Erythropoietin combined with traditional Chinese medicine for chemotherapy-induced anemias: A protocol of systematic review and meta-analysis.

BACKGROUND: As far as we know, several systematic review and meta-analysis have assessed the safety and efficacy of erythropoiesis-stimulating agents (ESAs) in the patients with chemotherapy-induced anemia (CIA). But no study assesses the safety and efficacy of ESAs combined with traditional Chinese medicine (TCM). The aim of our study is to assess the efficacy and safety of ESAs combination with TCM for patients with CIA and will provide a higher level of evidence for clinical applications. METHODS: This protocol adheres to the preferred reporting items for systematic reviews and meta-analysis protocol statement. The source of literature will be a structured search of the following 7 electronic databases: PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, and Wanfang Database. Records will be independently evaluated by 2 reviewers. Disagreements will be resolved through consensus or third-party adjudication. Review Manager 5.3 software (Cochrane Collaboration, Copenhagen Denmark) will be used to perform meta-analysis. For dichotomous variables, odds ratio with 95% confidence intervals will be obtained by the Mantel-Haenszel method. For continuous data, mean difference with 95% confidence intervals will be used. P < 0.05 will be considered to be statistically significant. RESULTS: This study will be performed to test the efficacy and safety of ESAs combined with TCM for CIA in patients with cancer. CONCLUSIONS: The result of this study will be promoted mainly in 2 ways: publish in peer-reviewed journals in the fastest way; and promotion in domestic and foreign conferences. INPLASY REGISTRATION NUMBER: INPLASY202080041.

Quality assessment and its impact on clinical performance of a biosimilar erythropoietin: A simulated case study.

The case study described in this paper was developed for the purpose of training for a better understanding of principles relating especially to a comprehensive evaluation of multiple quality attributes as outlined in the WHO guidelines on evaluation of similar biotherapeutic products. It is also to emphasize the importance of an understanding of the critical quality attributes and a risk assessment of the impact on clinical performance. It was prepared to mimic a real situation in which regulators need to evaluate the differences in quality attributes known to have potential impact on clinical activity. Erythropoietin has been identified as one of the important glycosylated therapeutic proteins and a good example to illustrate how structural characteristics would affect product efficacy and safety. The case study illustrates biosimilarity assessment of a candidate of erythropoietin biosimilar and the important quality attributes that need to be considered in order to understand the importance of structure-function relationships as they contribute to the stepwise evaluation of biosimilarity. This paper reflects the outcomes of the case study exercise and discussion from two WHO implementation workshops held in Ghana (September 2015) and Denmark (July 2017).

Collaborative study for the establishment of erythropoietin BRP batch 5.

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 Ph. Eur. monograph 1316 'Erythropoietin concentrated solution'. BRP batch 4 (BRP4) was calibrated in 2014 and its stocks are depleted. The European Directorate for the Quality of Medicines and HealthCare (EDQM) thus endorsed a project (BSP147) to calibrate a replacement batch in International Units against the 3rd WHO International Standard (IS) for erythropoietin, recombinant, for bioassay (11/170). The amount of material contained in the vial of BRP4 greatly exceeded the amount needed for one bioassay, sometimes leading to considerable waste. It was thus decided to prepare a candidate material with a lower EPO content. The collaborative study involved eight laboratories in Europe, the USA and Australia. Based on the outcome of the study, the Ph. Eur. Commission adopted the proposed standard as Erythropoietin BRP batch 5 in June 2018 for use as a reference preparation solely for the polycythaemic and normocythaemic mouse bioassays, with an assigned potency of 2000 IU/ampoule. Furthermore, the potency of BRP batch 4 was confirmed during the study thus warranting a good continuity of the International Unit.

Efficacy and safety of erythropoietin in patients with traumatic brain injury: A systematic review and meta-analysis.

