A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation.

An evaporative light scattering detection (ELSD) based high-performance liquid chromatography (HPLC) method is developed for the determination of polysorbate 80 (tween 80) in therapeutic protein formulations. The method is simple and overcomes the difficulties associated with specificity and sensitivity. The method is suitable for the quantitation of polysorbate 80 in the usual formulation range (0.01-0.1%) as well as in trace amounts ≥13 µg/mL. The analysis is based on the removal of protein first by solid-phase extraction using Oasis HLB cartridges followed by HPLC analysis using Inertsil ODS-3 C 18 column (4.6×150 mm, 5 µm) using reversed-phase conditions. The detector response changes exponentially with an increase in polysorbate concentration. A very good linear fit of log ELSD response against log polysorbate 80 concentration is observed. The specificity, sensitivity, precision, and accuracy of the method are suitable for the quantitation of polysorbate 80 in protein formulations.

Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance—possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics.

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants.To date, only a few studies on its side reactions have been published.We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine.The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS).Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini.Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon-carbon bonds in cysteine.(

S-thiolation is a reversible post-translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S-thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S-thiolation in recombinant proteins. In this work, reversed-phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4-phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S-thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S-thiolation in recombinant proteins.

The glycosylation state of individual antibodies was imaged using an atomic force microscope with a probe modified with lectins and an image acquisition system that permits simultaneous acquisition of sample topography data along with a map of lectin binding sites.

Journal Article Atomic Force Microscopy - Visualizing Proteins and Their Aggregates Under Natural Conditions Get access L Obenauer-Kutner, L Obenauer-Kutner Bristol-Myers Squibb Search for other works by this author on: Oxford Academic Google Scholar M Lin, M Lin Bristol-Myers Squibb Search for other works by this author on: Oxford Academic Google Scholar S Gozo, S Gozo Bristol-Myers Squibb Search for other works by this author on: Oxford Academic Google Scholar A Flesher, A Flesher Bristol-Myers Squibb Search for other works by this author on: Oxford Academic Google Scholar MJ Grace MJ Grace Bristol-Myers Squibb Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 13, Issue S02, 1 August 2007, Pages 1686–1687, https://doi.org/10.1017/S1431927607071085 Published: 05 August 2007

Pegylated interferon (PEG-IFN) has become standard therapy for hepatitis C virus (HCV) infection. We evaluated whether PEG-IFN pharmacodynamics and pharmacokinetics account for differences in treatment outcome and whether these parameters might be predictors of therapeutic outcome. Twenty-four IFN-naïve, HCV/human immunodeficiency virus–coinfected patients received PEG-IFN α-2b (1.5 μg/kg) once weekly plus daily ribavirin (1,000 or 1,200 mg) for up to 48 weeks. HCV RNA and PEG-IFN α concentrations were obtained from samples collected frequently after the first 3 PEG-IFN doses. We modeled HCV kinetics incorporating pharmacokinetic and pharmacodynamic parameters. Although PEG-IFN concentrations and pharmacokinetic parameters were similar in sustained virological responders (SVRs) and nonresponders (NRs), the PEG-IFN α-2b concentration that decreases HCV production by 50% (EC50) was lower in SVRs compared with NRs (0.04 vs. 0.45 μg/L [P = .014]). Additionally, the median therapeutic quotient (i.e., the ratio between average PEG-IFN concentration and EC50[C̄/EC50]), and the PEG-IFN concentration at day 7 divided by EC50 (C(7)/EC50) were significantly increased in SVRs compared with NRs after the first (10.1 vs. 1.0 [P = .012], 2.8 vs. 0.3 [P = .007], respectively) and second (14.0 vs. 1.1 [P = .016], 5.4 vs. 0.4 [P = .02], respectively) PEG-IFN doses. All 3 parameters may be used to identify NRs. In conclusion, PEG-IFN concentrations and pharmacokinetic parameters do not differ between SVRs and NRs. In contrast, pharmacodynamic measurements—namely EC50, the therapeutic quotient, and C(7)/EC50—are different in coinfected SVRs and NRs. These parameters might be useful predictors of treatment outcome during the first month of therapy. (HEPATOLOGY 2006;43:943–953.)

