Ronald Bordens

Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications

The type I interferon alpha family consists of small proteins that have clinically important anti-infective and anti-tumor activity. Interferon alpha-2b (Intron A) combination therapy with ribavirin is the current standard of care for the treatment of chronic hepatitis C virus infection. A drawback to the therapy however, is the short serum half-life and rapid clearance of the interferon alpha protein. Schering-Plough has developed a semi-synthetic form of Intron A by attaching a 12-kDa mono-methoxy polyethylene glycol to the protein (PEG Intron) which fulfills the requirements of a long-acting interferon alpha protein while providing significant clinical benefits. A detailed physicochemical and biological characterization of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Though pegylation appeared to decrease the specific activity of the interferon alpha-2b protein, the potency of PEG Intron, independent of protein concentration, was comparable to the Intron A standard at both the molecular and cellular level. Importantly, PEG Intron has demonstrated an enhanced pharmacokinetic profile in both animal and human studies. Recently, PEG Intron in combination with ribavirin has been shown to be very effective in reducing hepatitis C viral load and maintaining effective sustained viral suppression in patients. Because of the improved clinical benefits, it is anticipated that the PEG Intron plus ribavirin combination therapy will become the new standard of care for the treatment of chronic hepatitis C.

Interferon-α as an immunotherapeutic protein

Interferon‐α (IFN‐α) has proven to be a clinically effective antiviral and antineoplastic therapeutic drug for more than 16 years. During this time, evidence from in vitro laboratory studies and the clinical arena has supported the concept that IFN‐α is an immunotherapeutic drug. By regulating a diverse set of cytokines and their receptors, IFN‐α is uniquely positioned to prime the host immune response and provide an effective antineoplastic‐ and antiviral‐immune response. IFN‐α stimulates the innate cell‐mediated response and then participates in the transition of the initial host innate response into an effective adaptive‐immune response. IFN‐α also drives the adaptive cell‐mediated CD8+ T‐cell response and helps to maintain a CD4+ Th1‐cell population balance for an effective antineoplastic and antiviral host defense. This review will describe the current state of knowledge of IFN‐α as an immunoregulatory protein and address specific issues of IFN‐α as an immunotherapeutic for antineoplastic and antiviral diseases.

Structural and Biologic Characterization of Pegylated Recombinant IFN-α2b

The type I interferon-α (IFN-α) family is a family of natural small proteins that have clinically important anti-infective and antitumor activity. We have developed a semisynthetic protein-polymer conjugate of IFN-α2b (Intron® A) by attaching a 12,000-Da monomethoxypolyethylene glycol (PEG-12000) polymer to the protein. PEG conjugation is thought to increase the serum half-life and thereby prolong patient exposure to IFN-α2b without altering the biologic potency to the protein. Matrix-assisted laser desorption ionization/mass spectrometry (MALDI-MS), high-performance size exclusion chromatography (HPSEC), circular dichroism (CD) analysis and tryptic digestion peptide analysis of PEG Intron demonstrated that the IFN-α2b protein was approximately 95% monopegylated and that the primary, the secondary, and the tertiary structures were unaltered. Pegylation did not affect the epitope recognition of antibodies used for Intron A quantitation. An extensive analysis of the pegylated positional isomers revealed tha...

