An exploratory study on the effect of mechanical stress on particle formation in monoclonal antibody infusions.

Monoclonal antibody infusions (mAb-i) are administered for the treatment of various diseases. They are often transported over long distances from the compounding site to the site of administration. However, transport studies are typically carried out with the original drug product but not with compounded mAb-i. To address this gap, the impact of mechanical stress on the formation of subvisible/nanoparticles in mAb-i was investigated by dynamic light scattering and flow imaging microscopy. Different mAb-i concentrations were subjected to vibrational orbital shaking and stored at 2-8°C up to 35 days. The screening revealed that pembrolizumab and bevacizumab infusions show the highest propensity for particle formation. Especially bevacizumab at low concentrations exhibited an increase in particle formation. Because of the unknown health risks associated with the long-term application of subvisible particles (SVPs)/nanoparticles in infusion bags, stability studies carried out in the frame of licensing application procedures should also focus on SVP formation in mAb-i. In general, pharmacists should minimize the time of storage and mechanical stress during transport, especially in the case of low-concentrated mAb-i. Moreover, if siliconized syringes are used, they should be washed once with saline solution to minimize particle entry.

[Changes and developments in emergency medical services: key challenges for rescue management]. / Veränderungen und Entwicklungen in der präklinischen Notfallversorgung: Zentrale Herausforderungen für das Rettungsdienstmanagement.

Emergency medical services (EMS) and pre-hospital rescue management in Germany as a whole are currently confronted with numerous challenges. In the last decade, the number of calls has risen in almost every EMS area, resulting in a significant increase of ambulance provision. However, human resources management is becoming increasingly difficult and technical and medical requirements are rising steadily. Increasingly, voices are calling for a fundamental reform of emergency care.The aim of this article is to give an overview of the current developments, trends and future challenges in order to illustrate the requirements for present and prospective rescue management. Different topics and areas of action were identified using a non-systematic literature search. The requirements for rescue management are brought together iteratively.The challenges facing EMS are versatile and complex, as are the demands on its management. The heterogeneous and small-scale organisational structure of emergency medical services in Germany poses a major challenge for reform processes. The lack of scientific competencies in EMS also complicates process optimisation. The increasing academisation and research in this field are to be welcomed.

Autophosphorylation activates c-Src kinase through global structural rearrangements.

The prototypical kinase c-Src plays an important role in numerous signal transduction pathways, where its activity is tightly regulated by two phosphorylation events. Phosphorylation at a specific tyrosine by C-terminal Src kinase inactivates c-Src, whereas autophosphorylation is essential for the c-Src activation process. However, the structural consequences of the autophosphorylation process still remain elusive. Here we investigate how the structural landscape of c-Src is shaped by nucleotide binding and phosphorylation of Tyr416 using biochemical experiments, hydrogen/deuterium exchange MS, and atomistic molecular simulations. We show that the initial steps of kinase activation involve large rearrangements in domain orientation. The kinase domain is highly dynamic and has strong cross-talk with the regulatory domains, which are displaced by autophosphorylation. Although the regulatory domains become more flexible and detach from the kinase domain because of autophosphorylation, the kinase domain gains rigidity, leading to stabilization of the ATP binding site and a 4-fold increase in enzymatic activity. Our combined results provide a molecular framework of the central steps in c-Src kinase regulation process with possible implications for understanding general kinase activation mechanisms.

Shedding of glycan-modifying enzymes by signal peptide peptidase-like 3 (SPPL3) regulates cellular N-glycosylation.

Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, ß-1,3 N-acetylglucosaminyltransferase 1 and ß-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes.

Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90.

Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated.

N-glycan PK Profiling Using a High Sensitivity nanoLCMS Work-Flow with Heavy Stable Isotope Labeled Internal Standard and Application to a Preclinical Study of an IgG1 Biopharmaceutical.

