Interleukin-1 reduces food intake and body weight in rat by acting in the arcuate hypothalamus.

A reduction in food intake is commonly observed after bacterial infection, a phenomenon that can be reproduced by peripheral administration of Gram-negative bacterial lipopolysaccharide (LPS) or interleukin-1beta (IL-1ß), a pro-inflammatory cytokine released by LPS-activated macrophages. The arcuate nucleus of the hypothalamus (ARH) plays a major role in food intake regulation and expresses IL-1 type 1 receptor (IL-1R1) mRNA. In the present work, we tested the hypothesis that IL-1R1 expressing cells in the ARH mediate IL-1ß and/or LPS-induced hypophagia in the rat. To do so, we developed an IL-1ß-saporin conjugate, which eliminated IL-R1-expressing neurons in the hippocampus, and micro-injected it into the ARH prior to systemic IL-1ß and LPS administration. ARH IL-1ß-saporin injection resulted in loss of neuropeptide Y-containing cells and attenuated hypophagia and weight loss after intraperitoneal IL-1ß, but not LPS, administration. In conclusion, the present study shows that ARH NPY-containing neurons express functional IL-1R1s that mediate peripheral IL-1ß-, but not LPS-, induced hypophagia. Our present and previous findings indicate that the reduction of food intake after IL-1ß and LPS are mediated by different neural pathways.

Development of a stable chemically cross-linked erythropoietin dimer for use in the quality control of erythropoietin therapeutic products.

Erythropoietin (EPO) is a glycoprotein hormone which promotes red cell replenishment and is also a global biotherapeutic medicine widely used to treat anaemia resulting, for example, from chemotherapy. Requirements of the European Pharmacopoeia stipulate that the level of dimer must be quantified in clinical EPO products (with a limit of 2%). Quantification is hampered by the lack of reference preparations containing stable measurable levels of EPO dimer, but the reproducible generation of a stable dimerised EPO preparation is challenging. We describe here the development of a lyophilised, chemically cross-linked EPO preparation, which has good stability and may be used for calibration and system suitability assurance for the size exclusion chromatographic separation of EPO preparations. Graphical abstract.

An ELISA method to estimate the mono ADP-ribosyltransferase activities: e.g in pertussis toxin and vaccines.

ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. This new approach is easy to perform and adaptable in most laboratories. In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. coli, Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates. Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes.

International standards for monoclonal antibodies to support pre- and post-marketing product consistency: Evaluation of a candidate international standard for the bioactivities of rituximab.

The intrinsic complexity and heterogeneity of therapeutic monoclonal antibodies is built into the biosimilarity paradigm where critical quality attributes are controlled in exhaustive comparability studies with the reference medicinal product. The long-term success of biosimilars will depend on reassuring healthcare professionals and patients of consistent product quality, safety and efficacy. With this aim, the World Health Organization has endorsed the need for public bioactivity standards for therapeutic monoclonal antibodies in support of current controls. We have developed a candidate international potency standard for rituximab that was evaluated in a multi-center collaborative study using participants' own qualified Fc-effector function and cell-based binding bioassays. Dose-response curve model parameters were shown to reflect similar behavior amongst rituximab preparations, albeit with some differences in potency. In the absence of a common reference standard, potency estimates were in poor agreement amongst laboratories, but the use of the candidate preparation significantly reduced this variability. Our results suggest that the candidate rituximab standard can support bioassay performance and improve data harmonization, which when implemented will promote consistency of rituximab products over their life-cycles. This data provides the first scientific evidence that a classical standardization exercise allowing traceability of bioassay data to an international standard is also applicable to rituximab. However, we submit that this new type of international standard needs to be used appropriately and its role not to be mistaken with that of the reference medicinal product.

Tm-Values and Unfolded Fraction Can Predict Aggregation Rates for Granulocyte Colony Stimulating Factor Variant Formulations but Not under Predominantly Native Conditions.