Background/Aims: To compare the pharmacokinetics, pharmacodynamics, and antiviral activity of peginterferon alfa-2b and peginterferon alfa-2a in patients with chronic hepatitis C virus genotype 1.Methods: Thirty-six patients were randomised to peginterferon alfa-2b (1.5 mu g/kg/week) or peginterferon alfa-2a (180 mu g/week) for 4 weeks, then in combination with ribavirin (13 mg/kg/day) for a further 4 weeks. The pharmacokinetic profile of both peginterferons, mRNA expression of a selected group of interferon-induced gene transcripts, and serum HCV-RNA levels were assessed.Results: Patients receiving peginterferon alfa-2b had significantly greater up-regulation of interferon-alfa response genes compared with those receiving peginterferon alfa-2a. Correspondingly, patients treated with peginterferon alfa-2b also had a significantly greater loglo maximum and loglo time-weighted average decrease in serum HCV-RNA. A greater proportion of peginterferon alfa-2b patients achieved a >=, 2.0 log(10) reduction in serum HCV-RNA levels by week 8 (72% vs 44% of peginterferon alfa-2a patients, P = 0.09). There was an approximately 16-fold greater exposure to peginterferon in the serum of patients treated with peginterferon alfa-2a.Conclusions: These findings suggest that the biological activity, measured by early interferon-induced gene transcripts and early antiviral responsiveness, may have been greater in patients treated with peginterferon alfa-2b despite their lower exposure to the drug compared with patients treated with peginterferon alfa-2a. (c) 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Differential scanning calorimetry was used to identify the thermal stability profile of the replication deficient and protein IX deleted recombinant adenovirus type 5 that contains the p53 transgene (rAd/p53) in phosphate buffered saline (vPBS) or 10% glycerol (TRIS/phosphate buffer). The wildtype adenovirus (Ad/WT) and purified hexon protein (major capsid protein) were also evaluated in 10% glycerol (TRIS/phosphate buffer) as controls. The thermal profile of rAd/p53 revealed three endothermic transitions (T1, T2 and T3) occurring between 25 °C and 90 °C. T1, which occurred at 46.7 °C in vPBS and 49.4 °C in TRIS/PO4 10% glycerol buffer, was irreversible following repeated scanning and attributed to the degradation of the intact vector. The latter two endothermic transitions, T2 and T3, occurring at 69 °C and 78 °C, respectively, corresponded with the two transitions of purified hexon in temperature and amount of heat absorbed. The thermal profile of Ad/WT revealed four endothermic transitions at 51.5 °C (T1), 70.5 °C (T2A), 73.6 °C (T2B), and 77.4 °C (T3). The higher temperature of degradation as well as additional transition was attributed to the presence of protein IX associated with the hexon. The positions and excess molar heat capacities of the intact rAds were found to be affected by pH, glycerol, vector concentration and the presence or absence of protein IX in the capsid. Irreversibility of T1 implied that the degradation of the intact virus may follow first-order kinetics. The thermal scan rate dependence of T1 further confirmed that degradation of the intact virus may be first-order. The apparent activation energies for the degradation of the intact vectors were determined from the scan rate dependence of T1 and shown to be affected by protein IX in the capsid and solution conditions. Analysis of rAd samples incubated at 45 °C by Field Emission Electron Microscopy (FESEM) confirmed that loss of single particles was first-order. Although aggregates were observed in the samples, degradation appeared to be the dominant reaction leading to disappearance of single virions from the aqueous matrix. Based on thermal and FESEM analysis, an empirical model was proposed that accounted for the disappearance of single rAd particles. At or near T1, degradation of rAd particles followed a unidirectional, pseudo-first order reaction. However, at lower temperatures, disappearance of single virions resulted from competing irreversible degradation and aggregation reactions.