Pegylated Interferons for the Treatment of Chronic Hepatitis C Infection

BACKGROUND Interferon (IFN) alfa is a clinically effective therapy used in a wide range of viral infections and cell-proliferative disorders. Combination therapy with IFN alfa-2b and ribavirin is the current standard of care for the treatment of chronic hepatitis C (CHC) infection. However, standard IFN alfa has the drawbacks of a short serum half-life and rapid clearance. To overcome this problem, 2 pegylated forms of IFN have been developed and tested clinically. OBJECTIVE This article reviews the development and properties of pegylated IFN alfa-2b and pegylated IFN alfa-2a, and presents safety and efficacy data from recent clinical trials. METHODS Relevant clinical studies were identified through a MEDLINE search from 1966 through the present using the key words hepatitis C and interferon. Studies of the pegylated IFNs in humans were then selected. RESULTS Pegylated IFN alfa-2b is formed by covalent conjugation of a 12-kd mono-methoxy polyethylene glycol (PEG) molecule to IFN alfa-2b, and pegylated IFN alfa-2a by covalent conjugation of a 40-kd branched mono-methoxy PEG molecule to IFN alfa-2a. The 2 pegylated IFNs differ in the mixture of pegylation isomers resulting from their conjugation chemistry. Pegylated IFN alfa-2b has a prolonged serum half-life (40 hours) relative to standard IFN alfa-2b (7-9 hours). The greater polymer size of pegylated IFN alfa-2a acts to reduce glomerular filtration, markedly prolonging its serum half-life (72-96 hours) compared with standard IFN alfa-2a (6-9 hours). In clinical studies, once-weekly dosing of the pegylated IFNs was associated with a sustained virologic response in patients infected with hepatitis C virus (HCV). Once-weekly dosing with either of the pegylated IFNs was more effective than the respective thrice-weekly regimen of IFN alfa, with a comparable safety profile. The combination of once-weekly pegylated IFN and ribavirin effectively reduced HCV viral load and sustained viral suppression. CONCLUSIONS Once-weekly dosing with either pegylated IFN alfa-2b or pegylated IFN alfa-2a has been shown to produce significantly higher rates of viral eradication than standard thrice-weekly IFN alfa therapy without compromising safety. With respect to the treatment of CHC, the greatest anti-HCV efficacy has been achieved with the combination of once-weekly pegylated IFN and ribavirin.

Characterization of Replication-Competent Adenovirus Isolates from Large-Scale Production of a Recombinant Adenoviral Vector

Replication-deficient adenoviral vectors have been developed for the delivery of DNA sequences encoding a variety of proteins intended for the management of disease through gene therapy. One concern is the occurrence of replication-competent adenovirus (RCA) in the population of replication-deficient adenoviral vectors as a result of recombination or contamination. To address this concern, it is necessary to determine the frequency of occurrence and to fully characterize the molecular structure and biological infectivity of RCA. rAd/p53 is a pIX-deleted p53 gene therapy vector that is designed to lower the RCA occurrence and to deliver the tumor suppressor gene p53 for treatment of various cancers. Multiple preparations of the replication-deficient adenoviral vector rAd/p53 were tested for the presence of RCA, employing a sensitive biological assay. Single plaques from RCA-positive preparations of rAd/p53 were isolated for molecular characterization. All of the RCA isolates displayed a single unique structure that contains the complete E1 sequence of adenovirus type 5 but lacks the p53 sequence. The detailed sequence analysis of the RCA suggests that it is most likely generated as a result of recombination events between the rAd/p53 DNA and the 293 host adenoviral sequence. Results from viral infectivity analysis by flow cytometry demonstrate no substantial difference in infectivity of RCA, rAd/p53, and wild-type adenovirus type 5 in 293 cells.

Validation parameters for a novel biosensor assay which simultaneously measures serum concentrations of a humanized monoclonal antibody and detects induced antibodies

This report documents the validation of an assay using BIAcore 2000 that, with a single injection of mouse serum, allows the quantitation of a humanized monoclonal antibody and can also detect the presence of antibodies directed against this humanized antibody. The important components required for the validation of biosensor assays including precision, accuracy, linearity, stability of the immobilized ligand, specificity and sensitivity are addressed. The tandem assay that is used as a model for biosensor validations is accomplished by flowing each sample across the surface of two flowcells in sequence. The first flowcell has the antigen that the humanized mAb was generated against immobilized while the humanized mAb itself is immobilized on the second flowcell. The quantitation component of this assay is precise and accurate with a limit of quantitation of 1 microg/ml in mouse serum samples. Any antibodies directed against the humanized mAb can be detected and also characterized as to isotype. This unique assay can be performed with as little as 10 microl of serum which is then diluted ten-fold prior to analysis. The small sample requirement allows analysis of individual mouse serum samples rather than requiring the use of pooled samples from multiple mice. A further advantage of this assay is the automation capability which allows unattended operation.

