WHO/KFDA joint workshop on implementing WHO guidelines on evaluating similar biotherapeutic products, Seoul, Republic of Korea 24-26 August, 2010

In August 2010, the World Health Organization and the Korea Food & Drug Administration jointly organized the first implementation workshop of WHO guidelines on evaluating similar biotherapeutic products (SBPs) at the global level. The objective of the Workshop was to facilitate implementation of the newly adopted WHO Guidelines into the practice of national regulatory authorities (NRAs). WHO Guidelines were recognized by the workshop participants as a tool for harmonizing regulatory requirements worldwide. By reviewing and practicing several case studies, better understanding and consensus on the principles of clinical trial designs were reached. However, variations in terms of the national requirements for quality, safety and efficacy of these products revealed diversity in the regulatory expectations in different countries and regions. In addition, lack of terminology for the products developed as copy products (so called "me too" products) with a partial comparability to an RBP, led to a great diversity in evaluating as well as naming these products. The workshop participants proposed the following actions: a) NRAs should make efforts to build their capacities for regulation of SBPs; b) WHO should revise WHO Guidelines for assuring the quality of products prepared by recombinant DNA technology (WHO TRS 814) and continue monitoring progress with the implementation of the Guidelines on evaluating SBPs. Publication of the outcome of the Workshop was recognized as another action that WHO should coordinate.     

Evaluation of similar biotherapeutic products (SBPs): scientific principles and their implementation

The availability of biotherapeutic products to patients has a major impact on the success in treating many life-threatening and chronic diseases. These products are often derived by recombinant DNA technology and are expensive for the majority of patients who need them most. Increasing numbers of patents/data protection are now expiring and biologicals "similar" to the originators (innovative products) are coming to the market. This process is seen as a mechanism for increasing the access to biotherapeutic products which are very much needed for the treatment of chronic diseases worldwide. The emergence of Similar Biological Products (SBPs) has created numerous challenges in developing, licensing, and using these important products. From a public health perspective, the overall expectation is that similar products will be available at an affordable price and will increase patients' access to the therapy. In response to the requests for assistance in defining regulatory requirements for SBPs, the WHO Expert Committee on Biological Standardization (ECBS) adopted the new WHO Guidelines for evaluation of SBPs in October 2009. This article provides a brief account of the WHO initiative to assist its member states to establish national requirements for the regulatory oversight of SBPs. The aim of the article is to inform its readers of the current status of WHO Guidelines on the evaluation of similar biotherapeutic products and of the plan to strengthen national regulatory requirements to assure quality, safety and efficacy of similar biotherapeutic products at the global level.     

Biosimilars clinical development program: confirmatory clinical trials: a virtual/simulated case study comparing equivalence and non-inferiority approaches

As part of long term commitment of the Biologicals and Vaccines Committee (B&V) of the International Federation of Pharmaceutical Manufacturers and Association (IFPMA) to provide expert input to the WHO on their recently finalized GUIDELINES ON EVALUATION OF SIMILAR BIOTHERAPEUTIC PRODUCTS (SBPs), and in response to WHO's request, the IFPMA B&V prepared a clinical case study at a recent WHO workshop in Seoul, Korea. The case study, presented by Mark Fletcher on behalf of B&V, involved a model scenario for a clinical efficacy trial to support the approval of a Similar Biotherapeutic Product (SBP) as part of the required comparative clinical program against a Reference Biotherapeutic Product (RBP). A key goal was to understand and illustrate key clinical and statistical principles, and considerations described in the WHO Guidance for regulatory authorities when designing and implementing WHO guidelines and post-approval regulatory oversight for SBPs. Using this model SBP/RBP pair, an interactive discussion was carried out among the workshop participants on the pros and cons of using equivalence vs. non-inferiority designs to assess the two products' similarity. Through discussion of the case, the complexity of demonstrating similar efficacy and safety of a SBP vs. RBP for biotherapeutic products is outlined and discussed in the context of the key principles laid out in the recently published WHO GUIDELINES ON EVALUATION OF SIMILAR BIOTHERAPEUTIC PRODUCTS (SBPs). The exercise illustrates the need for a case-by-case approach when interpreting clinical data from SBP dossiers to adequately assure similar efficacy and safety of SBPs for any studied indication.     