Electron microscopy has a pivotal role as an analytical tool in pharmaceutical research. However, digital image data have proven to be too large for efficient quantitative analysis. We describe here the development and application of an automated image processing (AIP) program that rapidly quantifies shape measurements of recombinant adenovirus (rAd) obtained from digitized field emission scanning electron microscope (FESEM) images. The program was written using the macro-recording features within Image-Pro Plus software. The macro program, which is linked to a Microsoft Excel spreadsheet, consists of a series of subroutines designed to automatically measure rAd vector objects from the FESEM images. The application and utility of this macro program has enabled us to rapidly and efficiently analyze very large data sets of rAd samples while minimizing operator time.

Therapeutic pegylated interferon-αs (IFN-α) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-α molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-α molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-α2b that were monopegylated using 5–30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys1, His34, Lys31, Lys83, Lys121, Lys131, and Lys134. The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-α2b control. The highest residual activity was observed with the His34 positional isomers, and the lowest was observed with the Cys1 positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys134 > Lys83 ∼ Lys131 ∼ Lys121 > Lys31. The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-α to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His34 positional isomer suggests that this site may be favorable for pegylating IFN-α2b molecules.

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.

Type I interferon-α(IFN-α) has proven to be a clinically effective antiviral and antineoplastic drug for 20 yr (1). Recently, pegylated forms of IFN-α have been commercially produced and have shown superior clinical efficacy to unpegylated IFN-α for reducing HCV viral load with less frequent dosing required for the patient (2,3). The superior clinical efficacy is probably derived from the enhanced serum half-life of the pegylated IFN-α in patients. However, pegylation also reduces the in vitro activity of the core IFN-α protein (4,5). Understanding the structural implications of pegylation on IFN-α activity is critical for biologically characterizing the commercial drug product. In vitro characterization provides a basis for establishing consistency in the manufacturing process for the precursor IFN-α and the final pegylated product. The better characterized the product is, the higher the confidence is for assessing and demonstrating comparability between pegylated products when comparing changes in process of manufacture, site of manufacture, or like-product from different sources.

Pegylated interferon alpha-2, alone or in combination with ribavirin, has become the standard therapy for patients with chronic hepatitis C infection. Pegylation of interferon alpha-2 results in a substantially extended half-life that permits once-weekly dosing, because of reduced clearance and more sustained absorption. The size of PEG moiety appears to influence the relative antiviral activities of peginterferon alpha-2. Increasing the size of the polyethylene glycol (PEG) moiety results in a reduction of the specific antiviral activity of the pegylated protein. For example, peginterferon alpha-2b (12 kDa) has an in vitro antiviral specific activity 25–35-fold higher than peginterferon alpha-2a (40 kDa). The antiviral activity of pegylated interferon alpha-2 is also governed by the site of pegylation of the interferon alpha core proteins. Interferon alpha-2a is monopegylated at four major positional lysine (Lys) residues. The major site of interferon alpha-2b monopegylation is histidine (His34), with additional pegylation sites at lysine and cysteine residues. The 12 kDa pegylated His34 positional isomer of peginterferon alpha-2b has the highest antiviral and antiproliferative in vitro specific activity compared with both the 12 kDa Lys positional isomers and the 40 kDa Lys positional isomers. The correlative effects of size and site of pegylation on the JAK/STAT signalling pathway, as evidenced by differences in the formation of the Stat1 homodimer complex, are suggestive of a receptor-mediated mechanism that governs the antiviral activity of pegylated interferons. The elucidation of the in vitro effects of pegylation is important and this will ultimately have a positive impact on the in vivo efficacy of treatment for patients with hepatitis C.

