[Show abstract][Hide abstract] ABSTRACT: Protein glycosylation is among the most common and well defined post-translational modifications due to its vital role in protein function. Monitoring variation in glycosylation is necessary for producing more effective therapeutic proteins. Glycans attached to glycoproteins interact highly specific with lectins, natural carbohydrate-binding proteins, which property is used in the current label-free methodology. We have established a lectin micro-array for label-free detection of lectin-carbohydrate interactions allowing us to study protein glycosylation directly on unmodified glycoproteins. The method enables simultaneous measurement of up to 96 lectin-carbohydrate interactions on a multiplex surface plasmon resonance imaging platform within 20 minutes. Specificity determination of lectins succeeded by analysis of neoglycoproteins and enzymatically remodeled glycoproteins to verify carbohydrate binding. We demonstrated the possibilities for glycosylation fingerprinting by comparing different Erythropoietin sources without the need for any sample pretreatment and we were able to accurately quantify relative sialylation levels of Erythropoietin.
[Show abstract][Hide abstract] ABSTRACT: The European Union (EU) was the first region to establish a regulatory framework for biosimilars, in which animal studies are required to confirm similarity to a reference product. However, animal studies described in European public assessment reports (EPARs) or marketing authorisation applications (MAAs) did not identify clinically or toxicologically relevant differences despite differences in quality, suggesting that animal studies lack the sensitivity to confirm biosimilarity. Scientific advice provided learning opportunities to evolve existing guidance. Altogether, the data support a step-wise approach to develop biosimilars that focuses on quality and clinical efficacy of biosimilar. This approach might be more effective and does not necessarily require animal studies, which is also reflected in new EU draft guidance.
Drug Discovery Today 11/2014; 20(4). DOI:10.1016/j.drudis.2014.11.009 · 5.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conjugation of polyethylene glycol (PEG) to therapeutics has proven to be an effective approach to increase the serum half-life. However, the increased use of PEGylated therapeutics has resulted in unexpected immune-mediated side-effects. There are claims that these are caused by anti-PEG antibodies inducing rapid clearance. These claims are however hampered by the lack of standardized and well-validated antibody assays. PEGylation has also been associated with the activation of the complement system causing severe hypersensitivity reactions. Here, we critically review the clinical and analytical tools used. In addition, we propose an explanation of the immune-mediated side-effects of PEGylated products based on the haptogenic properties of PEG, responsible for complement activation and the induction of anti-PEG antibodies.
Drug Discovery Today 09/2014; 19(12). DOI:10.1016/j.drudis.2014.08.015 · 5.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Understanding the mechanism of aggregation of a therapeutic protein would not only ease the manufacturing processing but could also lead to a more stable finished product. Aggregation of recombinant interferon (IFNβ-1b) was studied by heating, oxidizing, or seeding of unformulated monomeric solution. The formation of aggregates was monitored by dynamic light scattering (DLS) and UV spectroscopy. The autocatalytic monomer loss model was used to fit the data on aggregation rates. The influence of pre-nucleation on aggregation step was demonstrated by inducing the liquid samples containing a monomer form of folded IFNβ-1b by heat and also an oxidizing agent. Results tend to suggest that the nucleus includes a single protein molecule which has been probably deformed. Seeding tests showed that aggregation of IFNβ-1b was probably initiated when 1.0% (w/w) of monomers converted to nucleus form. Chemiluminescence spectroscopy analysis of the sample indicated the generation of 3.0 μM of hydrogen peroxide (H2O2) during nucleation stage of IFNβ-1b aggregation. Arginine with a concentration of 200 mM was sufficient to suppress aggregation of IFNβ-1b by decreasing the rate of pre-nucleation step. We proposed the formation of pre-nucleus structures prior to nucleation as the mechanism of aggregation of IFNβ-1b. Furthermore, we have showed the positive anti-aggregation effect of arginine on pre-nucleation step.
[Show abstract][Hide abstract] ABSTRACT: Aggregation often occurs during manufacturing and storage of protein drugs. Detergents such as sodium dodecyl sulfate are commonly used to prevent aggregation but need to be eliminated before final formulation for safety reasons. We studied the ability of dodecylmaltoside (DDM), a nontoxic alkyl saccharide surfactant, to reduce aggregation and increase the stability of interferon beta-1b (IFN)-β-1b. An increase of 8°C in the Tm of IFN-β-1b was observed when 0.1% of DDM was present in the protein solution. The absorption of DDM on hydrophobic surfaces of IFN-β-1b enables the surface to become hydrophilic and non-ionic, and increases the stability of the protein. 0.1% DDM also results in a 62% increase in helical and a 25% decrease in β-sheet structures. 0.1% DDM not only suppresses aggregate formation but also improves IFN-β-1b solubilization. Furthermore, we have showed the protective effect of DDM on the anti-viral activity of IFN-β-1b in solution.