Clinical programs in the development of similar biotherapeutic products: rationale and general principles

Similar biotherapeutic products (SBPs) or biosimilars are biologics developed by pharmaceutical manufacturers to match originator biologics that have been on the market for a long time and lost their exclusivity (patent and market protection). The recently issued WHO guidelines on evaluation of SBPs provide clear guidance for manufacturers and regulators on how to develop and gain approval for these products. The present contribution illustrates the rationale for and general principles of the clinical programs used in the development of SBPs, taking the example of the three biosimilar products developed and marketed in Europe by Sandoz, namely growth hormone (Omnitrope?, the first ever EU biosimilar approval), erythropoietin α (Binocrit?), and filgrastim (Zarzio?).     

Towards regulation of similar biotherapeutic products: Thailand's perspective

The implementation of universal health coverage scheme in Thailand allows quality, equitable and accessible health care for all. Patients with life threatening and chronic diseases can get access to biotherapeutic products to treat their ailments. This triggered a major impact on the need for specific guidelines in evaluation of similar biotherapeutic products in order to standardize the regulatory pathway to license this class of products ensuring that the products meet acceptable levels of quality, safety and efficacy. The development of similar biotherapeutic products (SBP) should be considered to ensure therapeutic equivalence of biotherapeutics products at more affordable prices. This will lead to greater ease and speed of approval and assurance of the quality, safety and efficacy of these products. Therefore, we report herein the SBP situation in Thailand.     

China's perspective on similar biotherapeutic products

In order to ensure most Chinese patients, particularly in the population with relatively low incomes, have access to safe, low cost, effective and quality-assured medicines, a number of "stand-alone" biological products, which have good quality, safety and efficacy have been marketed in China. Many countries and regions' regulatory agencies are actively engaging in the development of bio-similar guidance and documents, which is being coordinated by WHO. As a major developing country of new drug development, China is now working hard to promote the process of new similar biotherapeutic products (SBPs) approval and also actively involved in developing and updating technical documents.     

Biosimilars--global issues, national solutions

Biotechnology derived medicinal products are presently the best characterized biologicals with considerable production and clinical experience, and have revolutionized the treatment of some of the most difficult-to-treat diseases, prolonging and improving the quality of life and patient care. They are also currently one of the fastest growing segments of the pharmaceutical industry market. The critical challenge that the biopharmaceutical industry is facing is the expiry of patents for the first generation of biopharmaceuticals, mainly recombinant DNA derived products, such as interferons, growth hormone and erythropoetin. The question that immediately arose was how should such copies of the originator products be licensed, bearing in mind that they are highly complex biological molecules produced by equally complex biological production processes with their inherent problem of biological variability. Copying biologicals is much more complex than copying small molecules and the critical issue was how to handle the licensing of products if relying in part on data from an innovator product. Since 2004 there has been considerable international consultation on how to deal with biosimilars and biological copy products. This has led to a better understanding of the challenges in the regulatory evaluation of the quality, safety and efficacy of "biosimilars", to the exchange of information between regulators, as well as to the identification of key issues. The aim of this article is to provide a brief overview of the scientific and regulatory challenges faced in developing and evaluating similar biotherapeutic products for global use. It is intended as an introduction to the series of articles in this special issue of Biologicals devoted to similar biotherapeutic products.     

