Addressing the challenges of biomarker calibration standards in ligand-binding assays: a European Bioanalysis Forum perspective.

The analysis of biomarkers by ligand-binding assays offers significant challenges compared with the bioanalysis of small and large molecule drugs. The presence of endogenous analyte is a commonly cited issue. Also the sourcing and application of appropriate calibration or reference standards can present many issues. One of the main challenges is ensuring the continuity and validity of biomarker data when the source or lot number of calibration standard changes within or between studies. Several strategies exist in attempting to deal with this and standardize the biomarker data through the assay life or looking for ways to compare and normalize biomarker data. In this manuscript, the European Bioanalysis Forum view on dealing with calibration standards in biomarker assays is described.

Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European Bioanalysis Forum.

Flow cytometry is a powerful tool that can be used for the support of (pre)clinical studies. Although various white papers are available that describe the set-up and validation of the instrumentation (the flow cytometer) and validation of flow cytometry methods, to date no guidelines exist that address the requirements for performing flow cytometry in a regulated environment. In this manuscript, the European Bioanalysis Forum presents additional practice guidance on the use of flow cytometry in the support of drug development programs and addresses areas that are not covered in the previous publications. The concepts presented here are based on the consensus of discussions in the European Bioanalysis Forum Topic Team 32, in meetings in Barcelona, Limelette and multiple telephone conferences.

EBF recommendation for stability testing of anti-drug antibodies; lessons learned from anti-vaccine antibody stability studies.

Long- and short-term stability testing of the analyte is one of the key parameters in bioanalytical method validation in support of pharmacokinetics. However, for immunogenicity testing, the scientific rationale for long- and short-term stability testing on quality control samples most often spiked with polyclonal antibody raised in a different species should be questioned. Therefore, the European Bioanalysis Forum (EBF) formed a Topic Team to discuss the scientific rationale for stability testing of anti-drug antibodies (ADAs). A review of EBF member companies' experience on ADA stability and on anti-vaccine antibodies from vaccine projects was the basis of this discussion. EBF recommends to perform short-term stability testing of the positive control, but not to perform long-term stability testing of ADAs in nonclinical and clinical studies.

Conference report: AAPS and US FDA Crystal City V meeting on Quantitative Bioanalytical Method Validation and Implementation: feedback from the EBF.

Crystal City V meeting on Quantitative Bioanalytical Method Validation and Implementation: 2013 Revised US FDA Guidance 3-5 December 2013, Hilton Baltimore, MD, USA The meeting provided an opportunity for Industry and regulators from the US FDA to discuss the recently published revised draft FDA Guidance for Industry on Bioanalytical Methods Validation during the 90 day review period. Key perspective and philosophical positions were shared leading to a healthy exchange of views and ideas on topics in the revised document. Discussions covered all aspects of bioanalytical method validation and method utilization. However, the main dialogue was focused on chromatographic methods, ligand-binding assay methods and biomarker analysis. The resulting open debate led to greater understanding of the document, but also provided clear feedback, including the request on harmonization with approved Bioanalytical Methods Validation guidance's release from other health authorities, as well as the consensus view between industry and the FDA. Members of the European Bioanalysis Forum summarized prospective discussions during the meeting in Baltimore; however, this Report is not intended to constitute the official proceedings from the meeting, which are expected to be published later this year.

The European Bioanalysis Forum community's evaluation, interpretation and implementation of the European Medicines Agency guideline on Bioanalytical Method Validation.

The European Medicines Agency's (EMA) 2011 guideline on bioanalytical method validation (BMV) was evaluated and subsequently intensely discussed by the European Bioanalysis Forum (EBF) during a 2-day workshop (EBF Workshop on the implementation of the EMA guideline on BMV, Château de Limelette, Limelette, Belgium, 15-16 March 2012). The goal of the evaluation and discussions was to come to a uniform interpretation of the guideline and thus to help facilitate a smooth implementation at our laboratories. Up front preparations for the workshop by dedicated teams concentrated on challenges on implementation: ambiguities, technical or operational challenges and issues in general. In addition, common understandings were identified as well as main differences to the 2011 US FDA guideline. The guideline was perceived as being well written with a clear structure, separating method validation from sample analysis and treating all relevant aspects one-by-one in a logical order. It is the first BMV guideline clearly addressing the specifics for ligand binding assays and it shows a good match with current scientific thinking. The EBF community considers the EMA BMV guideline an excellent basis for countries that are in the process of developing or updating their own BMV guideline.