Protein engineering and formulation optimization strategies can be taken to minimize protein aggregation in the biopharmaceutical industry. Short-term stability measures such as the midpoint transition temperature (Tm) for global unfolding provide convenient surrogates for longer-term (e.g., 2-year) degradation kinetics, with which to optimize formulations on practical time-scales. While successful in some cases, their limitations have not been fully evaluated or understood. Tm values are known to correlate with chemical degradation kinetics for wild-type granulocyte colony stimulating factor (GCSF) at pH 4-5.5. However, we found previously that the Tm of an antibody Fab fragment only correlated with its rate of monomer loss at temperatures close to the Tm. Here we evaluated Tm, the fraction of unfolded protein (fT) at temperature T, and two additional short-term stability measures, for their ability to predict the kinetics of monomer and bioactivity loss of wild-type GCSF and four variants, at 37 °C, and in a wide range of formulations. The GCSF variants introduced one to three mutations, giving a range of conformational stabilities spanning 7.8 kcal mol-1. We determined the extent to which the formulation rank order differs across the variants when evaluated by each of the four short-term stability measures. All correlations decreased as the difference in average Tm between each pair of GCSF variants increased. The rank order of formulations determined by Tm was the best preserved, with R2-values >0.7. Tm-values also provided a good predictor (R2 = 0.73) of the aggregation rates, extending previous findings to include GCSF variant-formulation combinations. Further analysis revealed that GCSF aggregation rates at 37 °C were dependent on the fraction unfolded at 37 °C (fT37), but transitioned smoothly to a constant baseline rate of aggregation at fT37 < 10-3. A similar function was observed previously for A33 Fab formulated by pH, ionic strength, and temperature, without excipients. For GCSF, all combinations of variants and formulations fit onto a single curve, suggesting that even single mutations destabilized by up to 4.8 kcal mol-1, are insufficient to change significantly the baseline rate of aggregation under native conditions. The baseline rate of aggregation for GCSF under native conditions was 66-fold higher than that for A33 Fab, highlighting that they are a specific feature of each native protein structure, likely to be dependent on local surface properties and dynamics.

Circulating endothelial cell-derived extracellular vesicles mediate the acute phase response and sickness behaviour associated with CNS inflammation.

Brain injury elicits a systemic acute-phase response (APR), which is responsible for co-ordinating the peripheral immunological response to injury. To date, the mechanisms responsible for signalling the presence of injury or disease to selectively activate responses in distant organs were unclear. Circulating endogenous extracellular vesicles (EVs) are increased after brain injury and have the potential to carry targeted injury signals around the body. Here, we examined the potential of EVs, isolated from rats after focal inflammatory brain lesions using IL-1ß, to activate a systemic APR in recipient naïve rats, as well as the behavioural consequences of EV transfer. Focal brain lesions increased EV release, and, following isolation and transfer, the EVs were sequestered by the liver where they initiated an APR. Transfer of blood-borne EVs from brain-injured animals was also enough to suppress exploratory behaviours in recipient naïve animals. EVs derived from brain endothelial cell cultures treated with IL-1ß also activated an APR and altered behaviour in recipient animals. These experiments reveal that inflammation-induced circulating EVs derived from endothelial cells are able to initiate the APR to brain injury and are sufficient to generate the associated sickness behaviours, and are the first demonstration that EVs are capable of modifying behavioural responses.

An autologous endothelial cell:peripheral blood mononuclear cell assay that detects cytokine storm responses to biologics.

There is an urgent unmet need for human tissue bioassays to predict cytokine storm responses to biologics. Current bioassays that detect cytokine storm responses in vitro rely on endothelial cells, usually from umbilical veins or cell lines, cocultured with freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers. These assays therefore comprise cells from 2 separate donors and carry the disadvantage of mismatched tissues and lack the advantage of personalized medicine. Current assays also do not fully delineate mild (such as Campath) and severe (such as TGN1412) cytokine storm-inducing drugs. Here, we report a novel bioassay where endothelial cells grown from stem cells in the peripheral blood (blood outgrowth endothelial cells) and PBMCs from the same donor can be used to create an autologous coculture bioassay that responds by releasing a plethora of cytokines to authentic TGN1412 but only modestly to Campath and not to control antibodies such as Herceptin, Avastin, and Arzerra. This assay performed better than the traditional mixed donor assay in terms of cytokine release to TGN1412 and, thus, we suggest provides significant advancement and a definitive system by which biologics can be tested and paves the way for personalized medicine.

Establishment of the first international standard for PEGylated granulocyte colony stimulating factor (PEG-G-CSF): report of an international collaborative study.