Structure, biology, and therapeutic implications of pegylated interferon alpha-2b.

Derivatization of protein-based therapeutics with polyethylene glycol (pegylation) can often improve pharmacokinetic and pharmacodynamic properties of the proteins and thereby, improve efficacy and minimize dosing frequency. This review will provide an overview of pegylation technology and pegylated protein-based drugs being used or investigated clinically. The novel therapeutic, PEG Intron(R), formed by attaching a 12-kDa mono-methoxy polyethylene glycol (PEG) to the interferon alpha-2b protein, will be discussed in detail in terms of its structure, biological activities, pharmacokinetic properties, and clinical efficacy for the treatment of chronic hepatitis C. Detailed physicochemical and biological characterization studies of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Pegylation of Intron A at pH 6.5 results in a mixture of > or = 95% mono-pegylated isoforms with the predominant species (approximately 50%) derivatized to the His(34) residue with the remaining positional isomers pegylated at various lysines, the N-terminal cysteine, as well as serine, tyrosine, and another histidine residue. The anti-viral activity for each pegylated isomer showed that the highest specific activity (37%) was associated with the His(34)-pegylated isomer. Though pegylation decreases the specific activity of the interferon alpha-2b protein in vitro, the potency of PEG Intron was comparable to the Intron A standard at both the molecular and cellular level. The substituted IFN had an enhanced pharmacokinetic profile in both animal and human studies, and, when combined with ribavirin, was very effective in reducing hepatitis C viral load and maintaining sustained viral suppression in patients.

A highly sensitive electrochemiluminescence immunoassay for interferon alfa-2b in human serum

A highly sensitive immunoassay for the quantitative measurement of recombinant interferon alfa-2b (IFN alfa-2b) in serum was developed using the ORIGEN electrochemiluminescence (ECL) detection system. The ECL assay developed uses a ruthenylated mouse monoclonal anti-IFN-alfa antibody and a sheep polyclonal anti-IFN-alfa antibody that has been biotinylated. An immune complex, formed between the antibodies and the IFN in the serum, is captured by streptavidin coated paramagnetic beads. The assay is sensitive and can accurately measure 4 IU/ml in undiluted serum samples. In addition, this assay requires less labor than classical ELISAs and is not significantly affected by individual serum factors. The total assay variance for both intra- and inter-assay precision is < 10%. The assay is specific for the analyte. Mean percent recoveries in pooled and individual donor serum samples range from 84 to 114%. In addition, results of the ECL assay correlate well with a bioassay and were more accurate that an ELISA for the detection of interferon alfa-2b in individual human serum samples. The assay can be modified to measure other forms of the analyte and/or interferon in other matrices.

The Use of Field Emission Scanning Electron Microscopy to Assess Recombinant Adenovirus Stability

A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples.

Pegylated IFNs for chronic hepatitis C: an update.

For over a decade, IFN-α2 has been the standard treatment for chronic hepatitis C. However, the drug’s rapid clearance and short half-life have led to low rates of sustained virological response. Pegylation is a well-established method of modifying the pharmacological properties of IFNs, causing significant improvements in pharmacokinetics, which in turn lead to improved efficacy. Two pegylated forms of IFN-α2 have been developed: PEG-IFN-α2b and PEG-IFN-α2a, and their efficacy has been established in randomised, -controlled trials. However, the two differ significantly in structure, invitro activity and pharmacological properties, and this may translate into -differences in clinical efficacy. Comparative trials have been initiated that will provide insight into relative importance of pharmacokinetics, bioactivity and dosing regimen.