OBJECTIVE: The purpose of this study was to evaluate the effects of erythropoietin (EPO) on mortality and neurological outcomes in patients with traumatic brain injury (TBI). MATERIALS AND METHODS: Electronic databases of studies published up to January 5, 2017 were searched to retrieve relevant investigations comparing the outcomes of EPO-treated patients and untreated patients following TBI. We calculated the relative risk (RR) of mortality, neurologic outcomes, and deep vein thrombosis (DVT) with corresponding 95% confidence interval (CI) using meta-analysis. RESULTS: Six randomized controlled clinical trials met the eligibility criteria. In total, 1041 patients were included among the studies. EPO was found to significantly reduce the occurrence of mortality (RR 0.68 [95% CI 0.50-0.95]; P = 0.02), but did not significantly reduce poor functional outcome (RR 1.22 [95% CI 0.82-1.81]; P = 0.33). There were no significant differences in the occurrence of complications, such as DVT, between the treatment groups (RR -0.02 [95% CI -0.06-0.02]; P = 0.81). CONCLUSIONS: Results of the present meta-analysis suggest that the use of EPO may prevent death following TBI without causing adverse events, such as deep vein thrombosis. However, the role of EPO in improving neurological outcome(s) remains unclear. Further well-designed, randomized controlled trials using modified protocols and involving specific patient populations are required to clarify this issue, and to verify the findings.

[Improvement of erythropoietin bioassay.]

The relevance of bioassay standardization results from the lack of consistent national regulatory requirements for evaluation of recombinant human erythropoietin quality and the need to harmonize these requirements with international ones. Precision studies were carried out in 6 experiments on Balb/C mice. The factors that can influence the accuracy of the method were altered during the experiments. Each experiment included three levels: 20, 40 and 80 IU/ml, and 8 replicates for the reference and test samples. The trueness was estimated by bias relative to the reference value at 5 levels: 10, 20, 40, 80 and 160 IU/ml, and 4 replicates for the reference and test samples at each level. The test samples were prepared by a series of independent dilutions of the reference standard. Reticulocyte count was performed using a flow cytometer. 5 µmol acridine orange solution was used as a dye. Experimental study of accuracy and optimization of erythropoietin bioassay procedure helped to obtain two validation characteristics (trueness and precision). It was shown that logarithms of erythropoiesis registered values could reasonably be used in statistical calculations of erythropoietin specific activity and evaluation of the method's validation parameters. The theoretically and experimentally justified test procedure includes three levels of doses: 20, 40 and 80 IU/ml, and 8 animals for each level, which is consistent with the international requirements for accuracy. According to the results of experimental studies, the trueness is characterized by a bias of no more than 9 % and does not exceed the range of the calculated activity (80-125 %). Statistical processing of the test results by the parallel-line method makes it possible to check the assumption of equivalence of the test and reference samples and to calculate the test sample activity. The confidence limit of the calculated activity for intra-laboratory precision of 5.6 % is equal to 76-131 % which complies with the proposed range (64-156 %, P=0.95).

Quality and Batch-to-Batch Consistency of Original and Biosimilar Epoetin Products.

Comprehensive physicochemical characterization and biological assays are essential parts in assessing quality attributes of biologicals. Here, we compared the quality of different marketed recombinant human erythropoietin (epoetin) products: originators, Eprex and NeoRecormon as well as 2 biosimilars, Retacrit and Binocrit. In addition, assessment of batch-to-batch variability was included by collecting 2 or more batches of each product. Common assays which included sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance size-exclusion chromatography, asymmetrical flow field-flow fractionation, capillary zone electrophoresis, and potency testing were used. Of the tested products and among batches of single products, variations in epoetin content, isoform profiles, and potency were found. Ultimately, this study demonstrated the high quality of epoetin products with some degree of variation among products and batches, confirming the "similar but not identical" paradigm of biologicals.

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.

Analytical platform for glycomic characterization of recombinant erythropoietin biotherapeutics and biosimilars by MS.

BACKGROUND: Erythropoietin is a therapeutic glycoprotein that stimulates red blood cell production. The quality, safety and potency of recombinant erythropoietins are determined largely by their glycosylation. Small variations in cell culture conditions can significantly affect the glycosylation, and therefore the efficacy, of recombinant erythropoietins. Thus, detailed glycomic analyses are necessary to assess biotherapeutic quality. We have developed a platform for qualitative and quantitative glycomic analysis of recombinant erythropoietins. RESULTS: The platform was used to profile native N-glycans from three production batches of darbepoetin alfa (also known as NESP), a common form of recombinant erythropoietin. Darbepoetin alfa was found to contain an abundance of large, multi-antennary N-glycans with high levels of sialylation, O-acetylation and dehydration. Results were verified by independent orthogonal analysis with both MALDI-TOF and nano-LC/Q-TOF MS. CONCLUSION: This platform may be applied to QC and batch analysis of not only recombinant erythropoietin, but also other complex, glycosylated biotherapeutics and biosimilars.