We assessed the feasibility of developing a suitable international reference standard for determination of in vitro biological activity of human sequence recombinant PEG-G-CSF products with a 20kD linear PEG linked to the N-terminal methionyl residue of G-CSF (INN Filgrastim), produced using a conjugation process and coupling chemistry similar to that employed for the lead PEGfilgrastim product. Based on initial data which showed that the current WHO 2nd international standard, IS for G-CSF (09/136) or alternatively, a PEG-G-CSF standard with a unitage traceable to the G-CSF IS may potentially serve as the IS for PEG-G-CSF products, two candidate preparations of PEG-G-CSF were formulated and lyophilized at NIBSC. These preparations were tested by 23 laboratories using in vitro bioassays in a multi-centre collaborative study. Results indicated that on the basis of parallelism, the current WHO 2nd IS for G-CSF or any of the PEG-G-CSF samples could be used as the international standard for PEG-G-CSF preparations. However, because of the variability in potency estimates seen when PEG-G-CSF preparations were compared with the current WHO 2nd IS for G-CSF, a candidate PEG-G-CSF was suitable as the WHO IS. The preparation 12/188 was judged suitable to serve as the WHO IS based on in vitro biological activity data. Therefore, the preparation coded 12/188 was established by the WHO Expert Committee on Biological Standardization (ECBS) in 2013 as the WHO 1st IS for human PEGylated G-CSF with an assigned in vitro bioactivity of 10,000IU per ampoule.

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist.

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1. We hypothesized that the uncontrolled activation of CD28SA-stimulated T cells is due to both the enhanced expression of activation molecules and the lack of or reduced inhibitory signals. In this study, we show that anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation and interleukin-2 secretion, accompanied by PD-1 expression. In contrast, CD28SA-activated T cells demonstrate uncontrolled activation parameters including enhanced expression of LFA-1 and CCR5 but fail to express PD-1 on the cell surface. We demonstrate the functional relevance of the lack of PD-1 mediated regulatory mechanism in CD28SA-stimulated T cells. Our findings provide a molecular explanation for the dysregulated activation of CD28SA-stimulated T cells and also highlight the potential for the use of differential expression of PD-1 as a biomarker of safety for T cell immunostimulatory biologics.

Role of public standards in the safety and efficacy of biologic medicines.

In this report, we emphasize the importance of public monographs with reference materials, coupled with careful process and change control and attention to GMPs, as a means of advancing access to good quality, safe, and effective medicines, with emphasis on available and incoming biologic medicines. With adequate control of articles covered by a monograph, these public standards can form the basis for a global public quality platform that covers reference products, non-interchangeable reference products, biosimilars, and interchangeable biosimilars. Working collaboratively with all stakeholders, new approaches allow these public standards to emerge nationally and globally in a timely way. Yet, there are increasing limitations in the availability of public standards for biologic medicines, which may reverse many decades of progress. Solutions are considered in this report.

Antibody C region influences TGN1412-like functional activity in vitro.

The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab')(2) and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants. Superagonistic activity, as measured by cytokine release and proliferation, was assessed by exposing PBMCs to immobilized mAbs/fragments or to aqueous mAbs/fragments in the presence of HUVEC monolayers. Removing the Fc generally curtailed or abolished PBMC activation. However, eliminating detectable FcγR-binding of the IgG4 by mutation (L235E) did not abrogate activity. Stabilizing the "wild-type" IgG4 hinge (S228P) enhanced activity without increasing FcγR binding, which could only partially be explained by inhibition of Fab arm-exchange. Subclass switching the IgG4 mAb to IgG1 decreased activity, whereas switching to IgG2 markedly increased activity. We conclude that the C region strongly influences in vitro CD28-mediated superagonistic signaling. Superagonism requires an intact Fc, as shown by the absence of activity of TGN1412 Fab and F(ab')(2) fragments, but, notably, appears to be relatively independent of FcγR-binding properties. We propose that the Fc, potentially through restricting flexibility, maintains a favorable V region conformation to allow superagonistic activity. These findings have important implications for Ab design strategies.

Glycan analysis of glycoprotein pharmaceuticals: Evaluation of analytical approaches to Z number determination in pharmaceutical erythropoietin products.

N-Glycosylation of many glycoprotein drugs is important for biological activity and should therefore be the target of specific and quantitative analytical methods. In this study, we focus on the two N-glycan mapping approaches that are used in pharmacopoeial monograph to analyse N-glycans released from fifteen preparations of recombinant human erythropoietin supplied by ten Chinese manufacturers. Underivatised N-glycans were analysed by high performance anion-exchange chromatography with pulsed amperometric detection and fluorophore-labelled N-glycans were analysed by weak anion-exchange and normal-phase high performance liquid chromatography. N-glycans were also analysed by matrix assisted laser desorption ionisation mass spectrometry. The release of N-glycans by PNGase F was shown to be consistent. Z number, a mathematical expression of the total negatively charged N-glycans composition has provided a convenient way to summarise the complex dataset and it might be suitable for product consistency monitoring. However, this Z number reduces the information of individual acidic N-glycan structure and is also found to be method dependent. Therefore, its use requires clear specification and validation. In this study, we only found weak but positive correlation between the Z number and its bioactivity. Wide range of N-glycans yields were obtained from the fifteen preparations but the significance of their differences is unclear.