Managing scientific, technical and regulatory innovation in regulated bioanalysis: a discussion paper from the European Bioanalysis Forum.

On 12-13 June 2012, the European Bioanalysis Forum hosted its third Focus Meeting in Brussels (Belgium). At the meeting, a panel discussion was held on the hurdles that the bioanalytical community encounters when adopting new technologies or managing regulated bioanalysis expectations around emerging technologies. Over the last few years, the industry has seen many new technologies maturing. As they became available, the bioanalytical scientist has observed that implementing these technologies in the regulated environment has become increasingly challenging. For one, scientific developments and regulatory expectations may not go hand in hand. At the same time, the pharmaceutical industry has become increasingly risk averse in their response to these real or perceived higher expectations in regulated bioanalysis. As a downstream consequence, the potential result of overinterpretation of guidance or occasional widespread and premature implementation of responses to health authority inspections, industry may be contributing significantly to raising the bar on some processes related to day-to-day practices in the bioanalytical laboratory. Last but not least, with the community being satisfied with the performance of the current tools, potential complacency can be observed in the regulated bioanalytical community because existing technologies, such as LC-MS/MS and ligand-binding assays, have served and still are serving them extremely well. Hence, the question 'what's next after LC-MS/MS or ELISA?' is not resonating with many scientists as pertinently compared with 'What's next after RIA, GC or LC-UV?', which was the key question in the 1990s, certainly in the context of an increasing effort needed to validate these new tools. With this article, the European Bioanalysis Forum aims to stimulate an open dialogue between all stakeholders in regulated bioanalysis to positively influence how we balance science, process and regulations in day-to-day work. This discussion should facilitate the evaluation and the subsequent implementation of innovative techniques for the benefit of the patient, while stimulating our community to raise the bar on added-value science, but at the same time removing the bar on processes with limited or no added value.

Conference Report: the 3rd EBF Focus Meeting: 'Hatching'--emerging technologies approaching the regulated bioanalysis laboratory. Brussels, Belgium, 12-13 June 2012.

One hundred and eighty scientists from industry and academia discussed the progress in emerging technologies approaching regulated bioanalysis, with a focus on what potential hurdles prevent them becoming broadly accepted by industry and regulators. The conference delegates agreed that moving innovative technologies forward can only be achieved by providing solid data to support the application. In addition, also establishing an open dialogue with health authorities is key for success. By successfully integrating new technologies in the bioanalytical laboratory we contribute to bringing more safe and efficacious therapies faster to patients.

European Bioanalysis Forum recommendation on method establishment and bioanalysis of biomarkers in support of drug development.

Biomarkers have become increasingly important in drug development and many bioanalysts are getting involved. Consequently, different views on how to approach the bioanalysis of biomarkers have been published or are being developed. The European Bioanalysis Forum has intensively discussed this topic since 2010 and is ready with their recommendation on method establishment and bioanalysis of biomarkers. Acknowledging that the challenges step outside the bioanalytical laboratory is a cornerstone of our recommendation. The importance of integrating all scientific aspects, from purely analytical aspects, all the way to understanding the biology and effects of the biomarker, prior to embarking on method establishment or sample analysis, cannot be underestimated. Close and iterative interactions with the teams requesting the data is imperative to develop a bioanalytical strategy that combines science, analytical performance and regulations. The European Bioanalysis Forum developed a straightforward decision tree to help the scientific community in developing a bioanalytical strategy for any biomarker in drug development.

Validation of the rapid fluorescent focus inhibition test for rabies virus-neutralizing antibodies in clinical samples.

Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vaccinated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentrations can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples.

'Less is More': defining modern bioanalysis.

The 4th Open Symposium of the European Bioanalytical Forum entitled 'Less is More' was held on 16-18 November 2011 at the Hesperia Tower Hotel, Barcelona, Spain. More than 50 interesting presentations were delivered covering areas with interest for the small- and large-molecule community - biomarker validation; regulations, including an update on new and emerging guidelines and on Global harmonization; technology updates; incurred sample stability; microdosing; dried blood spots and microsampling; challenges of 'free' and 'total' macromolecule quantification; stability issues in ligand binding assays or anomalous results. In excess of 450 delegates from more than 170 institutes and companies (industry, regulators and academia) from all global regions participated in the open and stimulating discussions. This manuscript provides an overview of the highlights discussed at the meeting.