Biopharmaceuticals and biosimilars in psoriasis: what the dermatologist needs to know.

The entry of biosimilar forms of biopharmaceutical therapies for the treatment of psoriasis and other immune-mediated disorders has provoked considerable interest. Although dermatologists are accustomed to the use of a wide range of generic topical agents, recognition of key differences between original agent (ie, the name brand) and the generic or biosimilar agent is necessary to support optimal therapy management and patient care. In this review we have summarized the current state of the art related to the impending introduction of biosimilars into dermatology. Biosimilars represent important interventions that are less expensive and hence offer the potential to deliver benefit to large numbers of patients who may not currently be able to access these therapies. But the development of biosimilars is not equivalent to that of small molecule generic therapies because of differences in molecular structure and processes of manufacture. The planned regulatory guidelines and path to approval may not encompass all of these potentially important differences and this may have clinical relevance to the prescriber and patient. Consequently, we have identified a series of key issues that should be considered to support the full potential of biosimilars for the treatment of psoriasis; ie, that of increased access to appropriate therapy for the psoriasis population worldwide.

Randomised, phase III trial of epoetin-ß to treat chemotherapy-induced anaemia according to the EU regulation.

BACKGROUND: Erythropoietin-stimulating agents (ESAs) effectively decrease the transfusion requirements of patients with chemotherapy-induced anaemia (CIA). Recent studies indicate that ESAs increase mortality and accelerate tumour progression. The studies also identify a 1.6-fold increased risk of venous thromboembolism. The ESA labelling was thus revised in Europe and the United States in 2008. This is the first randomised, phase III trial evaluating the efficacy and safety of epoetin-ß (EPO), an ESA, dosed in accordance with the revised labelling, which specifies that ESAs should be administered to CIA patients with a haemoglobin level of ≤ 10 g dl⁻¹ and that a sustained haemoglobin level of > 12 g dl⁻¹ should be avoided. METHODS: A total of 186 CIA patients (8.0 g dl⁻¹ ≤ haemoglobin ≤ 10.0 g dl⁻¹) with lung or gynaecological cancer were randomised to receive EPO 36,000 IU or placebo weekly for 12 weeks. RESULTS: The proportion of patients receiving transfusions or with haemoglobin < 8.0 g dl⁻¹ between week 5 and the end of the treatment period as the primary end point was significantly lower in the EPO group (n=89) than in the placebo group (n=92; 10.0% vs 56.4%, P < 0.001). The proportion receiving transfusions was significantly lower in the EPO group (4.5% vs 19.6%, P=0.002). Changes in quality of life were not different. No significant differences in adverse events - for example, the incidence of thromboembolic events was 1.1% for each group - or the 1-year overall survival were observed between groups. CONCLUSION: Weekly EPO administered according to the revised labelling approved by the European Medicines Agency is effective and well tolerated for CIA treatment. Further investigations are needed on the effect of ESAs on mortality.

Fluctuations of haemoglobinaemia in chronic haemodialysis patients.

In March 2008 and June 2009, an ad hoc working group of nephrologists discussed the status of anaemia therapy with erythropoiesis-stimulating agents [ESA] in patients on chronic haemodialysis, the phenomenon of fluctuations of haemoglobinaemia, and the need for individualisation of ESA treatment. The working group put together the following statements: (1) ESAs increase the haemoglobin concentration and adaptations of the ESA dose adjust the response according to a negative-feedback loop. The long lag time between an ESA dose change and its effect on erythropoiesis is cumbersome. The optimal haemoglobin target concentration is different for every haemodialysis patient; the lowest haemoglobin concentration upon which one could consistently demonstrate a positive subjective and objective clinical benefit in chronic dialysis is 11 g/dL, in contrast to the lowest haemoglobin concentration of 10 g/dL recommended in the current EMEA label for ESAs. (2) Intra-individual fluctuation of haemoglobinaemia over time is unavoidable, not only due to the ESA dose/haemoglobin response interaction, but also, and more importantly, due to the occurrence of acute illnesses and exacerbations of co-morbid conditions. Many different methodologies to characterise haemoglobin variability have been described but there is currently no universally applied definition of the phenomenon. (3) An impact of the haemoglobin level and the amplitude of the haemoglobin fluctuations on patient outcome has been observed. Without disclosing any causal relationship, worse outcomes were associated with haemoglobin fluctuations around the lower target level, but later on, more simply linked to the relative time spent below the haemoglobin concentration of 11 g/dL and to the administration of inappropriately high ESA doses in order to achieve the recommended haemoglobin target range. A plausible mechanism might be that acute illnesses blunt the patients' basal ESA sensitivity; this leads to subnormal and/or varying haemoglobin levels, currently initiating an ESA dose increase. The longer it takes the patient to recover from the acute illness, the more the prolongation of the clinically poor condition is to some extent maintained by the persistence of low haemoglobinaemia and/or by the administration of high ESA doses, and, as such, on their turn possibly contributing to an ultimate poor outcome. In the absence of clinical trials, recommendations should be offered how to proceed with the administration of ESAs as optimal as possible in periods of clinical instability.