The role of the quality assessment in the determination of overall biosimilarity: A simulated case study exercise

A determination of biosimilarity is based on a thorough characterization and comparison of the quality profiles of a similar biotherapeutic product and its reference biotherapeutic product. Although the general principles on the role of the quality assessment in a biosimilar evaluation are widely understood and agreed, detailed discussions have not been published yet. We try to bridge this gap by presenting a case study exercise based on fictional but realistic data to highlight key principles of an evaluation to determine the degree of similarity at the quality level. The case study comprises three examples for biosimilar monoclonal antibody candidates. The first describes a highly similar quality profile whereas the second and third show greater differences to the reference biotherapeutic product. The aim is to discuss whether the presented examples can be qualified as similar and which additional studies may be helpful in enabling a final assessment. The case study exercise was performed at the WHO implementation workshop for the WHO guidelines on quality assessment of similar biotherapeutic products held in Xiamen, China, in May 2012. The goal was to illustrate the interpretation of the comparative results at the quality level, the role of the quality assessment in the entire biosimilarity exercise and its influence on the clinical evaluation. This paper reflects the outcome of the exercise and discussion from Xiamen.     

Variability of intended copies for etanercept (Enbrel®): Data on multiple batches of seven products

Fusion protein and monoclonal antibody-based tumor necrosis factor (TNF) inhibitors represent established treatment options for a range of inflammatory diseases. Regulatory authorities have outlined the structural characterization and clinical assessments necessary to establish biosimilarity of a new biotherapeutic product with the innovator biologic drug. Biologic products that would not meet the minimum World Health Organization's standard for evaluation of similar biotherapeutic products are available in some countries; in some cases relevant data to assess biosimilarity and appropriate regulatory approval pathways are lacking.

Batches of seven intended copy (IC) products for etanercept (Enbrel®) were subjected to a subset of test methods used in the routine release and heightened characterization of Enbrel®, to determine key attributes of identity, quality, purity, strength, and activity. While a number of quality attributes of the IC lots tested met the release specifications for Enbrel®, none fell within these limits across all methods performed, and there were no IC lots that satisfied the criteria typically applied by the innovator to support comparability with Enbrel®. Although the consequences of these differences are largely unknown, the potential for unanticipated clinical outcomes should not be overlooked.     

黑腹果蝇头部中RNA结合蛋白RBP9的互作蛋白筛选

【目的】筛选RNA结合蛋白RBP9在黑腹果蝇Drosophila melanogaster头部中的互作蛋白。【方法】利用CRISPR/Cas9基因组编辑技术,分别将3×Flag和V5标签编码序列插入到黑腹果蝇成虫头中RBP9和FNE基因起始密码子ATG的后面,构建黑腹果蝇转基因品系3×Flag-RBP 9/3×Flag-RBP9(3FRBP9)和V5-FNE/V5-FNE(VFNE);利用免疫沉淀和质谱法鉴定黑腹果蝇3FRBP9和野生型品系成虫头部中RBP9的互作蛋白并进行生物信息学分析。利用免疫共沉淀方法检测RBP9与FNE蛋白之间的相互作用。【结果】质谱法从黑腹果蝇成虫头部共鉴定了190个与RBP9相互作用的蛋白,其中包括ELAV和FNE。KEGG富集分析显示这些蛋白的基因主要参与核糖体、碳代谢、三羧酸循环、氨基酸生物合成等通路。免疫共沉淀实验结果验证了RBP9与FNE蛋白之间存在相互作用。【结论】RNA结合蛋白ELAV家族成员在黑腹果蝇成虫头部中具有相互作用。本研究为RBP9在果蝇神经系统发育中的功能研究提供了重要实验依据。     

Visualization of the stop of microtubule depolymerization that occurs at the high-density region of microtubule-associated protein 2 (MAP2).