Impact of recombinant adenovirus serotype 35 priming versus boosting of a Plasmodium falciparum protein: characterization of T- and B-cell responses to liver-stage antigen 1.

Prime-boost vaccination regimens with heterologous antigen delivery systems have indicated that redirection of the immune response is feasible. We showed earlier that T-cell responses to circumsporozoite (CS) protein improved significantly when the protein is primed with recombinant adenovirus serotype 35 coding for CS (rAd35.CS). The current study was designed to answer the question whether such an effect can be extended to liver-stage antigens (LSA) of Plasmodium falciparum such as LSA-1. Studies with mice have demonstrated that the LSA-1 protein induces strong antibody response but a weak T-cell immunity. We first identified T-cell epitopes in LSA-1 by use of intracellular gamma interferon (IFN-gamma) staining and confirmed these epitopes by means of enzyme-linked immunospot assay and pentamer staining. We show that a single immunization with rAd35.LSA-1 induced a strong antigen-specific IFN-gamma CD8(+) T-cell response but no measurable antibody response. In contrast, vaccinations with the adjuvanted recombinant LSA-1 protein induced remarkably low cellular responses but strong antibody responses. Finally, both priming and boosting of the adjuvanted protein by rAd35 resulted in enhanced T-cell responses without impairing the level of antibody responses induced by the protein immunizations alone. Furthermore, the incorporation of rAd35 in the vaccination schedule led to a skewing of LSA-1-specific antibody responses toward a Th1-type immune response. Our results show the ability of rAd35 to induce potent T-cell immunity in combination with protein in a prime-boost schedule without impairing the B-cell response.

High-level expression from two independent expression cassettes in replication-incompetent adenovirus type 35 vector.

Replication-incompetent adenovirus type 35 (rAd35) represents a potent vaccine carrier that elicits strong, antigen-specific T- and B-cell responses in diverse preclinical models. Moreover, Ad35 is rare in human populations, resulting in the absence of neutralizing antibodies against this carrier, in contrast to the commonly used rAd5. Therefore, rAd35 is being investigated as a vaccine carrier for a number of diseases for which an effective vaccine is needed, including malaria, AIDS and tuberculosis. However, it can be perceived that effective immunization will require insertion of multiple antigens into adenoviral vectors. We therefore wanted to create rAd35 vectors carrying double expression cassettes, to expand within one vector the number of insertion sites for foreign DNA encoding antigenic proteins. We show that it is possible to generate rAd35 vectors carrying two cytomegalovirus promoter-driven expression cassettes, provided that the polyadenylation signals in each expression cassette are not identical. We demonstrate excellent rAd35 vector stability and show that expression of a transgene is not influenced by the presence of a second expression cassette. Moreover, by using two model vaccine antigens, i.e. the human immunodeficiency virus-derived Env-gp120 protein and the Plasmodium falciparum-derived circumsporozoite protein, we demonstrate that potent T- and B-cell responses are induced to both antigens expressed from a single vector. Such rAd35 vectors thus expand the utility of rAd35 vaccine carriers for the development of vaccines against, for example, malaria, AIDS and tuberculosis.

Increased immunogenicity of recombinant Ad35-based malaria vaccine through formulation with aluminium phosphate adjuvant.

Previously, we have shown the potency of recombinant Adenovirus serotype 35 viral vaccines (rAd35) to induce strong immune response against the circumsporozoite protein (CS) of the plasmodium parasite. To further optimize immunogenicity of Ad35-based malaria vaccines we formulated rAd35.CS vaccine with aluminium phosphate adjuvant (AlPO(4)). In contrast to the conventional protein based vaccines no absorption to aluminium adjuvant was observed and rAd35 viral in vitro infectivity in mammalian cells was preserved. Immunization with Ad35.CS formulated with AlPO(4) resulted in significantly higher CS specific T and B cell responses in mice upon either single or prime-boost vaccination regimens as compared to rAd35.CS alone. With these results we report for the first time the feasibility of using an AlPO(4) adjuvant to increase the potency of a live adenovirus serotype 35-based vaccine.

