Assessment of methods for determination of glycan composition of erythropoietin.

Erythropoietin (EPO) is a monomeric, highly glycosylated, protein hormone (molecular size around 30-35 kD), produced mainly in adult kidneys, which acts principally on red blood cell progenitors and precursors to promote red cell production. Therapeutic EPO products are widely used biotherapeutics. They are mainly produced by recombinant DNA technology in mammalian cells and their biological activity is closely linked to the degree of N-glycan sialylation. Determination of the sialic acids' content and complexity by glycan mapping therefore appears critical to ensure the quality and efficacy of the EPO therapeutic products. The European Directorate for the Quality of Medicines & HealthCare organised a study (BSP144) under the aegis of the Biological Standardisation Programme to assess N-glycan mapping tests with the aim of incorporating a standard method into the European Pharmacopoeia monograph 'Erythropoietin concentrated solution' (1316). The use of a 'reagent panel' consisting of six EPO preparations with a range of iso-electric properties facilitated comparison between laboratories and methodologies. Based on the study results, a robust and repeatable HPAEC-PAD chromatographic method was identified and work to introduce it in the monograph as an example method has been initiated.

Establishment of an erythropoietin CRS with stable measurable dimer content for SEC system suitability qualification

The European Pharmacopoeia (Ph. Eur.) monograph 1316 'Erythropoietin concentrated solution' prescribes that the dimer content of therapeutic erythropoietin (EPO) preparations must not exceed 2% as determined by Size-Exclusion Chromatography (SEC). This report describes the evaluation of a candidate Chemical Reference Substance (cCRS) to serve as system suitability reference material for the qualification of SEC systems used to assess dimer and oligomer content in EPO solutions. The study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) was performed with the participation of six European laboratories which tested the candidate material and the EPO for physicochemical tests CRS batch 1. The candidate material was shown to be a suitable reference material for the determination of the resolving capability of the SEC system for separation of dimer and higher oligomers from monomeric EPO. The cCRS was adopted by the Ph. Eur. Commission as Erythropoietin for SEC system suitability CRS batch 1 following consideration of the report. The importance of the resolving capability of the SEC system, as defined by the peak ratios or the peak-to-valley resolution, together with the asymmetry of the peaks eluted, and the linear response of the UV detector were all seen as critical parameters. Therefore, the monograph Erythropoietin concentrated solution (1316) was revised concomitantly to take account of the CRS and to set acceptance criteria for these critical parameters..

Quantification of low levels of methionine oxidation in erythropoietin drug substance.

A pharmacopoeial monograph under development for recombinant human erythropoietin (rhEPO) drug substance is likely to contain a specification limit for the proportion of the methionine-oxidised variant. Methionine oxidation has no effect on the folded structure and global thermodynamic stability of rhEPO but can decrease biological activity [1]. We describe here the development of a reference standard, a calibrated mixture of the native and oxidised tryptic peptides which contain methionine-54, and an optimised peptide mapping procedure to support this assay. The approach may be developed for analysis of drug product or generalised for other assays in which product-related impurities are quantified by peptide mapping.

Establishment of the Ph. Eur. erythropoietin chemical reference substance batch 1.

The Erythropoietin (EPO) European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 3 was calibrated in 2006 by in vivo bioassay and was used as a reference preparation for these assays as well as for the physicochemical methods in the Ph. Eur. monograph Erythropoietin concentrated solution (1316). In order to avoid the frequent replacement of this standard and thus reduce the use of animals, a new EPO Chemical Reference Substance (CRS) was established to be used solely for the physicochemical methods. Here we report the outcome of a collaborative study aimed at demonstrating the suitability of the candidate CRS (cCRS) as a reference for the physicochemical methods in the Ph. Eur. monograph. Results from the study demonstrated that for the physicochemical methods currently required in the monograph (capillary zone electrophoresis (CZE), polyacrylamide gel electrophoresis (PAGE)/immunoblotting and peptide mapping), the cCRS is essentially identical to the existing BRP. However, data also indicated that, for the physicochemical methods under consideration for inclusion in a revised monograph (test for oxidised forms and glycan mapping), the suitability of the cCRS as a reference needs to be confirmed with additional work. Further to completion of the study, the Ph. Eur. Commission adopted the cCRS as "Erythropoietin for physicochemical tests CRS batch 1" to be used for CZE, PAGE/immunoblotting and peptide mapping.

Collaborative study for the establishment of erythropoietin BRP batch 4.