Individual microtubules (MTs) repeat alternating phases of polymerization and depolymerization, a process known as dynamic instability. Microtubule-associated proteins (MAPs) regulate the dynamic instability by increasing the rescue frequency. To explore the influence of MAP2 on in vitro MT dynamics, we correlated the distribution of MAP2 on individual MTs with the dynamic phase changes of the same MTs. MAP2 was modified selectively on its projection region by X-rhodamine iodoacetamide without altering the MT-binding activity. When the labeled MAP2 was added to MTs, the fluorescence was distributed along almost the entire length of individual MTs. However, the inhomogeneity of the distribution gradually became obvious due to the fluorescence bleaching, and the MTs appeared to consist of rapidly bleached portions (RBPs) and slowly bleached portions (SBPs), which were distributed randomly along the MT. By measuring the duration of fluorescence bleaching, the density of MAP2 in SBP was estimated to be approximately 2.5 times higher than the RBP. The average tubulin:MAP2 ratio in SBP was calculated to be 16. When the MT dynamics were observed by dark-field microscopy after determining the MAP2 distribution, rescues were always found to occur only at the SBPs. MTs also displayed intermittent shortening by repeated depolymerization phases separated by pause phases. In these cases, depolymerization phases stopped only at the SBPs. Not every SBP stopped depolymerization, but depolymerization always stopped at an SBP. Taken together, we suggest that there is a minimum density of MAP2 that is necessary to stop depolymerization.     

Retinol-binding protein messenger RNA levels in the liver and in extrahepatic tissues of the rat

A retinol-binding protein (RBP) cDNA clone was used to examine the effect of retinol status on the level of RBP mRNA in the liver, and to explore whether extrahepatic tissues contain RBP mRNA. In the first series of experiments, poly(A+) RNA was isolated from the livers of normal, retinol-depleted, and retinol-repleted rats and the levels of RBP mRNA in these samples were determined by both Northern blot and RNA Dot blot analyses. The levels of RBP mRNA in liver were similar in all three groups of rats. These findings confirm and extend previous studies which showed that retinol did not alter the in vivo rate of RBP synthesis or the translatable levels of RBP mRNA. In a second series of experiments, the RBP cDNA clone was used to survey poly (A+) RNA isolated from 12 different rat tissues for RBP mRNA by Northern blot analysis. We found that, along with the liver, many extrahepatic tissues contained RBP mRNA. Kidney contained RBP mRNA at a level of 5-10% of that of the liver, and the lungs, spleen, brain, stomach, heart, and skeletal muscle contained 1-3% of that of the liver. Translation of kidney poly (A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-RBP antiserum resulted in a protein band of the same size as liver preRBP. These data suggest that RBP is synthesized in many extrahepatic tissues.It is possible that this extra-hepatically synthesized RBP may function in the recycling of retinol from these tissues back to the liver or to other target organs.     

Exposure limits: the underestimation of absorbed cell phone radiation, especially in children

The existing cell phone certification process uses a plastic model of the head called the Specific Anthropomorphic Mannequin (SAM), representing the top 10% of U.S. military recruits in 1989 and greatly underestimating the Specific Absorption Rate (SAR) for typical mobile phone users, especially children. A superior computer simulation certification process has been approved by the Federal Communications Commission (FCC) but is not employed to certify cell phones. In the United States, the FCC determines maximum allowed exposures. Many countries, especially European Union members, use the "guidelines" of International Commission on Non-Ionizing Radiation Protection (ICNIRP), a non governmental agency. Radiofrequency (RF) exposure to a head smaller than SAM will absorb a relatively higher SAR. Also, SAM uses a fluid having the average electrical properties of the head that cannot indicate differential absorption of specific brain tissue, nor absorption in children or smaller adults. The SAR for a 10-year old is up to 153% higher than the SAR for the SAM model. When electrical properties are considered, a child's head's absorption can be over two times greater, and absorption of the skull's bone marrow can be ten times greater than adults. Therefore, a new certification process is needed that incorporates different modes of use, head sizes, and tissue properties. Anatomically based models should be employed in revising safety standards for these ubiquitous modern devices and standards should be set by accountable, independent groups.     

Comparison of the periplasmic receptors for L-arabinose, D-glucose/D-galactose, and D-ribose. Structural and Functional Similarity.