An alternative to animal testing in the quality control of erythropoietin.

A physico-chemical method has been developed as an alternative to the current bioassay in normocythaemic mice for estimating the biological activity of erythropoietin batches. Capillary zone electrophoresis was used for quantification of the isoforms and their substructures were further elucidated by N-glycan mapping techniques. The analytical study was carried out on a total of 40 batches of epoetin beta which were selected to cover an adequate range of precisely established potency values. The relationship between the biological and chemical parameters was evaluated statistically in order to identify suitable covariates for the prediction of the biological activity. Out of several alternatives, a prediction model which is based on the percentages of isoforms per batch and the degree of sialidation was selected and tested. This model is comparable in terms of accuracy to the established in vivo bioassay, but is far superior in terms of precision. Further advantages of the method are improved animal welfare and savings in time and effort. The question whether the prediction model already meets the requirements for replacing the bioassay according to the ICH guideline Q6B is discussed.

2D-LC analysis of BRP 3 erythropoietin N-glycosylation using anion exchange fractionation and hydrophilic interaction UPLC reveals long poly-N-acetyl lactosamine extensions.

Post-translational modifications, in particular glycosylation, represent critical structural attributes that govern both the pharmacodynamic and pharmacokinetic properties of therapeutic glycoproteins. To guarantee safety and efficacy of recombinant therapeutics, characterization of glycosylation present is a regulatory requirement. In the current paper, we applied a multidimensional strategy comprising a shallow anion exchange gradient in the first dimension, followed by analysis using the recently introduced 1.7 µm HILIC phase in the second dimension for the comprehensive separation of complex N-glycans present on the European Biological Reference Preparation (BRP) 3 erythropoietin standard. Tetra-antennary glycans with multiple sialic acids and poly-N-acetyl lactosamine extensions were the most abundant oligosaccharides present on the molecule. Site-specific glycan analysis was performed to examine microheterogeneity. Tetra-antennary glycans with up to four sialic acids and up to five poly-N-acetyl lactosamine extensions were observed at asparagine 24 and 83, while biantennary glycans were the major structures at asparagine 38. The combined AEC x UPLC HILIC allows for the rapid and comprehensive analysis of complex N-glycosylation present on therapeutic glycoproteins, such as BRP3 erythropoietin.

Today's challenges in pharmacovigilance: what can we learn from epoetins?

Highly publicized safety issues of medicinal products in recent years and the accompanying political pressure have forced both the US FDA and the European Medicines Agency (EMA) to implement stronger regulations concerning pharmacovigilance. These legislative changes demand more proactive risk management strategies of both pharmaceutical companies and regulators to characterize and minimize known and potential safety concerns. Concurrently, comprehensive surveillance systems are implemented, intended to identify and confirm adverse drug reactions, including the creation of large pharmacovigilance databases and the cooperation with epidemiological centres. Although the ambitions are high, not much is known about how effective all these measures are, or will be. In this review we analyse how the pharmacovigilance community has acted upon two adverse events associated with the use of erythropoiesis-stimulating agents: the sudden increase in pure red cell aplasia and the possible risk of tumour progression associated with these products. These incidents provide important insight for improving pharmacovigilance, but also pose new challenges for regulatory decision making.

Optimization and qualification of capillary zone electrophoresis method for glycoprotein isoform distribution of erythropoietin for quality control laboratory.