The pleiotropic biologic effects of interferon (IFN) are mediated through regulation of the expression of numerous IFN-sensitive genes. Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were analyzed to study the immunoregulatory and antiviral messenger RNAs (mRNAs) and proteins regulated by pegylated IFN-alpha2b (PEG-IFN-alpha2b) and IFN-alpha2b. A dose-dependent and time-dependent response for multiple IFN-regulated genes was observed. IFN-dependent protein production and secretion were correlated with IFN-regulated mRNA induction. Overall regulation of gene expression patterns for PEG-IFN-alpha2b and IFN-alpha2b was comparable, even though the antiviral activity of PEG- IFN-alpha2b demonstrated a longer biologic half-life in vitro compared with IFN-alpha2b. To study the heterogeneity of responses, PBMCs obtained from over 25 healthy donors were analyzed. Within a particular donor dataset, gene-specific and dose-dependent responses to PEG- IFN-alpha2b treatment, demonstrated in both the amplitude of transcriptional upregulation and the duration of sustained mRNA upregulation, were observed. However because of donor heterogeneity, the amplitude of a given transcriptional response could not be predicted for a specific dose of PEG- IFN-alpha2b. Notably, mRNA levels of oligoadenylate synthetase (OAS), double-stranded RNA (dsRNA)-activated protein kinase (PKR), IP-10, IFN-stimulated gene 54 (ISG54), and ISG15 were upregulated after 120 h of continuous PEG-IFN-alpha2b treatment. These results suggest that the use of antiviral and immunoregulatory protein mRNA levels as markers to assess the therapeutic efficacy of IFN-alpha2b and PEG-IFN-alpha2b against viral and neoplastic diseases in clinical trials is promising but will require further analysis using clinical patient samples.

A cohort study was designed to evaluate the efficiency of gene transfer and whether biological activity from the expressed therapeutic gene resulted after administration of a recombinant adenovirus containing the human wild-type p53 (p53wt) gene (rAd-p53 SCH 58500). The cohort study was conducted in five trial subjects with recurrent ovarian cancer. Each trial subject received multiple cycles of rAd-p53 SCH 58500, each cycle comprised of doses of 7.5 × 1013 particles on each of five consecutive days. Subjects were treated with rAd-p53 SCH 58500 alone during Cycle 1 and in combination with gemcitabine during the subsequent cycles. Both tumor biopsies and peritoneal aspirates were collected and evaluated for gene transfer and evidence of the biological activities of the expressed p53wt gene. Using quantitative PCR and RT-PCR, and in situ PCR, gene transfer and expression were documented in tumor biopsies (four of five patients) collected from Cycle 1. Furthermore, upregulation of p21/WAF1, bax and mdm-2, and downregulation of survivin were observed in these same tumor biopsy samples, suggesting that intraperitoneal administration of rAd-p53 SCH 58500 leads to detectable p53 biological activity in target tumor tissue. In addition, gene transfer and its expression were observed in cells obtained from peritoneal aspirates. These fluids were mainly comprised of polymorphonuclear neutrophils, indicating that successful gene transfer can be achieved by multiple cycle intraperitoneal administration of recombinant adenovirus.

The relative activities of interferon-alpha2b (IFN-alpha2b) and polyethylene glycol(12000)-IFN-alpha2b (PEG-IFN-alpha2b) were assessed in cell culture studies using WM9 melanoma or ACHN renal cell carcinoma cell lines. Interferon-alpha2b and PEG-IFN-alpha2b had identical antiproliferative activities when tested in cell proliferation studies conducted with equivalent antiviral units of each IFN preparation. Neither IFN formulation was effective in inducing apoptosis in WM9 melanoma cells, but both increased slightly the percentage of ACHN cells undergoing apoptosis as assessed by Annexin V staining. Interferon-alpha2b and PEG-IFN-alpha2b both activated signal transducer and activator of transcription complexes, and the duration of complex activation was similar for both IFN formulations. Induction of different IFN-stimulated genes was assessed by Northern blotting and the quantitative real-time reverse transcription-coupled polymerase chain reaction (RT-PCR) in WM9 melanoma, ACHN renal cell carcinoma, U937 lymphoma, and MOLT-4 and Mono Mac 6 leukemia cell lines. Interferon-alpha2b and PEG-IFN-alpha2b had equivalent gene-modulatory activities within each of these tumor cell lines, although cell line-specific induction patterns were observed. When compared with the antiviral 50% inhibitory concentration (IC(50)) values, the dose-dependent gene expression data correlated with cell sensitivity to IFN treatment. Together, the drug comparability and cell sensitivity data suggest a predictive relation between dose, time, antiviral activity, and gene transcription effects. Therefore, although the specific activity of IFN-alpha2b is approximately three times greater than PEG-IFN-alpha2b, the two preparations have identical in vitro biologic activities when applied to cells at equivalent antiviral units.