The primary sequence of the receptor for L-arabinose or Ara-binding protein (ABP) composed of 306 residues is very different from the D-glucose/D-galactose-binding protein (GGBP) which consists of 309 residues. Nevertheless, superimpositioning of the well-refined high resolution structures of ABP in complex with D-galactose and the GGBP in complex with D-glucose shows very similar structures; 220 of the residues (or about 70%) have a root mean square deviation of 2.0 A. From the superpositioning, nine pairs of continuous segments (consisting of 8-51 residues), mainly alpha-helices and beta-strands that form the core of the two lobes of the bilobate proteins were found to exhibit strong sequence homology. The equivalenced structures and aligned sequences show that many of the polar, as well as aromatic residues, in the sugar-binding sites located in the cleft between the two lobes are highly conserved. Surprisingly, however, the exact mode of binding of the D-galactose in ABP is totally different from that of the D-glucose in GGBP. Using the structurally aligned sequences of the ABP and GGBP as a template, we have matched the sequence of the ribose-binding protein (RBP) which consists of 271 residues with the ABP/GGBP pair. Although the nine aligned segments of all three proteins show little sequence identity, they have significant homology. Four additional segments of RBP were matched only with GGBP, leading to the alignment of about 90% of the RBP sequence with the GGBP sequence. Many of the conserved residues in the binding sites of ABP and GGBP matched with similar residues in RBP. Additional observations indicate that the GGBP/RBP pair is more closely related than the ABP/RBP or ABP/GGBP pair. All three binding proteins, which may have diverged from a common ancestor, serve as primary receptors for bacterial high affinity active transport systems. Moreover, GGBP and RBP, but not ABP, also act as receptors for chemotaxis. An exposed site located in one domain, which includes Gly74, for interacting with the trg transmembrane signal transducer that is involved in triggering chemotaxis has been located in the structure of GGBP (Vyas, N.K., Vyas, M.N., and Quiocho, F.A. (1988) Science 242, 1290-1295). Whereas the site is absent in the structure of ABP, it is strongly predicted to be present in RBP which shares the same trg transducer with GGBP. The knowledge-based alignment of RBP further revealed two possible additional peripheral chemotactic sites that show high structural and sequence similarity between GGBP and RBP only. At least one of these sites, together with the one proven to exist in the other domain, could be used by the signal transducer with which both binding proteins interact in a way which the substrate-loaded "closed cleft" structure could be discriminated from the unliganded "open cleft" form by the transducer.     

Crystal Structure and Function of a DARPin Neutralizing Inhibitor of Lactococcal Phage TP901-1: COMPARISON OF DARPin AND CAMELID VHH BINDING MODE*