Journal of Interferon & Cytokine Research 06/2014; 34(11):894-901. DOI:10.1089/jir.2013.0131 · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Drug development has become the exclusive activity of large pharmaceutical companies. However, the output of new drugs has been decreasing for the past decade and the prices of new drugs have risen steadily, leading to access problems for many patients. By analyzing the history of drug development and the pharmaceutical
industry, we identified the main factors causing this system failure. Although many solutions have been
suggested to fix the drug development system, we believe that a combination of reforms of the regulatory and
patent systems is necessary to make drug development sustainable. These reforms must be combined with a
larger, open-access role for public research institutes in the discovery, clinical evaluation and safety evaluation of new drugs.
Drug Discovery Today 02/2014; 19(11). DOI:10.1016/j.drudis.2014.03.004 · 5.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein aggregates are a major risk factor for immunogenicity. Until now most studies on aggregate-driven immunogenicity have focused on linking physicochemical features of the aggregates to the formation of anti-drug antibodies. Lacking is however, basic knowledge on the effect of aggregation on the biodistribution and clearance of therapeutic proteins in vivo. The aim of current study was to get insight into the effect of aggregation on biodistribution in mice using different routes of administration. Fluorescently labeled stressed and unstressed mouse serum albumin was injected via different routes in mice and detected via in vivo fluorescence imaging up to 48 hrs post-injection. We found that biodistribution of stressed MSA significantly differed from its unstressed counterpart. Subcutaneous and intramuscular administration resulted in accumulation of protein at the site of injection, from which clearance of stressed MSA was considerably slower than clearance of unstressed MSA. Upon intravenous and intraperitoneal injection of stressed MSA, fluorescent "hotspots" were observed in the spleens, livers and lungs. Further and more detailed examination of biodistribution after intraperitoneal injection showed higher fluorescence in most of tested organs suggesting more efficient diffusion and/or lymphatic uptake from peritoneum of unstressed MSA than the stressed formulation.
PLoS ONE 01/2014; 9(1):e85281. DOI:10.1371/journal.pone.0085281 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: When the patent of a classical small molecule drug expires, generics can be marketed if their therapeutic equivalence to the original drug has been established. 1,2 Conventional generics for an orally administered drug are considered to be therapeutically equivalent to a reference once pharmaceutical equivalence (that is, identical active substances) and bioequivalence (that is, comparable pharmacokinetics essentially on healthy volunteers) have been established in a crossover study, and do not require formal clinical efficacy and safety studies. The therapeutic equivalence allows therapeutic interchangeability. 3 The acceptance intervals to show that bioequivalence for the logarithm transformed are under the curve and C max ratios lie within an acceptance range of 0.80–1.25 for the 90% confidence intervals. In some cases, the acceptance interval needs to be tightened, and in other cases a wider acceptance may be acceptable. The classical generic approach based on showing pharmaceutical equivalence and bioequivalence has been the basis of the introduction of many safe and effective alternatives to innovative medicines. However, this approach has so far only been applied to products that can be fully characterised. For more complex molecules that are difficult to characterise, such as proteins, the demonstration of bioequivalence requires an alternative approach. Therapeutic proteins are the most widely used class of drugs for which the classical generic paradigm cannot be used. 4,5 The molecular mass of most of the therapeutic proteins varies from 5 to 150kDa, which is 25–1000-times greater than the average small drug molecule. Nearly all therapeutic proteins are produced by living cells and these cells in general make mixtures of different proteins, for example, by variation in the process of post-translation modification, such as glycosylation. There are many tools to analyse the structure and in vitro activity of therapeutic proteins (biologics). However, because of their complexity there is no combination of techniques that can completely describe
[Show abstract][Hide abstract] ABSTRACT: The high prevalence of pure red cell aplasia in Thailand has been associated with the sharp increase in number of recombinant human erythropoietin (rhEPO) copy products, based on a classical generic regulatory pathway, which have entered the market. This study aims to assess the quality of rhEPO copy products being used in Thailand.
Twelve rhEPO copy products were purchased from pharmacies in Thailand, shipped under controlled cold chain conditions to the Netherlands and characterized using (1) high performance size-exclusion chromatography, (2) asymmetrical flow field-flow fractionation, (3) sodium dodecyl sulfate polyacrylamide gel electrophoresis in combination with (4) Western blotting and additionally tested for (5) host cell protein impurities as well as (6) endotoxin contamination.
Some of the tested rhEPO copy products showed high aggregate levels and contained a substantial amount of protein fragments. Also, one of rhEPO copy products had a high endotoxin level, exceeding the FDA limit.
