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.

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.

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.

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.

A Cell-Based Strategy for Bioactivity Determination of Long-Acting Fc-Fusion Recombinant Human Growth Hormone.

The long-acting growth hormone (LAGH) is a promising alternative biopharmaceutical to treat growth hormone (GH) deficiency in children, and it was developed using a variety of technologies by several pharmaceutical companies. Most LAGH preparations, such as Fc fusion protein, are currently undergoing preclinical study and clinical trials. Accurate determination of bioactivity is critical for the efficacy of quality control systems of LAGH. The current in vivo rat weight gain assays used to determine the bioactivity of recombinant human GH (rhGH) in pharmacopoeias are time-consuming, expensive, and imprecise, and there are no recommended bioassays for LAGH bioactivity in pharmacopoeias. Therefore, we developed a cell-based bioassay for bioactivity determination of therapeutic long-acting Fc-fusion recombinant human growth hormone (rhGH-Fc) based on the luciferase reporter gene system, which is involved in the full-length human GH receptor (hGHR) and the SG (SIE and GAS) response element. The established bioassay was comprehensively validated according to the International Council for Harmonization (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia, and is highly precise, time-saving, simple, and robust. The validated bioassay could be qualified for bioactivity determination during the research, development, and manufacture of rhGH-Fc, and other LAGH formulations.

Pharmacokinetics of PEGylated recombinant human endostatin (M2ES) in rats.

AIM: M2ES is PEGylated recombinant human endostatin. In this study we investigated the pharmacokinetics, tissue distribution, and excretion of M2ES in rats. METHODS: (125)I-radiolabeled M2ES was administered to rats by intravenous bolus injection at 3 mg/kg. The pharmacokinetics, tissue distribution and excretion of M2ES were investigated using the trichloroacetic acid (TCA) precipitation method. RESULTS: The serum M2ES concentration-time curve after a single intravenous dose of 3 mg/kg in rats was fitted with a non-compartment model. The pharmacokinetic parameters were evaluated as follows: Cmax=28.3 µg·equ/mL, t1/2=71.5 h, AUC(0-∞)=174.6 µg·equ·h/mL, Cl=17.2 mL·h(-1)·kg(-1), MRT=57.6 h, and Vss=989.8 mL/kg for the total radioactivity; Cmax=30.3 µg·equ/mL, t1/2=60.1 h, AUC(0-∞)=146.2 µg·equ·h/mL, Cl=20.6 mL·h(-1)·kg(-1), MRT=47.4 h, and Vss=974.6 mL/kg for the TCA precipitate radioactivity. M2ES was rapidly and widely distributed in various tissues and showed substantial deposition in kidney, adrenal gland, lung, spleen, bladder and liver. The radioactivity recovered in the urine and feces by 432 h post-dose was 71.3% and 8.3%, respectively. Only 0.98% of radioactivity was excreted in the bile by 24 h post-dose. CONCLUSION: PEG modification substantially prolongs the circulation time of recombinant human endostatin and effectively improves its pharmacokinetic behavior. M2ES is extensively distributed in most tissues of rats, including kidney, adrenal gland, lung, spleen, bladder and liver. Urinary excretion was the major elimination route for M2ES.

[Characterization and comparison of interferon reference standards using UPLC-MS].

The study aims to characterize and compare interferon reference standards from 5 manufacturers. By testing molecular mass and trypsin-digested peptide mass mapping, the amino acid sequence was verified and post-translational modifications such as disulfide bond were identified. Results show that the molecular mass and amino acid sequence were consistent with theory; the disulfide bonds of 4 lots of interferon were Cys1-Cys98/Cys29-Cys138, 1 lot was Cys29-Cys139/Cys86-Cys99; N-terminal "+Met", acetyl N-terminal and Met oxidation were identified in part of the sample. UPLC-MS can be used to characterize and compare interferon reference standards from different manufacturers.

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.

Expression, purification, and characterization of recombinant mouse nerve growth factor in Chinese hamster ovary cells.