The European Pharmacopoeia (Ph. Eur.) monograph for Erythropoietin Concentrated Solution describes a capillary zone electrophoresis method for identification of recombinant human erythropoietin. However, this method has shown poor reproducibility due to inadequate capillary conditioning. We have modified the Ph. Eur. method to make it more robust and suitable for the quality control laboratory for the analysis of epoetin alfa and epoetin alfa after formulation with polysorbate 80. This study qualified the modified method by showing improved robustness and reproducibility. The study also characterized and qualified a secondary standard of epoetin alfa as a substitute for the primary standard, Ph. Eur. erythropoietin Biological Reference Preparation, which is available in limited supply. Four sets of analyses were performed to assess repeatability, intermediate precision, and the secondary standard. The results showed that the modified method is suitable for its intended purpose to test epoetin alfa and formulated epoetin alfa samples. The epoetin alfa secondary standard is a suitable substitute for the primary standard. Further, we developed a procedure for the removal of polysorbate 80 from formulated epoetin alfa, allowing the material to be analyzed by the modified Ph. Eur. method.

Disposable bioreactors: maturation into pharmaceutical glycoprotein manufacturing.

Modern biopharmaceutical development is characterised by deep understanding of the structure activity relationship of biological drugs. Therefore, the production process has to be tailored more to the product requirements than to the existing equipment in a certain facility. In addition, the major challenges for the industry are to lower the high production costs of biologics and to shorten the overall development time. The flexibility for providing different modes of operation using disposable bioreactors in the same facility can fulfil these demands and support tailor-made processes.Over the last 10 years, a huge and still increasing number of disposable bioreactors have entered the market. Bioreactor volumes of up to 2,000 L can be handled by using disposable bag systems. Each individual technology has been made available for different purposes up to the GMP compliant production of therapeutic drugs, even for market supply. This chapter summarises disposable technology development over the last decade by comparing the different technologies and showing trends and concepts for the future.

Impact of illegal trade on the quality of epoetin alfa in Thailand.

BACKGROUND: Reports from the World Health Organization have suggested that counterfeit medicines pose a serious problem in developing countries. An investigation of anti-erythropoietin antibody-mediated pure red cell aplasia in Thailand found evidence of drug smuggling, which may have serious safety implications. OBJECTIVE: This study assessed the authenticity and quality of epoetin alfa samples in Thailand. METHODS: Samples of epoetin alfa-prefilled syringes were collected from the pharmacies at 2 major hospitals (62 samples), 8 retail pharmacies (41 samples), and Thai authorities (30 samples confiscated from smugglers at 2 airports, and 6 samples from a condominium used by smugglers). These samples were tested against the European Union Pharmacopeia specifications for aggregate content in epoetins of <2%. The integrity of epoetin alfa distribution channels, coldchain processes (maintenance at 2 degrees C-8 degrees C), primary and secondary packaging components (eg, batch number, expiration date, appearance, letter size), and company's confidential features (eg, nature of the ink, type and quality of the paper, other covered features) were also investigated. The main outcome measures were protein aggregate content, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting; and isoform distribution, assessed by isoelectric focusing and Western blotting. RESULTS: Epoetin alfa samples obtained from the company's cold-chain and authorized distribution channels met all quality standards, as did all epoetin alfa samples obtained from the hospital pharmacies. However, evidence showed that some samples were being smuggled or sold illegally through certain unauthorized retail pharmacies. The epoetin alfa samples obtained from both airports and the condominium were stored improperly at room temperature. Aggregate levels exceeded the specification of <2% in 11 samples from 2 of the retail pharmacies (range, 1.2%-3.1%), 15 samples from the Dongmuang Airport (range, 2.2%-17.0%), and all 6 samples from the condominium (range, 10.5%-19.8%). All samples from the 2 hospitals, 8 retail pharmacies, and Suvarnabhumi Airport had the authentic 6 isoform bands. Samples from Dongmuang Airport and the condominium appeared to have the 6 characteristic bands, but positive confirmation was difficult because of band smearing caused by a high level of aggregates. All features of primary and secondary packaging were found to be authentic. CONCLUSIONS: This investigation found evidence that some epoetin alfa samples were smuggled into Thailand without proper cold chain, contained high levels of protein aggregates, and were sold illegally through certain retail pharmacies. The Thai authorities have intervened to stop such unauthorized products from reaching patients. Strenuous efforts must be made to prevent illegal cross-border smuggling of biopharmaceuticals without proper cold chain because of the serious safety implications for patients in developing countries.