The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for erythropoietin (EPO) is used as a working standard for potency determination of EPO preparations by in vivo bioassay as prescribed in the Ph. Eur. monograph Erythropoietin concentrated solution (1316). The BRP batch 3 was calibrated in 2006 and its stocks are depleted. The European Directorate for the Quality of Medicines & HealthCare (EDQM) thus initiated a project to calibrate a replacement batch in International Units against the WHO 3(rd) International Standard (IS) for Erythropoietin, recombinant, for bioassay (11/170). A Ph. Eur. Chemical Reference Substance (CRS) was established recently for use as reference in some of the physicochemical tests prescribed in the monograph. Therefore, the EPO BRP batch 4 was only calibrated for the normocythaemic and polycythaemic mouse in vivo bioassays described in the Assay section of the Ph. Eur. monograph (1316). The collaborative study involved seven laboratories from Europe, the USA and South America. The results confirmed that the candidate BRP (cBRP) is suitable for use as a reference preparation in the potency determination of EPO medicinal products by bioassay (using the normocythaemic or polycythaemic mouse methods). The outcome of the study enabled the Ph. Eur. Commission to establish the proposed standard as erythropoietin BRP batch 4 in November 2014 for use as a reference preparation solely for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 13 000 IU/vial. Furthermore, the potency of BRP3 was confirmed during the study, thus warranting a good continuity of the IU.

Regulatory requirements for therapeutic proteins: the relationship between the conformation and biological activity of filgrastim.

Higher order structure, including conformation, is considered a critical quality parameter of therapeutic proteins, and is mandatory information in development of first use and bio-similar therapeutic protein drugs, the assumption being that the biological activity of a protein is directly dependent on its adoption of a 'correct' conformation. Studies on the relationship between conformation and activity depend on the ability to induce conformational changes in proteins, and conventional approaches such as thermal or chemical denaturation are incompatible with bioactivity measurements. To explore the relationship between bio-activity and conformational studies, we have studied variants of the therapeutic protein filgrastim (rec met huGCSF) which have been mutated by the replacement of helical alanine residues with glycine, to destabilise the conformation of the molecule. In the GCSF A-G mutant series studied, single conformation-destabilising amino-acid substitutions significantly reduced the biological activity. These effects were not, however correlated with changes in secondary structure measurable by far-UV Circular Dichroism (CD) spectroscopy. Only the more extensively mutated double and triple substitutions showed measurable reductions in alpha-helical structure by CD. We conclude that in this system, GCSF does not readily adopt a reduced-activity altered conformational state which can be detected by low resolution techniques such as CD. In contrast, reductions in biological activity do reflect reductions in conformational stability, possibly caused by time-dependent degradation of the protein in the cell-proliferation bioassay. Although not a formal model of biosimilarity, we suggest that our results could inform the regulatory process in determining appropriate experimental approaches to meeting regulatory requirements for higher order structural analysis of therapeutic proteins.

The role of the vagus nerve in mediating the long-term anorectic effects of leptin.

Leptin, the product of the obese (ob) gene, is mainly known for its regulatory role of energy balance by direct activation of hypothalamic receptors. Recently, its function in the acute control of food intake was additionally attributed to activation of the vagus nerve to regulate meal termination. Whether vagal afferent neurones are involved in longer term effects of leptin on food intake, however, remains undetermined. Using vagotomised (VGX) rats, we sought to clarify the contributions of vagal afferents in mediating the long-lasting effect of leptin on appetite suppression. Intraperitoneal (i.p.) injection of leptin (3.5 mg/kg) attenuated food intake at 4, 6, 8 and 24 h and body weight at 24 h postinjection in SHAM-operated rats; however, this response was not abrogated by vagotomy. In a separate study using immunohistochemistry, we observed leptin-induced Fos expression in the nucleus tractus solitarii, a brain structure where vagal afferent fibres terminate. This signal was not attenuated in VGX animals compared to the SHAM group. Moreover, leptin treatment led to a similar level of nuclear STAT3 translocation, a marker of leptin signalling, in the hypothalami of SHAM and VGX animals. In addition to the effects of leptin, vagotomy surgery itself resulted in a decrease of 24 h food intake. Analyses of brains from saline-treated VGX animals revealed a significant induction of Fos in the nucleus tractus solitarii and changes in agouti-related peptide and pro-opiomelanocortin mRNA expression in the hypothalamus compared to their SHAM counterparts, indicating that the vagotomy surgery itself induced a modification of brain activity in areas involved in regulating appetite. Collectively, our data suggest that vagal afferents do not constitute a major route of mediating the regulatory effect of leptin on food intake over a period of several hours.