Assignment of quantities to biological medicines: an old problem re-discovered.

A distinction exists between 'chemical' and 'biological' medicines. While, from antiquity, both organic and inorganic substances had been used in therapy, developing chemical sciences were inapplicable to materials extracted from natural sources, and the active principles could be neither identified nor characterized. The distinction between biological medicines or 'biologicals' grew out of this realization. Such 'biologicals' in clinical use were, however, variable in efficacy and in safety, and controlling the strength or quality was necessary. Without information on what biological medicines are, it was necessary to quantify what they do, and such medicines were quantified using systems based on biological responses (bioassays) in animals, organs or cells. Bioassays are defined in terms of an external standard rather than in absolute terms, and depend on a number of key assumptions: the need to assay 'like against like', the desirability of making the assay principle relevant to the intended clinical effect in man, and the importance of appropriate statistical models of design and analysis. The science of 'biological standardization' has kept pace with developments in medicine and continues to allow the use of biological medicines in man to be controlled on the basis of common measurement systems.

Sickness behaviour is induced by a peripheral CXC-chemokine also expressed in multiple sclerosis and EAE.

Non-CNS chemokine production may contribute to previously unrecognised components of Multiple Sclerosis (MS) pathology. Here we show that IL-8, a neutrophil chemoattractant, is significantly increased in serum from individuals with MS, and that the rodent homolog of IL-8 (CXCL1) is expressed in the liver in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. The hepatic expression of CXCL1 in EAE is accompanied by neutrophil recruitment to the liver, and we show that this recruitment is a feature of post mortem liver tissue from MS patients, which is a previously unrecognised phenomenon. We speculated that the presence of peripheral CXC-chemokine expression might contribute to the sickness behaviours associated with MS, which are a significant contributor to morbidity. Peripheral, but not central, administration of CXCL1 to Wistar rats inhibited spontaneous activity in the open field and burrowing behaviour in a dose-dependent manner (5-45 microg). The expression of CXCL1 by the liver and the recruitment of neutrophils can be modelled by the intracerebral injection of IL-1beta. Here, we found that interferon-beta (IFN-beta) pretreatment significantly inhibited hepatic CXCL1 production and neutrophil recruitment to the liver induced by the microinjection of IL-1beta into the brain. Thus while the mechanism by which IFN-beta therapy suppresses disease in MS remains unclear, the data presented here suggests that the inhibition of hepatic chemokine synthesis may be a contributing factor.

A novel bioassay for B-cell activating factor (BAFF) based on expression of a BAFF-receptor ectodomain-tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2 endodomain fusion receptor in human rhabdomyosarcoma cells.

B-cell activating factor (BAFF) is a type II transmembrane glycoprotein belonging to the tumour necrosis factor ligand superfamily. Active soluble forms of BAFF are generated either by cleavage of the extracellular domain or by recombinant DNA technology. The current bioassay for measuring the activity of soluble BAFF involves stimulation of the proliferation of mouse splenic B-cells in the presence of goat anti-mouse IgMmicro chain which is rather cumbersome and lengthy and yields variable results. We have therefore developed an alternative functional assay which relies on the ability of BAFF to induce an apoptotic response in human rhabdomyosarcoma cells. For this, we constructed a chimeric receptor containing the ectodomain of the MuBAFF-R--the major cell receptor for BAFF--and the endodomain of the HuTRAIL-R2--one of the two functional receptors for TRAIL--which is known to contain a death domain and trigger apoptosis. When the chimeric receptor was expressed in the TRAIL-sensitive human rhabdomyosarcoma cell line KD4 clone 21, recombinant BAFF of either human or mouse sequence stimulated apoptosis, similar to TRAIL, in a dose-dependent manner. The transfected cell population, called FL17, expressing the MuBAFF-R/ HuTRAIL-R2 thus provided the basis of a novel functional bioassay for BAFF that is simple and relatively fast to perform. The construction of the chimeric receptor, development of the transfected cells expressing this receptor and the development of sensitive and reproducible bioassays for BAFF and anti-BAFF neutralising antibodies are described.

Anti-D and anti-i activities are inseparable in V4-34-encoded monoclonal anti-D: the same framework 1 residues are required for both reactivities.