Our observations show that some of the tested copy products on the Thai market differ significantly from the originator rhEPO product, Epogen®. This comparison study supports a link between the quality attributes of copy rhEPO products and their immunogenicity.
Pharmaceutical Research 11/2013; 31(5). DOI:10.1007/s11095-013-1243-9 · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this critical review is to reach a global consensus regarding the introduction of follow-on versions of nonbiological complex drugs (NBCD). A nonbiological complex drug is a medicinal product, not being a biological medicine, where the active substance is not a homo-molecular structure, but consists of different (closely related and often nanoparticulate) structures that cannot be isolated and fully quantitated, characterized and/or described by state of the art physicochemical analytical means and where the clinical meaning of the differences is not known. The composition, quality and in vivo performance of NBCD are highly dependent on manufacturing processes of both the active ingredient as well as in most cases the formulation. The challenges posed by the development of follow-on versions of NBCD are illustrated in this paper by discussing the 'families' of liposomes, iron-carbohydrate ('iron-sugar') drugs and glatiramoids. It is proposed that the same principles for the marketing authorization of copies of NBCD as for biosimilars be used: the need for animal and/or clinical data and the need to show similarity in quality, safety and efficacy. The regulatory approach of NBCD will have to take into consideration the specific characteristics of the drugs, their formulation and manufacturing process and the resulting critical attributes to achieve their desired quality, safety and efficacy. As with the biosimilars, for the NBCD product, family-specific methods should be evaluated and applied where scientifically proven, including sophisticated quality methods, pharmacodynamic markers and animal models. Concerning substitution and interchangeability of NBCD, it is also advisable to take biosimilars as an example, i.e. (1) substitution without the involvement of a healthcare professional should be discouraged to ensure traceability of the treatment of individual patients, (2) keep an individual patient on a specific treatment if the patient is doing well and only switch if unavoidable and (3) monitor the safety and efficacy of the new product if switching occurs.
The AAPS Journal 09/2013; 16(1). DOI:10.1208/s12248-013-9533-z · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the last decade, discussions on the development of the regulatory framework of generic versions of complex drugs such as biologicals and non-biological complex drugs have attracted broad attention. The terminology used is far from harmonized and can lead to multiple interpretations of legal texts, reflection papers, and guidance documents regarding market introduction as well as reimbursement. This article describes the meaning of relevant terms in different global regions (Europe, USA, WHO) and offers a proposal for a globally accepted terminology regarding (non-) biological complex drugs.
The AAPS Journal 09/2013; 16(1). DOI:10.1208/s12248-013-9532-0 · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advanced therapy medicinal products (ATMPs) are the cutting edge of drug innovation. ATMPs have different challenges than other drug classes. To accommodate these challenges and facilitate science-driven development, flexibility in the requirements to demonstrate the safety and efficacy of this rapidly evolving drug class is necessary. To create flexibility, the European Union introduced the risk-based approach. This approach provides the possibility of omitting guideline-based studies based on risk analyses. To gain insight into the effect of the risk-based approach on the non-clinical development of ATMPs, two questions are addressed in this paper. Firstly, "Do companies use a risk-based approach for the non-clinical development of ATMPs?" and, secondly, "Does the Committee for Medicinal Products for Human Use(CHMP) of the European Medicines Agency (EMA) accept non-clinical development programs based on the risk-based approach?". Scientific advice letters formulated by the CHMP were analyzed. The risk-based approach was used to justify deviations from the guidelines in the majority (75%) of the cases. The CHMP accepted 40% of the proposals to omit studies and stated that additional data was necessary to make an informed decision for 35% of the proposals. This indicates that the risk-based approach facilitates the science-driven development of ATMPs.
[Show abstract][Hide abstract] ABSTRACT: The immunogenicity of biopharmaceuticals used in clinical practice remains an unsolved challenge in drug development. Non-human primates (NHPs) are often the only relevant animal model for the development of monoclonal antibodies (mAbs), but the immune response of NHPs to therapeutic mAbs is not considered to be predictive of the response in humans because of species differences. In this study, we accessed the drug registration files of all mAbs registered in the European Union to establish the relative immunogenicity of mAbs in NHPs and humans. The incidence of formation of antidrug-antibodies in NHPs and patients was comparable in only 59% of the cases. In addition, the type of antidrug-antibody response was different in NHP and humans in 59% of the cases. Humanization did not necessarily reduce immunogenicity in humans. Immunogenicity interfered with the safety assessment during non-clinical drug development when clearing or neutralizing antibodies were formed. While important to interpret the study results, immunogenicity reduced the quality of NHP data in safety assessment. These findings confirm that the ability to compare relative immunogenicity of mAbs in NHPs and humans is low. Furthermore, immunogenicity limits the value of informative NHP studies.