Collaborative study for the establishment of erythropoietin BRP batch 3.

The European Pharmacopoeia (Ph. Eur.) monograph on Erythropoietin concentrated solution (1316) specifies that identification and assay are performed using pharmacopoeial methods requiring the use of a reference preparation. To replace the current erythropoietin Biological Reference Preparation (BRP) of Ph. Eur., in 2006, the European Directorate for the Quality of Medicines undertook a collaborative study designed to establish a replacement batch. In order to guarantee continuity, the formulation of the candidate batch was similar to that of previous batches (1 and 2). The methods chosen to qualify the new standard were those included in the current monograph. The study was defined to allow calibration of the candidate by in vivo bioassay in terms of the current World Health Organization (WHO) International Standard (IS) and to assign a unitage. The suitability of the candidate preparation to serve as a reference standard for the other pharmacopoeial analytical procedures was also investigated. The collaborative study involved 16 laboratories from Europe, Australia, Canada, China, Japan, South Korea and the United States of America. Participants carried out biological and physicochemical assays on the candidate erythropoietin BRP batch 3 (cBRP3), using batch 2 (BRP2) and where necessary the 2nd World Health Organization International Standard (WHO 2nd IS) for recombinant erythropoietin as the reference standards. It was demonstrated that the replacement batch is appropriate for use as erythropoietin BRP in the context of the control of erythropoietin concentrated solutions according to the Ph. Eur. monograph (1316). However as regards the potency of BRP2 and cBRP3 in the mouse bioassay unexpected observations were made. Direct calibration of BRP2 against the WHO 2nd IS yielded, in all laboratories, results that were systematically higher than the potency of 32,500 IU/vial assigned by direct calibration against the WHO 2nd IS in the former study. It was therefore recommended to assign the potency of cBRP3 against BRP2, using the average of all results that were not considered as outlying obtained in the collaborative study, in order to guarantee continuity of unitage between the successive BRP batches. The outcome of the study enabled the Ph. Eur. Commission to establish the proposed standard as 'erythropoeitin BRP batch 3' (BRP3). BRP3 was established in June 2007 for use as a reference preparation for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 35,280 IU/vial, the identification by capillary zone electrophoresis, by polyacrylamide gel electrophoresis, immunoblotting and peptide mapping and as a reference for checking the system suitability of size-exclusion chromatographic procedures used in the test for dimers and related substances of higher molecular mass in the Ph. Eur. monograph (1316).

Collaborative study for the establishment of erythropoietin BRP batch 2.

The preparation and establishment of the 2nd European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for erythropoietin was the goal of a project run within the framework of the European Biological Standardisation Programme. The project, coded BSP062, was carried out between October 2002 and July 2003. The candidate preparation (cBRP2) was prepared in a similar manner to the first BRP batch (BRP1), as follows: -50:50 (weight/weight) blending of the two erythropoietin preparations currently available on the European market (epoietin-alpha and epoietin-beta), -lyophilisation using a protein-free carrier formulation to allow use of the standard for both biological and physico-chemical assay methods, -each vial contains approximately 250 microg erythropoietin. The cBRP2 was analysed in a collaborative study, carried out with the following aims: -to calibrate cBRP2 by in vivo bioassay in terms of the International Standard for erythropoietin, and assign a unitage, -to demonstrate continuity of unitage with BRP1, -to evaluate the suitability of cBRP2 to serve as a reference material for physico-chemical tests of erythropoietin. The collaborative study involved 14 laboratories both from Europe, and from Australia, Canada, South-Korea and the United States of America. Participants carried out biological and physicochemical assays on the candidate BRP batch 2, using BRP 1 and the 2nd World Health Organization (WHO) International Standard (IS) for recombinant erythropoietin as the reference standards. It was demonstrated that: -an assigned potency of 32,500 U per vial would maintain continuity between BRP1 and BRP2 in terms of the IS for erythropoietin, -the replacement batch was appropriate for use as erythropoietin BRP in the context of the control of erythropoietin concentrated solutions according to the Ph. Eur. monograph 1316. In July 2003, the Ph. Eur. Commission established the proposed standard as 'Erythropoeitin BRP batch 2' for use as a reference preparation for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 32,500 U/vial, the identification by capillary zone electrophoresis (CZE), by polyacrylamide gel electrophoresis, immunoblotting and peptide mapping and as a reference for checking the system suitability of size exclusion chromatographic procedures used in the test for 'Dimers and related substances of higher molecular mass'.