Combinatorial libraries of designed ankyrin repeat proteins (DARPins) have been proven to be a valuable source of specific binding proteins, as they can be expressed at very high levels and are very stable. We report here the selection of DARPins directed against a macromolecular multiprotein complex, the baseplate BppU·BppL complex of the lactococcal phage TP901-1. Using ribosome display, we selected several DARPins that bound specifically to the tip of the receptor-binding protein (RBP, the BppL trimer). The three selected DARPins display high specificity and affinity in the low nanomolar range and bind with a stoichiometry of one DARPin per BppL trimer. The crystal structure of a DARPin complexed with the RBP was solved at 2.1 Å resolution. The DARPin·RBP interface is of the concave (DARPin)-convex (RBP) type, typical of other DARPin protein complexes and different from what is observed with a camelid VHH domain, which penetrates the phage p2 RBP inter-monomer interface. Finally, phage infection assays demonstrated that TP901-1 infection of Lactococcus lactis cells was inhibited by each of the three selected DARPins. This study provides proof of concept for the possible use of DARPins to circumvent viral infection. It also provides support for the use of DARPins in co-crystallization, due to their rigidity and their ability to provide multiple crystal contacts.Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry for the production of an array of fermented milk products. Several industrial strains are sensitive to various distinct bacteriophages, mostly belonging to the Siphoviridae family. The lactococcal phage population is divided in at least 10 genetically distinct groups, of which the 936, c2, and P335 groups are prominent (1, 2). These L. lactis-infecting phages are considerably problematic in causing milk fermentation failures and resulting in decreased yields as well as low quality products (3). Preventing these infections has proven to be difficult because of lactococcal phage ubiquity, biodiversity, and genomic plasticity (4).Phage infection is initiated by binding of the phage receptor-binding protein (RBP),⁵ located within the baseplate at the distal part of the tail, to its receptor on the host cell surface (5). We have previously solved the crystal structures of the three RBPs of the lactococcal phages p2 (936) (6), bIL170 (936) (7), TP901-1 (P335) (8), and their chimera (9) as well as characterized their saccharide binding sites (10). The RBPs of these phages have a similar homotrimeric architecture related by a 3-fold axis. They comprise three domains: the N terminus shoulder domain, the interlaced β-prism neck domain, and the jellyroll head domain at the C terminus. The head domain has a saccharide binding site likely involved in host recognition. The lactococcal phage TP901-1 contains a double-disk-shaped baseplate at the tip of its tail which is made of a lower baseplate protein (BppL) and an upper baseplate protein (BppU) (11).One strategy to minimize bacteriophage infections is to competitively block phage adsorption by adding a protein that specifically binds to the phage RBP. A neutralizing llama VHH domain recognizing the head domain of the phage p2 RBP has been used to block L. lactis phage infection in milk fermentation (12). Lactococcal phages could readily escape neutralization by generating mutations interfering with VHH binding over the large interaction surface while keeping the central polysaccharide receptor binding pocket intact (10). Designed ankyrin repeat proteins (DARPins) may be another tool to neutralize viral infection, as they display distinct characteristics from VHHs and contain the required properties in terms of stability and facility of expression (13).Ankyrin repeat proteins are found in virtually all phyla and mediate specific protein-protein interactions in all cell compartments (14). The ankyrin elementary module is composed of 33 amino acids structured as a β-turn followed by two antiparallel α-helices and a loop connected to the β-turn of the next repeat. The repeats are stacked in a rigid manner. In creating a DARPin library, residues in each repeat were subdivided in two groups; (i) randomized residues constituting potential target interaction points and (ii) framework residues, important for maintaining the ankyrin fold (13). Libraries with varying repeat numbers were assembled and named according to the constituent repeat number; N2C and N3C libraries were used in this study, with two and three internal repeats inserted between the N and C capping repeats, respectively. DARPins are a powerful alternative to the use of antibodies, notably because of their very high expression rates in Escherichia coli, their high stability paired with high affinity, and successful reports of their use in co-crystallization (15–19). Their architecture results in a very rigid structure that facilitates multiple crystal contacts and may promote crystal formation of the protein of interest by providing additional surfaces for such crystal contacts.We report here the selection and analysis of DARPin binders directed against a macromolecular multiprotein ensemble, the TP901-1 baseplate BppU·BppL protein complex. Ribosome display selection, ELISA screening, and surface plasmon resonance (SPR) measurements allowed us to isolate and characterize three N2C DARPins that recognized the RBP (BppL of the BppU·BppL complex) with high specificity and affinity. Further studies showed that the three DARPins bound to a unique area of the RBP at the tip of the head domain. QELS, MALS, UV, and refractometry coupled online with a size exclusion chromatography (SEC) column allowed us to monitor complex formation in solution as well as to estimate DARPin binding stoichiometry. Crystals of one of these selected DARPins in complex with the RBP were obtained, and the x-ray structure was solved at 2.1 Å resolution. This constitutes the first structure of a DARPin complex originating from the N2C library and the highest resolution for a DARPin complex structure reported to date. Finally, phage adsorption inhibition experiments demonstrated that the three N2C DARPins strongly inhibited L. lactis infection by TP901-1. We describe the DARPin·RBP interface and compare it to other DARPin interfaces. We also compare it to the p2 RBP·VHH5 complex, a previously selected llama VHH domain inhibiting p2 phage adsorption (12), to highlight the different binding mode of these two types of binders.     

Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Background

Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac).

Methodology/Principal Findings

We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.

Conclusions

We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described.     