Mouse NGF (mNGF) extracted from mouse submaxillary gland has been approved on the market in China for treating nerve damage caused by N-hexane poisoning for over a decade, and many researches showed the clinical effectiveness of mNGF for the treatment of other nerve system diseases. The extracted mNGF have risks of potential viral contamination due to the animal origin. Here, we report the successful expression, purification, and characterization of recombinant mNGF (rmNGF). An expression plasmid of mouse nerve growth factor (mNGF) was constructed and transfected into CHO-S cells. Stable transfectants were obtained using a two-phase selection scheme with the addition of different concentrations of methotrexate and puromycin. Recombinant mNGF (rmNGF) was purified from cell culture medium by a two-step procedure: cation exchange followed by size-exclusion chromatography. The purity of rmNGF was 98.6% determined by size exclusion high performance liquid chromatography (SEC-HPLC). The molecular weight, isoelectric point and N-terminal sequence of rmNGF were identical to the theoretical values entirely. In TF-1/MTS, the specific activity of the protein was approximately 1.7×10(6)U/mg against rhNGF (the reference standard). In DRGs, the specific activity was approximately 7.3×10(5)AU/mg against mNGF (the reference standard). Our results showed that a high quality of rmNGF with marked biological activity comparable with mNGF was produced, and laid the basis for further research and development of rmNGF.

[Characterization of the primary structure of TNK-tissue plasminogen activator using LC-MS].

The primary structure of TNK-tissue plasminogen activator (TNK-tPA) was characterized using liquid chromatography-mass spectrometry (LC-MS). Firstly, the molecular mass of deglycosylated protein was measured. Then peptide mass mapping and MS/MS of the reduced, alkylated and trypsin-digested sample were tested and analyzed so as to verify its amino acid sequence and identify post-translational modifications. Results show that the amino acid sequence was consistent with designed structure; about 5% of M207 was oxidized; T61 was fucosylated with -80% occupancy; N103, N448 and N184 (-15% occupancy) were glycosylated with complex-type oligosaccharides. LC-MS coupled with proper sample pretreatment is approved to be a rapid and powerful approach to characterize the primary structure of TNK-tPA.

Development and calibration of a standard for the protein content of granulocyte colony-stimulating factor products.

This collaborative study characterizes a homogeneous standard for the protein content determination of granulocyte colony-stimulating factor (G-CSF) products with traceability of the measurement. The Kjeldahl method was used to determine the average protein content of G-CSF bulk as 2.505 mg/ml (95% C.I: 2.467-2.543 mg/ml, GCV 4.0%). Using G-CSF bulk as a traceability benchmark, the protein content of the final freeze-dried standard using reverse phase HPLC (RP-HPLC) was 215.4 µg protein per ampoule (95% C.I: 212.407-218.486 µg/ampoule, GCV 3.4%). A comparative study showed that there was no difference between using Filgrastim CRS (European Pharmacopeia G-CSF reference standard) and freeze-dried homogeneous standard when quantifying G-CSF protein content by RP-HPLC (P > 0.05). However, there were significant differences in the G-CSF protein content obtained using a serum albumin standard by Lowry assay and a G-CSF standard with RP-HPLC. Therefore, use of RP-HPLC with a freeze-dried homogeneous standard would eliminate the systematic errors introduced when using a serum albumin standard because of the differences in protein composition between the standard and the sample. It would also be helpful to use this method to compare the quality of G-CSF biosimilar products in situations where the protein content has been calibrated using various standards.

[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.

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon α2b.

Recombinant human interferon α2b (rhIFNα2b) is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C. The current identification test for rhIFNα2b is complex. In this study, an anti-rhIFNα2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNα2b. RhIFNα2b was used to immunize an alpaca, which established a phage nanobody library. After five steps of enrichment, the nanobody I22, which specifically bound rhIFNα2b, was isolated and inserted into the prokaryotic expression vector pET28a. After subsequent purification, the physicochemical properties of the nanobody were determined. A semiquantitative detection and rapid identification assay of rhIFNα2b was developed using this novel nanobody. To develop a rapid test, the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips. The developed rhIFNα2b detection assay had a limit of detection of 1 µg/mL. The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products. The principle of this novel assay is generally applicable for the rapid testing of other commercial products, with a great potential for routine use in detecting counterfeit recombinant protein products.

[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.

Capillary isoelectric focusing with UV fluorescence imaging detection enables direct charge heterogeneity characterization of erythropoietin drug products.