Endogenous interleukin-10 is required for the defervescence of fever evoked by local lipopolysaccharide-induced and Staphylococcus aureus-induced inflammation in rats.

We tested the hypothesis that endogenous interleukin (IL)-10 limits the fever induced by a Gram-negative bacterial toxin (Escherichia coli lipopolysaccharide, LPS) and a Gram-positive bacterial toxin (Staphylococcus aureus), when these toxins are injected into a subcutaneous air pouch (I.PO.) in rats. Injection of LPS or S. aureus caused fevers that were reduced in amplitude and duration by simultaneous administration of rat recombinant IL-10. The inhibition of fever by IL-10 was accompanied by a significant reduction in the toxin-evoked increases in concentrations of immunoreactive IL-6 at the site of inflammation and of IL-6 and IL-1 receptor antagonist in the circulation. Conversely, neutralisation of endogenous IL-10 in the pouch increased the amplitude and dramatically increased the duration of toxin-evoked fever, and augmented toxin-induced increases in pouch tumour necrosis factor-alpha, IL-1beta, and especially IL-6. Our data support a crucial regulatory role for endogenous IL-10 in limiting the fever responses during both Gram-negative and Gram-positive infections.

Progress and problems in the replacement of in vivo pharmacopoeial bioassays for hormones.

In vivo bioassays for peptide and protein hormones have, until recently, represented a major use of laboratory animals. Advances in physico-chemical methodology, particularly HPLC, have moved most of these substances into the "non-biological" category. Newer soluble regulators (interferons, cytokines, growth factors), will probably be controlled by in vitro bioassays, obviating the need for validation of alternatives. Validation of in vitro alternatives for glycoproteins remains problematic, and is the subject of current research and development.

The Second International Standard for somatropin (recombinant DNA-derived human growth hormone): preparation and calibration in an international collaborative study.

A preparation of somatropin (recombinant DNA-derived human growth hormone) was prepared as lyophilised ampoules according to WHO procedures for international biological standards. The candidate preparation (98/574) was evaluated in an international collaborative study (16 laboratories, nine countries), with the following aims: (i) to determine the suitability of the preparation to serve as the International Standard for somatropin by studying its performance in the current range of physico-chemical and biological assay methods employed for somatropin; (ii) to assign a content in terms of the existing (first) International Standard for somatropin, using the currently recognised assay procedure (Size Exclusion High Performance Liquid Chromatography, SE HPLC); (iii) to confirm the specific biological activity of the candidate preparation; (iv) to confirm the stability of the candidate preparation. On the basis of the collaborative study WHO agreed that: the preparation in ampoules coded 98/574 is suitable to serve as the next WHO International Standard for somatropin; the preparation in ampoules coded 98/574 should be established as the second International Standard for somatropin, with a defined ampoule content of 1.95 mg total somatropin plus somatropin-related proteins per ampoule; the specific activity of the preparation should be defined as 3.0 IU/mg somatropin.

The rat interleukin 10 receptor: cloning and sequencing of cDNA coding for the alpha-chain protein sequence, and demonstration by western blotting of expression in the rat brain.

cDNA coding for the alpha chain of the rat interleukin 10 (IL-10) receptor was amplified by polymerase chain reaction (PCR), cloned and sequenced. The nucleic acid coding sequence exhibited 88% and 68% homology with the mouse and human IL-10 receptor sequences, respectively. The translated protein exhibited 83% and 61% homology with the mouse and human IL-10 receptor proteins. Specific antibodies were raised to the extracellular domain of the rat IL-10 receptor expressed as a secreted protein in recombinant Drosophila S2 cells. Western blotting using these antibodies demonstrated the presence of the IL-10 receptor in five major regions of the rat brain (cortex, cerebellum, hippocampus, hypothalamus and pituitary), supporting a role for IL-10 as a central regulator of inflammation.

Rat interleukin-10: production and characterisation of biologically active protein in a recombinant bacterial expression system.

Interleukin-10 is an anti-inflammatory Th1 immunosuppressive cytokine, the active form of which is a non-covalent homodimer, and which exhibits species-specificity both with respect to structure and biological activity. The rat homologue of IL-10 shares 73% identity with human IL-10 at the amino-acid sequence level, and has, in addition to the two disulphide bonds present in human IL-10, a fifth, unpaired cysteine (cys-149). Preparation of rat IL-10 by bacterial expression followed by solubilisation and refolding in a glutathione redox system, results in a molecule in which cys-149 is almost entirely oxidised, existing either as disulphide dimer or as a mixed disulphide with glutathione, and which has less than 1% of the activity of the native (cys-149-SH) form of the molecule. Site directed mutagenesis of rat IL-10 to replace cys-149 with tyrosine produces a molecule which readily adopts the active conformation upon solubilisation and refolding, and which is recoverable in good yield from bacterial expression systems. Comparison of the biological activities of rat IL-10tyr149 and commercial rat IL-10 preparations confirms that the activity of native-sequence rat IL-10 is either reduced or absent. It is proposed therefore that the biosynthetic analogue rat IL-10tyr149 is a more useful molecule to investigate the biological actions of IL-10 in the rat.