BACKGROUND: The heavy-chain V4-34 germline gene segment is mandatory for pathologic cold-reacting autoantibodies with anti-I/i specificity (cold agglutinins) and is also preferentially used by monoclonal immunoglobulin M alloantibodies against D and other Rh antigens. The use of the V4-34 segment by monoclonal anti-D has previously been shown to also confer anti-I/i reactivity (cold agglutinin activity), which has implications for the use of such antibodies for Rh blood typing. V4-34 framework 1 (FR1) sequence is believed to be critical for cold agglutinin activity of cold agglutinins. STUDY DESIGN AND METHODS: The aim of this investigation was to use site-directed mutagenesis of a recombinant V4-34-encoded anti-D to determine the contribution of V4-34 FR1 sequence to anti-D activity and whether mutational modifications in the FR1 region could separately alter anti-D and anti-i activities. RESULTS: The results show that amino acid changes in V4-34 FR1 at W7, A23, and Y25 have a profound effect on anti-D activity as well as on anti-i activity. It was not possible to substantially reduce or remove anti-i activity without reducing anti-D activity to a comparable extent. CONCLUSIONS: The same nonpolar hydrophobic amino acids in FR1 are critical for maintaining both anti-D and anti-i activity. It is proposed that these residues influence the conformation of the antigen-binding site.

"Cytokine storm" in the phase I trial of monoclonal antibody TGN1412: better understanding the causes to improve preclinical testing of immunotherapeutics.

The CD28-specific mAb TGN1412 rapidly caused a life-threatening "cytokine storm" in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.

Leptin induces cyclooxygenase-2 via an interaction with interleukin-1beta in the rat brain.

In addition to its central effects on appetite regulation, leptin has been implicated in immune function and inflammation. Previous data suggested that leptin acts as an inflammatory signal within the brain, as exogenously administered leptin induced fever, a typical brain-regulated inflammatory response. The present study aimed to delineate the inflammatory actions and cellular targets of leptin in the brain by examining its effects on the expression of interleukin (IL)-1beta and cyclooxygenase (COX)-2, two important inflammatory components of the fever response. Intracerebroventricular injection of leptin (5 microg/rat) induced IL-1beta and COX-2 mRNA and protein in the hypothalamus between 1 and 3 h after treatment as determined by reverse transcription-polymerase chain reaction and immunohistochemistry. Coinjection of IL-1 receptor antagonist (100 microg/rat, intracerebroventricular) attenuated leptin-induced COX-2, whereas IL-1 receptor antagonist had no effect on endogenous IL-1beta levels, suggesting that leptin induces COX-2 via, at least partly, IL-1beta action. IL-1beta protein expression was induced in macrophages in the meningis and perivascular space after leptin treatment, whereas COX-2 induction was observed in endothelial cells, indicating the roles for these non-neuronal cells in mediating inflammatory actions of leptin. In addition, neutralization of endogenous circulating leptin with anti-leptin antiserum attenuated intraperitoneal lipopolysaccharide (100 microg/kg)-induced brain IL-1beta and COX-2 upregulation, suggesting that leptin indeed acts as an inflammatory signal to the brain during systemic inflammation. These findings are in contrast to the effects of leptin on appetite regulation where it is believed to act primarily on neurons, thus presenting a distinct anatomical basis for the inflammatory and appetite regulatory actions of leptin in the brain.

Standardization of biological medicines: the first hundred years, 1900-2000.

The use of materials of biological origin in medicine has a long history. These materials, including tissue, organ and microbial extracts, blood and its derivatives, antibodies and hormones, share the feature that for much of the last century the ability to characterize and quantify the active substance was limited. Quantification of these substances depends on biological standardization, a discipline that was refined to a science by the Medical Research Council from the 1920s onwards, and which, with contributions from several prominent Fellows of the Royal Society, including principally Sir Henry Dale, the UK has led the world to the present date.

A sialylation-sensitive cell-based in vitro bioassay for erythropoietin; incorporation of the galactose-binding Erythrina crista-galli lectin.

The in vivo biological activity of erythropoietin (Epo) is dependent on its being adequately sialylated. Current in vitro bioassays for Epo do not correlate with the in vivo bioassays as the former do not take into account the role the liver plays in clearing desialylated glycoproteins from the circulation. Here we describe a sialylation-sensitive cell-based Epo bioassay. In the first instance, Epo activity in vitro was measured using proliferation of AS-E2 cells, and in vivo using the polycythaemic mouse bioassay. Activity in vivo was progressively abolished by controlled desialylation, whereas activity in vitro was essentially unaffected. Incorporation of an incubation step with a solid-phase galactose-binding lectin (Erythrina crista-galli), effectively mimicking passage through the liver in vivo, renders the in vitro bioassay sensitive to desialylation, such that Epo desialylated almost to completion had <10% of the activity of untreated Epo. These studies offer proof of principle, that rational manipulation of in vitro bioassays can allow prediction of activity in vivo without the use of live animals.