eg, the addition of methionine to enable expression in Escherichia coli or the removal of a cysteine to enhance stability. Other amino acid changes can be the result of mutations that occurred in the cell line from which the gene was cloned. These amino acids may or may not influence biological activity, and/or safety and antigenicity. The host cell also influences the nature of the product and its properties. Produc- tion in prokaryotics lacks post-translational modification such as glycosylation. The lack of sugar residues may influence stability or biological characteristics such as pharmaco- kinetic behaviour and receptor binding. Production in biological systems also leads inevitably to product heterogeneity. Mutation in the gene which is expressed may occur spontaneously. The fidelity of protein synthesis in bacterial hosts may be less than in mammalian systems. For instance in the case of interleukin-2 pro- duced in E coli, methionine is replaced by norleucine in about 20 per cent of the mole- cules. This may be related to an imbalance in available amino acids by the forced expression of a product which may consti- tute 40 per cent of the total protein synthesis of the host cells. The degree and type of glycosylation of the protein may also be variable due to the speed of production and the constituents of the production media. A number of other modifications may occur such as removal of N-terminal amino acids, abnormal folding by the formation of variant disulphide link- ages and de-sialation of sugar residues. Also a number of degradations may occur such as oxygenation, deamidation and aggregate formation.3

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.

Adenovirus-mediated gene therapy of bladder diseases has been limited by the inability to transduce the urothelium successfully using adenoviral vectors. We have sought to identify agents that would increase adenovirus-mediated transgene expression in the bladder. We have utilized a rat model to screen compounds for their ability to enhance viral transgene expression in the rat bladder. Rats received intravesical administration of replication-deficient adenovirus (rAd) formulated in various agents, and transgene expression was evaluated after 48 h by determining the amount of lacZ expression in the luminal epithelium of the bladder. We report the identification of two different polyamides, each capable of dramatically increasing viral transgene expression in the bladder without causing detectable alteration of the umbrella cell layer of the urothelium. We have utilized a carcinogen-induced rat bladder tumor model to demonstrate that these polyamides are also capable of enhancing viral transgene expression in tumor tissue. The identification of these polyamides potentiates the use of adenovirus-mediated gene therapy for the treatment of superficial bladder cancer or other bladder diseases.

The type I interferon-alpha (IFN-alpha) 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-alpha2b (Intron(R) 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-alpha2b 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-alpha2b 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 that approximately 50% of PEG Intron was monopegylated on the His(34) residue of the IFN-alpha2b protein. The highest antiviral activity of the pegylated positional isomers for PEG Intron was associated with the His(34) pegylated isomer. The specific activity for PEG Intron in an antiviral cytopathic protection assay was 28%, relative to Intron A. However, the potency of PEG Intron, defined as bioactivity independent of protein concentration, was comparable to Intron A at both the molecular and cellular levels in a battery of in vitro assays. Equivalent units of PEG Intron and Intron A were indistinguishable for the induction of several key IFN-induced genes, including 2',5'-oligoadenylate synthetase (2',5'-OAS) and protein kinase R (PKR), in Molt 4 cells. The antiviral dose-response curves revealed that there were no significant differences between PEG Intron and Intron A. This demonstrated that the introduction of more IFN-alpha2b protein associated with equivalent unit dosing of PEG Intron did not create any antagonism or agonism in the antiviral assay. In assays for the immune response, PEG Intron and Intron A displayed comparable potency for both natural-killer (NK) and lymphokine-activated killer (LAK) cell cytolytic activity and for the induction of class I major histocompatibility protein. These results demonstrate that PEG Intron maintains an in vitro biologic potency profile for both antiviral and immunotherapeutic activity that is highly comparable to that of Intron A.

Interferons display a wide range of antiviral, antiproliferative, and immunomodulatory activities on a variety of cell types and have been used to treat many diseases including hairy-cell leukemia and hepatitis B and C and have also been applied to other therapeutic areas. To improve the pharmacological properties of interferon (IFN) alpha-2b, a long-acting pegylated form (PEG-IFN) has been developed [PEG, monomethoxy poly(ethylene glycol) with average molecular mass of 12 000 Da]. PEG-IFN is a mixture of pegylated proteins with differing sites of PEG attachment. To identify the major positional isomer in the pegylated material [PEG-IFN(His-34)], NMR studies were conducted on a subtilisin-digested N-acetylated peptide of the major positional isomer [PEG-IFN(His-34)dig], synthetic peptide analogues containing His-34, as well as unmodified IFN and PEG-IFN(His-34). Our studies reveal a novel interferon-polymer attachment site as a histidine-linked interferon conjugate. We show that the major component of PEG-IFN is pegylated in the imidazole side chain of histidine-34. Chemical shift data suggest that pegylation occurs mainly at the N(delta)(1) position in the imidazole side chain of this residue. This positional isomer, PEG-IFN(His-34), comprises approximately 47% of the total pegylated species when PEG-IFN is synthesized under the current experimental conditions at pH 6.5 with an electrophilic derivative of PEG, succinimidyl carbonate PEG. The reversibility of the histidine modification was examined. The PEG-imidazole adduct in the intact protein, PEG-IFN(His-34), is labile but much more stable than in the peptide, PEG-IFN(His-34)dig. Apparently, the tertiary structure of the intact protein protects the His(34)-imidazole ring from depegylation.

HPLC analyses of GM-CSF in solution mixtures containing both GM-CSF and HSA showed losses of GM-CSF which could not be accounted for using conventional electrophoretic and/or RP-HPLC techniques. Further investigation of these mixtures by immunoblotting and by immunoaffinity chromatography demonstrated the presence of high molecular weight (>67,000) GM-CSF related species. No such species was detectable in solutions of GM-CSF alone. This experiment pointed to the formation of an adduct between GM-CSF and HSA in the solution mixtures. To probe further the hypothesis of a GM-CSF/HSA adduct, an immunologically based test was conceived which could react only with this type of hybrid molecule. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed using two antibodies, anti-GM-CSF (capture antibody) and anti-HSA (detection antibody), as part of the quantitation of GM-CSF/HSA adducts. After confirming its existence by ELISA, a GM-CSF/HSA adduct was isolated from the solution mixture containing both GM-CSF and HSA. This isolate served as a primary reference standard in the ELISA assay. The immunoassay has a subnanogram sensitivity and is highly specific for GM-CSF/HSA adducts in the presence of either free GM-CSF or free HSA. As a verification, conjugates of GM-CSF/HSA were synthesized using a cross-linking reagent. These covalent conjugates reacted positively in the ELISA and are employed as a convenient alternative reference standard.