: To establish methods and requirements for the quality control of recombinant human neu epitope peptide 12. METHODS: Biological activity of recombinant human neu epitope peptide 12 was evaluated in FVB/N transgenic mice (TgN MMTV neu 202 Mul, Jackson Lab., USA) administered with samples. The percentage of antibody-positive mice detected by ELISA was used in the biological activity evaluation. The peptide map was performed by peptic digestion. The antigen content was determined by SEC-HPLC. RESULTS AND CONCLUSION: The quality control methods, such as biological activity, peptide map, antigen content, and the requirements for the quality control of recombinant human neu epitope peptide 12 were established. The established methods and requirements were already used for the quality control of recombinant human neu epitope peptide 12 products.

[Study on methods and requirements for quality control of recombinant human tumor necrosis factor receptor Fc fusion protein].

AIM: To establish methods and requirements for quality control of recombinant human tumor necrosis factor receptor Fc fusion protein (rhTNFR-Fc). METHODS: Biological potency of rhTNFR-Fc was determined by neutralizing the bioactivity of TNF-alpha. rhTNFR-Fc samples were reduced by beta-mercaptoethanol and the peptide map was performed by tryptic digestion. Residual protein A and the host cell protein content were detected by ELISA. Anti-TNFR and anti-Fc antibodies were used in ELISA for detection of the rhTNFR-Fc content. RESULTS: The quality control methods, such as bioassay, peptide map, residual protein A detection, were established and used for quality control of rhTNFR-Fc. The unit of rhTNFR-Fc (AU) was defined according to the international unit of TNF-alpha. The specific activity was up to 8 x 10(4) AU.mg-1. The requirements for quality control of rhTNFR-Fc were established. CONCLUSION: The methods and requirement were used for quality control of rhTNFR-Fc products.

[Quality control of recombinant adeno-associated virus type 2/human blood coagulation factor IX].

AIM: To establish quality control requirements and methods for recombinant adeno-associated virus(rAAV) type 2/human blood coagulation factor IX (rAAV-2/hFIX). METHODS: Identification of rAAV genome fragments, potential contaminants including wild type AAV(wtAAV) and helper virus, were detected by PCR. Purity of rAAV-2/hFIX was analyzed by cation-exchange HPLC and SDS-PAGE. Virus partical numbers were performed by dot blot assay. hFIX expression was demonstrated by ELISA and potency of hFIX was verified by APTT. RESULTS: Identity of rAAV-2/hFIX was proved. Residues of wtAAV and helper virus were conformed to requirements. Purity of rAAV-2/hFIX were more than 98%. Partical numbers of rAAV-2/hFIX were more than 1.0 x 10(15) VG.L-1. hFIX expression was more than 20.0 micrograms.L-1. hFIX potency was verified by APTT following rAAV-2/hFIX injected to FIX gene knockout mice, potency results conformed to requirements. CONCLUSION: The methods and requirements had been established for quality control of rAAV-2/hFIX.

[Quality control for recombinant adenovirus-IL2].

AIM: To investigate the quality and optimized test methods and establish the quality specification of recombinant adenovirus-IL2. METHODS: The titer of Adv-IL2 was measured by cytopathic effect (CPE). Hela cells were infected with Adv-IL2 in vitro, the expressed IL-2 and its bioactivity in Hela cell were determined by enzyme-linked immunosorbent assay (ELISA) and 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) respectively. The purity of Adv-IL2 was analysed by UV and IE-HPLC method. The molecular weight and enzyme digestive map of Adv-IL2 genome were analysed by electrophoresis. The characteristic gene E2B, IL-2 expression casseter and foreign factors (RCV, HIV, HBV, HCV) were detected with polymerase chain reaction (PCR). Other tests were carried out according to the Chinese Requirements for Biological Products. RESULTS: Adv-IL2 was generated efficiently with a titer of 3 x 10(10) pfu.mL-1. The expressed IL-2 and its bioactivity were 25 ng.mL-1 and 700 u.mL-1 respectively. A260 nm/A280 nm was 1.23. The purity determined by IE-HPLC was higher than 98%. The molecular weight, enzyme digestive map of Adv-IL2 genome, the characteristic gene E2B and IL-2 expression casseter conformed to expected values. CONCLUSION: The specification for Adv-IL2 is established and can be used for the quality control of the product.