Recombinant Human Interferon Beta-1a Research Articles

Comparative study of commercial media to improve GMP manufacturing of recombinant human interferon β-1a by CHO cells in perfusion bioreactor.

Chinese hamster ovary cells are the main cellular factories for production of a wide range of recombinant proteins in biopharmaceutical industry. Recombinant human Interferon beta-1a (rh-IFN β-1a), as a cytokine is broadly used to treat multiple sclerosis. In this work, the cell line producing rh-IFN β-1a was studied to improve cell density along with the specific expression. For this reason different cell culture experiments were done using different commercial serum-free media to find the appropriate media providing higher cell density. It was shown DMEMF12, DMEM:ProCHO5, and CHO-S-SFM II led to higher cell density and shorter doubling time. Next, using these media, fed-batch, and perfusion culture with temperature shift were implemented to investigate the best condition for industrial-scale manufacturing of rh-IFN β-1a in terms of higher cell density and product expression yield. The results demonstrated that CHO-S-SFM II media and a thermally biphasic condition provide enhanced expression of rh-IFN β-1a in perfusion bioreactor.

Comparison of the efficacy and safety of FP-1201-lyo (intravenously administered recombinant human interferon beta-1a) and placebo in the treatment of patients with moderate or severe acute respiratory distress syndrome: study protocol for a randomized controlled trial

BackgroundAcute respiratory distress syndrome (ARDS) results in vascular leakage, inflammation and respiratory failure. There are currently no approved pharmacological treatments for ARDS and standard of care involves treatment of the underlying cause, and supportive care. The vascular leakage may be related to reduced concentrations of local adenosine, which is involved in maintaining endothelial barrier function. Interferon (IFN) beta-1a up-regulates the cell surface ecto-5′-nucleotidase cluster of differentiation 73 (CD73), which increases adenosine levels, and IFN beta-1 may, therefore, be a potential treatment for ARDS. In a phase I/II, open-label study in 37 patients with acute lung injury (ALI)/ARDS, recombinant human IFN beta-1a was well tolerated and mortality rates were significantly lower in treated than in control patients.Methods/designIn this phase III, double-blind, randomized, parallel-group trial, the efficacy and safety of recombinant human IFN beta-1a (FP-1201-lyo) will be compared with placebo in adult patients with ARDS. Patients will be randomly assigned to receive 10 μg FP-1201-lyo or placebo administered intravenously once daily for 6 days and will be monitored for 28 days or until discharged from the intensive care unit. Follow-up visits will then take place at days 90, 180 and 360. The primary endpoint is a composite endpoint including any cause of death at 28 days and days free of mechanical ventilation within 28 days among survivors. Secondary endpoints include: all-cause mortality at 28, 90, 180 and 360 days; organ failure-free days; length of hospital stay; pharmacodynamic assessment including measurement of myxovirus resistance protein A concentrations; and measures of quality of life, respiratory and neurological function at 180 and 360 days. The estimated sample size to demonstrate a reduction in the primary outcome between groups from 30% to 15% is 300 patients, and the study will be conducted in 70–80 centers in nine countries across Europe.DiscussionThere are no effective specific treatments for patients with ARDS and mortality rates remain high. The results from this study will provide evidence regarding the efficacy of a potential new therapeutic agent, FP-1201-lyo, in improving the clinical course and outcome for patients with moderate/severe ARDS.Trial registrationEuropean Union Clinical Trials Register, no: 2014-005260-15. Registered on 15 July 2017.

Identification of Oxidation Sites and Covalent Cross-Links in Metal Catalyzed Oxidized Interferon Beta-1a: Potential Implications for Protein Aggregation and Immunogenicity

Oxidation via Cu(2+)/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu(2+)/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl)benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated, and digested protein was employed to localize chemical degradation products. Oxidation products of methionine, histidine, phenylalanine (Phe), tryptophan, and Tyr residues were identified throughout the primary sequence. Covalent cross-links via 1,4- or 1,6-type addition between primary amines and DOCH (2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl)propanoic acid, an oxidation product of Phe and Tyr) were detected. There was no evidence of disulfide bridge, Schiff base, or dityrosine formation. The chemical cross-links identified in this work are most likely responsible for the formation of covalent aggregates of IFNβ1a induced by oxidation, which have previously been shown to be highly immunogenic.