Rat tumour necrosis factor-alpha: expression in recombinant Pichia pastoris, purification, characterization and development of a novel ELISA.

Tumour necrosis factor-alpha is a pro-inflammatory cytokine involved in many aspects of acute phase and immune responses. Species specificity in the biological action and receptor binding of TNF-alpha make it desirable to use homologous reagents in experimental models, both in vivo and in vitro. As the rat is the model of choice in many investigations on fever, trauma and pathology, there is a need for specific rat reagents. In this paper, we describe the production of recombinant rat TNF-alpha in milligram quantities, using a methylotrophic yeast expression system, Pichia pastoris. Recombinant TNF-alpha was produced intracellularly in a soluble form, cells were lysed and the protein purified by ammonium sulphate precipitation, Sephadex G75 fractionation and finally, ion-exchange chromatography. The purified recombinant rat TNF-alpha had a molecular mass of 17401.38 +/- 0.38 Da, which is within 1 Da of the value predicted by the sequence data, taking into account N-acetylation of the initial methionine residue and a single disulphide bridge between amino acids 70 and 101. Recombinant rat TNF-alpha was shown to be 20 x fold more biologically active in the WEHI cytotoxicity assay, than the human standard preparation. Polyclonal antibodies were raised against purified recombinant rat TNF-alpha, these reagents were used to develop a novel enzyme-linked immunosorbant assay (ELISA). The ELISA was sensitive to 10 pg.ml- 1 rat TNF-alpha and was specific for TNF-alpha, showing no cross-reactivity with rat IL-1alpha, rat IL-1beta, rat IL-1Ra or rat IL-6. The ELISA was used to measure TNF-alpha in the plasma of rats injected with bacterial endotoxin and in cultures of rat white blood cells. The ELISA was shown to be a robust method suitable for use in assaying samples generated in both in vivo or in vitro experiments.

International standards for growth hormone.

Since 1955, with the establishment by the World Health Organization of the first International Standard (IS) for bovine growth hormone (GH) for bioassay, there have been a number of developments in the standardization of GH. Two GH ISs are in current use: the IS for human GH established in 1982 and the IS for somatropin (recombinant DNA-derived GH) established in 1994. The availability of two such standards does not encourage reproducibility of GH estimates between different laboratories. The author proposes that the international validation and adoption of the IS for somatropin, which has the advantage of an internationally agreed specific activity of 3 IU/mg, as the primary reference standard for all GH immunoassays would finally eliminate this source of variation in GH estimates.

Rat interleukin 6: expression in recombinant Escherichia coli, purification and development of a novel ELISA.

Interleukin 6 (IL-6) is a cytokine involved in many aspects of the acute phase and immune responses. Cloning of rat IL-6 cDNA into the pET-21d expression plasmid under control of a bacteriophage T7 RNA polymerase promoter system allowed isopropylthio-galactopyranoside (IPTG)-inducible production of recombinant rat IL-6 in Escherichia coli. The cloning, expression and purification of rat IL-6 is described. In this expression system, rat IL-6 was produced in insoluble inclusion bodies. The protein was solubilized in 6 M guanidine hydrochloride and refolded in a glutathione redox system. Refolded rat IL-6 was purified to homogeneity using anion-exchange chromatography on SP-Trisacryl. The purified recombinant rat IL-6 had a molecular mass of 21 756.38+/-0.25 Da, which is within 0.01% of the predicted value, taking into account cleavage of the N-terminal methionine residue and the formation of two disulfide bridges. Recombinant rat IL-6 was 2-3-fold more bioactive than the human standard preparation in the B9 hybridoma bioassay. Purified rat IL-6 was used to raise polyclonal antibodies in sheep and these reagents were used to develop a novel rat IL-6 enzyme-linked immunosorbent assay (ELISA). The ELISA is sensitive to 10 pg/ml and has been shown to detect IL-6 in plasma from rats injected with lipopolysaccharide (LPS).