Expression and immunogenicity of the Plasmodium falciparum circumsporozoite protein: the role of GPI signal sequence.

Previous studies have shown that the immunogenicity of rodent malaria parasite-derived circumsporozoite protein (CS) can be improved by deleting the glycosyl-phosphatidyl-inositol (GPI) signal sequence. To study whether GPI signal sequence deletion would also improve immunogenicity of CS derived from the major plasmodium species causing mortality in humans (P. falciparum), we tested different variants of the P. falciparum CS protein in the context of a live vector-based vaccine carrier (rAd35). We demonstrate that deletion of the GPI signal sequence from CS did not result in altered expression or secretion. In contrast, cellular localization was clearly altered, which perhaps helps to explain the significant improvement of anti-CS antibody and T-cell responses observed in mice using deletion variants in the context of the rAd35 carrier. Our results show that rational design of antigens is warranted for further development of malaria vaccines.

In vivo evoked specific cell mediated cytotoxicity in carp (Cyprinus carpio L.) uses mainly a perforin/granzyme-like pathway.

A specific cytotoxicity assay has been developed for carp using the Epithelioma Papulosum Cyprini (EPC) cell line as target cells and the release of lactate dehydrogenase (LDH) as read out system. Non-specific cytotoxicity against EPC was not observed, but animals immunized with EPC clearly showed specific killing by effector cells present in kidney, blood and spleen. This killing was strongly calcium dependent, suggesting the utilization of a perforin/granzyme-like pathway. However, blocking with EGTA was not complete indicating the existence of other additional killing routes.

Elevated interleukin-18 protein expression in early active and progressive plaque-type psoriatic lesions.

Psoriasis is a T cell-mediated inflammatory skin disease characterized by an elevated IFN-gamma and IL-12p70 expression in skin lesions. Interleukin-18 (IL-18) synergizes with IL-12 to induce IFN-gamma production and a strong T-helper-1-mediated immune response, or to induce Th2 polarization depending on the immunological context. We have previously shown that keratinocytes in normal skin produce and store large amounts of pro-IL-18. In this study, we hypothesized that the expression of IL-18 in psoriatic lesional skin might be altered compared to normal skin. Therefore, IL-18 expression was assessed in psoriatic, stable, plaque-type lesions and early active and progressive lesions. IL-18 mRNA and protein concentrations were constitutively high, and did not differ between normal and stable, plaque-type epidermis. In active and progressive lesions an elevated expression of total IL-18 protein relative to normal and stable, plaque-type epidermis was detected using ELISA, while on Western blot, the differences in pro- or mature IL-18 were less clear. Our results indicate that the role of IL-18 in the pathogenesis of early phases of psoriasis may be more prominent than in established psoriatic lesions.

CD40 ligation-induced cytokine production in human skin explants is partly mediated via IL-1.

CD40 ligation by CD40 ligand(+) CD4(+) T cells has been claimed to be involved in inflammatory responses in human skin. However, these data are derived from in vitro cell culture systems and immunohistochemistry, and the mechanisms involved have not been fully elucidated. We previously observed that cells in intact normal human skin secrete high levels of IL-6 and IL-8 upon stimulation with IL-1 beta. In vitro studies have shown that CD40 ligation on human keratinocytes results in the production of IL-6 and IL-8 as well. We used a novel tissue culture system with intact normal human skin, and show that antibody ligation of CD40 results in the induction of several pro- and anti-inflammatory cytokines. IL-6, IL-8, tumor necrosis factor (TNF)-alpha, IL-12 and IL-1 beta were induced upon CD40 ligation and IFN-gamma stimulation, while IL-10 could be induced by CD40 ligation alone and was reduced again by the addition of IFN-gamma. Since CD40 ligation on monocytes and dendritic cells in vitro results in the secretion of IL-1, which is pre-stored in high concentrations in normal human keratinocytes, we subsequently investigated whether CD40 induced IL-6 and IL-8 production in skin is mediated via IL-1. Indeed IL-1 receptor antagonist inhibited the CD40 ligation-induced IL-6 and IL-8 production, while TNF-alpha and IL-10 production were not affected. These data show that CD40 ligation-induced secretion of IL-6 and IL-8, but not TNF-alpha and IL-10, is partially mediated via IL-1 and that IL-1 plays a prominent role in the inflammatory response initiated by CD40 ligation in intact human skin.