Immunogenicity of Recombinant Human Interferon Beta Interacting with Particles of Glass, Metal, and Polystyrene

Aggregates play a major role in the immunogenicity of recombinant human interferon beta (rhIFNβ), a protein used to treat multiple sclerosis. A possible cause of aggregation is interaction between therapeutic protein and surfaces encountered during processing, storage, and administration. Moreover, proteins may adsorb to particles shed from these surfaces. In this work, we studied the immunogenicity of recombinant human interferon beta-1a (rhIFNβ-1a) interacting with glass microparticles, stainless steel microparticles, and polystyrene nanoparticles. At physiological pH, rhIFNβ-1a readily adsorbed to the particles, while the degree of adsorption was influenced by the ionic strength of the phosphate buffer. Front-face fluorescence showed that the tertiary structure of rhIFNβ-1a slightly changed upon adsorption to glass. The interaction with stainless steel microparticles resulted in increased levels of aggregates in the free protein fraction. Furthermore, protein adsorbed to stainless steel microparticles was more difficult to desorb than protein adsorbed to glass. Incubation with stainless steel considerably enhanced the immunogenicity of rhIFNβ-1a in transgenic mice immune tolerant for human interferon beta. The protein fraction adsorbed on stainless steel particles was responsible for this. In conclusion, rhIFNβ-1a adsorbs to common hydrophilic surface materials, possibly increasing the immunogenicity of the protein.

Oxidized and Aggregated Recombinant Human Interferon Beta is Immunogenic in Human Interferon Beta Transgenic Mice

PurposeTo study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFNβ-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice.MethodsUntreated rhIFNβ-1a was degraded by metal-catalyzed oxidation, H2O2-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFNβ-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15× during 3 weeks. Both binding and neutralizing antibodies were measured.ResultsAll rhIFNβ-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFNβ-1a contained high levels of covalent aggregates as compared with untreated rhIFNβ-1a. H2O2-treated rhIFNβ-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFNβ-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFNβ-1a. All degraded samples showed alterations in tertiary protein structure. Wild-type mice showed equally high antibody responses against all preparations. Transgenic mice were discriminative, showing elevated antibody responses against both metal-catalyzed oxidized and H2O2-treated rhIFNβ-1a as compared to untreated and guanidine-treated rhIFNβ-1a.ConclusionsOxidation-mediated aggregation increased the immunogenicity of rhIFNβ-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic.

Phase II Radiation Therapy Oncology Group trial of conventional radiation therapy followed by treatment with recombinant interferon-β for supratentorial glioblastoma: Results of RTOG 9710

The aim of this study was to determine whether recombinant human interferon beta-1a (rhIFN-beta), when given after radiation therapy, improves survival in glioblastoma. After surgery, 109 patients with newly diagnosed supratentorial glioblastoma were enrolled and treated with radiation therapy (60 Gy). A total of 55 patients remained stable after radiation and were treated with rhIFN-beta (6 MU/day i.m., 3 times/week). Outcomes were compared with the Radiation Therapy Oncology Group glioma historical database. RhIFN-beta was well tolerated, with 1 Grade 4 toxicity and 8 other patients experiencing Grade 3 toxicity. Median survival time (MST) of the 55 rhIFN-beta-treated patients was 13.4 months. MST for the 34 rhIFN-beta-treated in RPA Classes III and IV was 16.9 vs. 12.4 months for historical controls (hazard ratio [HR] = 1.27, 95% confidence interval [CI] = 0.89-1.81). There was also a trend toward improved survival across all RPA Classes comparing the 55 rhIFN-beta treated patients and 1,658 historical controls (HR = 1.24, 95% CI = 0.94-1.63). The high rate of early failures (54/109) after radiation and before initiation of rhIFN-beta was likely caused by stricter interpretation of early radiographic changes in the current study. Matched-pair and intent-to-treat analyses performed to try to address this bias showed no difference in survival between study patients and controls. RhIFN-beta given after conventional radiation therapy was well tolerated, with a trend toward survival benefit in patients who remained stable after radiation therapy. These data suggest that rhIFN-beta warrants further evaluation in additional studies, possibly in combination with current temozolomide-based regimens.

Development of a Transgenic Mouse Model Immune Tolerant for Human Interferon Beta

Therapeutic proteins may induce antibodies that inhibit their efficacy or have other serious biological effects. There is a great need for strategies to predict whether a certain formulation will induce an immune response. In principle, conventional animals develop an immune response against all human proteins no matter how they are formulated, which restricts their use. The aim of this study was to develop a mouse model immune tolerant for human interferon beta (hIFNbeta). A transgenic mouse model immune tolerant for hIFNbeta was developed by making C57Bl/6 mice transgenic for the hIFNbeta gene. To evaluate the model, both wild-type and transgenic mice were immunized with recombinant human interferon beta 1a (rhIFNbeta-1a) and recombinant human interferon beta 1b (rhIFNbeta-1b). Serum antibodies against rhIFNbeta were detected by ELISA. The genetically modified mice were shown to be immune tolerant for mammalian cell-derived rhIFNbeta-1a, which has a relative low immunogenicity in patients. However, Escherichia coli-derived rhIFNbeta-1b, known to have a relatively high immunogenicity in patients, was shown not only to be immunogenic in the wild-type mice but could also break the immune tolerance of the genetically modified mice. This animal model offers the possibility to study the many factors influencing the immunogenicity of hIFNbeta and test new formulations before going into clinical trials. The model also provides the first evidence that the rhIFNbetas differ in the immunological mechanisms responsible for the development of antibodies.