An imaged capillary isoelectric focusing (icIEF) - UV fluorescence imaging detection method is described for the direct charge heterogeneity characterization of recombinant human erythropoietin (rhEPO) drug products (DPs). rhEPO is one of the most important protein therapeutics for biopharmaceutical industry worldwide. As a heavily glycosylated protein therapeutic, its charge heterogeneity must be carefully monitored in each step of manufacturing and storage. Current charge characterization methods suffer from challenges to characterize rhEPO DPs, due to low sensitivity of the method and potential for interference from the DP's formulation. The method described herein leverages the separation power of imaged cIEF separation combined with the increased sensitivity afforded by UV fluorescence imaging detection and requires no pre-treatment of the DP sample prior to analysis. The method was evaluated initially using a simulated DP, and subsequently a mini method validation was performed using a commercial rhEPO DP sample according to the guideline set by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The limit of quantitation (LOQ) of the method is validated to be 20.3 IU/mL (or 0.10 µg/mL), which is approximately 100 times more sensitive than CZE - UV absorption detection method. To demonstrate the applicability of the method for use, 8 different commercial rhEPO DPs with concentrations ranging from 2000 IU/mL - 10,000 IU/mL were successfully evaluated. This method allows for sensitive, rapid analysis of low concentration rhEPO drug products without sample pre-treatment to provide critical charge heterogeneity information.

[Structure verification of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody].

Structure of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody was verified by the application of high-performance liquid chromatography-mass spectrometry (HPLC-MS)and N-terminal sequencer. Molecular masses, N-terminal sequences and peptide maps of the antibody treated with different reagents and enzymes were measured. Results indicate that the amino acid sequences of light and heavy chains and 10 disulfide bonds were consistent with theoretical structure. By comparison of molecular masses and peptide maps for the fully glycosylated and deglycosylated samples, the N-linked glycosylation site was identified. The method is simple, rapid, precise, and could be referred to the quality control and structure determination of other IgG1 products.

UPLC-MS assessment on the structural similarity of recombinant human erythropoietin (rhEPO) analogues from manufacturers in China for attribute monitoring.

Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy. Since the expiration of the patent of the innovator epoetin alfa, numerous rhEPO products have emerged in global markets. As described here, multiple complementary analytical approaches are utilized for the extensive characterization of rhEPO molecules, and more importantly for the structural comparison of the rhEPO analogues on the Chinese market. The focus of this study is placed on the overall glycosylation profiling, O-glycan profiling, and N-glycan mapping by UPLC-MS with an aim to develop an effective analytical methodology to monitor the product quality attributes of rhEPO analogues. Two rhEPO analogues manufactured in China were analyzed to demonstrate the principle of the developed methods. Each rhEPO product showed a characteristic glycoform profile with respect to the distribution of sialic acids across multi-antennary structures, the occurrence of O-glycosylation, O-acetylation on sialic acids, and the extension of N-glycan antennae with N-acetyllactosamine units. The study demonstrates that UPLC-MS is an effective analytical tool to characterize and monitor the glycosylation profiles among rhEPO analogues in order to detect and account for the divergence between rhEPO products, as well as the presence of unusual or unexpected glycans.

Interlaboratory method validation of imaged capillary isoelectric focusing methodology for analysis of recombinant human erythropoietin.

Recombinant human erythropoietin (rhEPO) is one of the most important biopharmaceuticals worldwide, with global sales expected to reach US$11.9 billion in 2020. The charge heterogeneity of rhEPO must be monitored throughout the entire production process. Imaged capillary isoelectric focusing (icIEF) is a promising method for monitoring rhEPO charge heterogeneity, but it must be validated according to the ICH guideline (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use). Here, a multi-laboratory joint method validation of the icIEF method for rhEPO analysis was performed according to the ICH Harmonized Tripartite Guideline on Analysis Procedure. This guideline includes specificity, precision, accuracy, linearity, range, LOQ and robustness, whereby precision is defined by the repeatability, intermediate precision and reproducibility.

Responsive Cells for rhEGF bioassay Obtained through Screening of a CRISPR/Cas9 Library.

Bioassay of recombinant protein products is important tests to ensure protein effectiveness. Some recombinant protein products have no cells used in their bioassay but instead use animal models, while others have no suitable method. Here, we developed a method to obtain responsive cells used in bioassay of proteins. After screening of a CRISPR/Cas9 library, we obtained a responsive cell line that grew faster in the presence of rhEGF (recombinant human epidermal growth factor) than that of control cells. We used this cell line for bioassay of rhEGF. This cell line, compared with the control cells, had a 2 day shorter operation time and had lower interference. The responsive cell line is more suitable for use in bioassay of rhEGF.