O-carboxamidomethyl tyrosine as a reaction product of the alkylation of proteins with iodoacetamide

Iodoacetamide-1-¹⁴C was found to be rapidly incorporated into RBP, a protein devoid of free SH, under conditions similar to those normally employed for the derivatization of SH. The reaction extent is pH dependent and at pH 10.0 one mole of acetamide was incorporated per mole of tyrosine residue. The amino acid composition of the alkylated RBP was found to be identical to RBP except for a loss of tyrosine and the appearance of a new dicarboxylic acid peak which was converted to tyrosine by prolonged acid hydrolysisUnder similar reaction conditions, phenol and p-cresol reacted rapidly to form phenoxyacetamide and p-cresoxyacetamide. These phenolic ethers as well as anisole and phenetole were found to be readily hydrolized under the conditions normally used to hydrolyze protein.The incorporation of acetamide into RBP did not effect its riboflavinbinding capacity or its immunological reactivity to RBP antibody. The ¹⁴C alkylated RBP has been found to be a convenient tool for biological half-life studies.The tyrosine residues of glucagon react with iodoacetamide in a similar fashion and the use of ¹⁴C-iodoacetamide may prove to be a convenient means of introducing ¹⁴C into proteins. Iodoacetamide in the pH 7–10.0 range will derivatize the cysteine and tyrosine groups of proteins at comparable rates.     

Considerations in the development of live biotherapeutic products for clinical use

Food products in the United States (U.S.), including dietary supplements, may contain live microorganisms and can be promoted for general health, nutritional, or structure/function claims. In contrast, such preparations used with the intention of having a preventive or therapeutic effect in humans are regulated by the Food and Drug Administration (FDA) in the U.S. as biological products, specifically as live biotherapeutic products (LBPs). Discussion of considerations in the early development of LBPs may aid in preparation of an Investigational New Drug Application (IND) that is designed to collect clinical data to support marketing approval of a LBP in the U.S. for a specific clinical use. Product information is an important component of an IND to support a proposed clinical study.     

On the Shelf Life of Pharmaceutical Products

This article proposes new terminology that distinguishes between different concepts involved in the discussion of the shelf life of pharmaceutical products. Such comprehensive and common language is currently lacking from various guidelines, which confuses implementation and impedes comparisons of different methodologies. The five new terms that are necessary for a coherent discussion of shelf life are: true shelf life, estimated shelf life, supported shelf life, maximum shelf life, and labeled shelf life. These concepts are already in use, but not named as such. The article discusses various levels of "product" on which different stakeholders tend to focus (e.g., a single-dosage unit, a batch, a production process, etc.). The article also highlights a key missing element in the discussion of shelf life-a Quality Statement, which defines the quality standard for all key stakeholders. Arguments are presented that for regulatory and statistical reasons the true product shelf life should be defined in terms of a suitably small quantile (e.g., fifth) of the distribution of batch shelf lives. The choice of quantile translates to an upper bound on the probability that a randomly selected batch will be nonconforming when tested at the storage time defined by the labeled shelf life. For this strategy, a random-batch model is required. This approach, unlike a fixed-batch model, allows estimation of both within- and between-batch variability, and allows inferences to be made about the entire production process. This work was conducted by the Stability Shelf Life Working Group of the Product Quality Research Institute.     

Experience of reviewing the follow-on biologics including Somatropin and erythropoietin in Japan

To share the experience of reviewing clinical data required for the licensing of follow-on biologic products (biosimilar products and similar biotherapeutical products as EU and WHO terminology, respectively) in Japan, the data packages of two follow-on biologics, "Somatropin BS s.c. [Sandoz] (Omnitrope?)" and "Epoetin alfa BS [JCR]", which have been recently approved in Japan according to the "Guidelines for the Quality, Safety and Efficacy Assurance of Follow-on Biologics" published on March 4th 2009, are described. The clinical data package and indication of Somatropin BS/Omnitrope(?) were different in each country. In case of Epoetin alfa BS [JCR], non-clinical and clinical data-package was different from those of erythropoietin biosimilar products approved in EU. Submission of post-marketing surveillance plans for both products was required. Even though there seem to be differences in data requirements by each national regulatory authority, the accumulation of experience will provide the rationale and consensus on how to design the clinical trials for follow-on biologics.