General pharmacokinetic model for drugs exhibiting target-mediated drug disposition.

Drugs that bind with high affinity and to a significant extent (relative to dose) to a pharmacologic target such as an enzyme, receptor, or transporter may exhibit nonlinear pharmacokinetic (PK) behavior. Processes such as receptor-mediated endocytosis may result in drug elimination. A general PK model for characterizing such behavior is described and explored through computer simulations and applications to several therapeutic agents. Simulations show that model predicted plasma concentration vs. time profiles are expected to be polyexponential with steeper distribution phases for lower doses and similar terminal disposition phases. Noncompartmental parameters always show apparent Vss and CL(D) decreasing with dose, but apparent clearance decreases only when the binding process produces drug elimination. The proposed model well captured the time-course of drug concentrations for the aldose reductase inhibitor imirestat, the endothelin receptor antagonist bosentan, and recombinant human interferon-beta 1a. This type of model has a mechanistic basis and considerable utility for fully describing the kinetics for various doses of relevant drugs.

Comparative pharmacokinetics and pharmacodynamics of recombinant human interferon beta-1a after intramuscular and subcutaneous administration

Comparative pharmacokinetics and pharmacodynamics of recombinant human interferon beta-1a after intramuscular and subcutaneous administration

Comparative pharmacokinetics and pharmacodynamics of recombinant human interferon beta-1a after intramuscular and subcutaneous administration.

The pharmacokinetics and pharmacodynamics of recombinant human interferon beta (IFN-beta-1a) were compared after intramuscular administration of two preparations (Rebif(R) and AvonexTM) and subcutaneous administration of Rebif(R). Healthy volunteers (n = 30) received a single dose (60 µg) of each of the three treatments in a randomised crossover study. Serum concentrations of IFN-beta were measured by enzyme-linked immunosorbent assay over 24 h after dosing. Pharmacodynamics were assessed by measurement of intracellular 2',5'-oligoadenylate synthetase activity, and serum neopterin and beta2-microglobulin concentrations, over 144 h after dosing. There was no significant difference between the three treatments in peak serum IFN-beta concentrations (Cmax) or area under the concentration-time curve (AUC). No significant differences in pharmacodynamic measures were observed between the three treatments. It is concluded that the bioavailability of IFN-beta-1a is equivalent after subcutaneous or intramuscular administration of Rebif(R), and intramuscular administration of AvonexTM. Copyright Rapid Science Ltd

Impaired Bioavailability of Interferon Beta-1a when Administered Intramuscularly by Needle-Free Injection

Intramuscular (IM) or subcutaneous (SC) drug administration of small molecules and protein has been demonstrated by use of needle-free jet injection methods. One device that achieves needle-free parenteral administration, BIOJECTORr`, is commercially available and was evaluated for IM delivery of interferon beta-1a. Recombinant human interferon beta-1a (IFN β-1a) is a glycosylated protein containing 166 amino acids and has a molecular weight of 22.5 kDa. Needle-free jet injection of IFN β-1a with the BIOJECTORr` was assessed in a human Phase I trial. The study was a randomized, open-label crossover in which 12 healthy subjects each received 60 μ of IFN β-1a as an IM injection by standard needle administration and by needle-free jet injection. Blood samples for pharmacokinetic (serum activity, PK) and pharmacodynamic (serum neopterin, PD) determinations were collected through 144 hours post-dose. Mean serum antiviral activity AUC values for needle-free and standard needle injection were 218 and 531 U x h/ml, respectively; corresponding Cmax values were 19.7 and 29.0 U/ml. Median Tmax following both treatments was 12 hours. The relative bioavailability of IFN β-1a, needle-free to standard needle injection, was 41.1% with 90% confidence limits of 24.4% to 69.3%. Mean serum neopterin EAUC values for needle-free and standard needle injection were 114 and 325 ng x h/mL, respectively; corresponding Emax values were 2.3 and 5.6 ng/mL. The ratio of serum neopterin EAUC, needle-free to standard needle, was 34.9% with 90% confidence limits of 23.4% to 52.1%. Injection site reactions were substantially more frequent following needle-free injection; however, systemic side effects were less frequent. Intramuscular needle-free jet injection and needle-based injection of a 22.5-kDa glycoprotein do not produce equivalent systemic PK or PD responses.