PURPOSE: In this study an innovative, highly sensitive work-flow is presented that allows the analysis of a possible influence of individual glyco-variants on pharmacokinetics already during pre-clinical development. Possible effects on the pharmacokinetics caused by glyco-variants have been subject of several studies with in part contradictory results which can be related to differences in the set-up. METHODS: Using 96-well plate based affinity purification an IgG1 antibody was isolated from preclinical samples and glycans were analyzed individually by nanoLCMS. Prerequisite was a reference standard based on stable heavy isotope labeled glycans. The high sensitivity and low sample consumption enabled the integration into the preclinical development program. RESULTS: The data of an IgG1 biopharmaceutical from a preclinical rabbit study showed that some N-glycoforms have a different PK profile compared with the average of all molecule variants as determined by ELISA. IgG1 high mannose glycoforms M5 and M6 were removed from circulation at a higher rate. CONCLUSION: The results of the preclinical study demonstrated the applicability of the developed innovative workflow. The PK profile of glyco-variants could be determined individually. It was concluded that M6 was converted by mannosidases in circulation to M5 which in turn was selectively cleared by mannose receptor binding which is in-line with previously published results. Therefore the developed technology delivers reliable results and can be applied for PK profiling of other mAbs and other types of biopharmaceuticals.

Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals.

N-Glycosylation is a common post-translational modification of monoclonal antibodies with a potential effect on the efficacy and safety of the drugs; detailed knowledge about this glycosylation is therefore crucial. We have developed a reversed-phase liquid chromatographic-mass spectrometric method, with different fluorescent labels, for analysis of N-glycosylation, and compared the sensitivity and selectivity of the methods. Our work demonstrates that anthranilic acid as fluorescent label in combination with reversed-phase liquid chromatography-mass spectrometry is an advantageous method for identification and quantification of neutral and acidic N-glycans. Our results show that mass spectrometry-based quantification correlates with quantification by fluorescence. Chromatographic discrimination between several structural glycan isomers was achieved. The sharp peaks of the eluting anthranilic acid-labeled N-glycans enabled on-line mass spectrometric analysis of even low-abundance glycan species. The method is broadly applicable to N-glycan analysis and is an orthogonal analytical method to the widely established hydrophilic-interaction liquid chromatography of 2-aminobenzamide-labeled N-glycans for characterization of N-glycans derived from biopharmaceuticals.

Tungsten-induced denaturation and aggregation of epoetin alfa during primary packaging as a cause of immunogenicity.

PURPOSE: Following two cases of neutralizing antibodies to epoetin alfa in an investigational clinical study, a small number of individual syringes of two drug product batches were found to contain unusually high levels of aggregation at the end of the clinical trial. METHODS: We undertook an extensive analytical approach to determine the root-cause of the increased aggregation in the affected batches. RESULTS: Soluble tungsten was found in the syringes, most likely derived from the pins used to manufacture the syringes. Spiking of epoetin alfa with sodium polytungstate or an extract of tungsten pins used to manufacture the syringes induced the formation of aggregates, both dimers that appeared to be covalently linked by disulphide bonds as well as higher-order aggregates. Sodium polytungstate had also a strong denaturing effect on the protein. CONCLUSIONS: We propose tungsten-mediated unfolding and aggregation of epoetin alfa in pre-filled syringes as a potential root cause for increased immunogenicity. This finding may be more broadly applicable to this and other classes of therapeutic proteins.

Strategies for the assessment of protein aggregates in pharmaceutical biotech product development.

Within the European Immunogenicity Platform (EIP) ( http://www.e-i-p.eu ), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.

N-glycans of complex glycosylated biopharmaceuticals and their impact on protein clearance.

N-glycosylation is a common post-translational modification of biopharmaceutical products. Certain types of N-glycans have been shown to influence important properties of monoclonal antibody products including pharmacokinetics and effector functions. Complex biopharmaceuticals e.g. Fc fusion proteins may contain several N- and O-glycosylation sites. Domain specific characterization of two Fc fusion proteins showed an Fc N-glycosylation pattern comparable to IgG molecules. The receptor N-glycosylation was found to contain some larger and more complex N-glycans compared to the Fc part. Analyses of samples from non-clinical studies of the two studied fusion proteins indicate that their N-glycans impact pharmacokinetic properties. Interestingly, besides the type of N-glycan this influence on the pharmacokinetics depends also on the glycosylation site and thus the accessibility on the protein. The same type of N-glycan can influence the clearance of fusion proteins when located at the receptor part, but not if located at the Fc part. In this study, it is shown that N-glycans with terminal galactose or N-acetylglucosamine residues have a negative impact on serum half-life when located at the receptor part. Terminal sialylation of galactose residues prevents this faster clearance even when only one sialic acid is present. O-acetylation, a modification of sialic acids does not impact pharmacokinetics. Thus, type and accessibility of N-glycan moieties of fusion proteins both play an important role in pharmacokinetics. Finally, detailed site specific analysis is critical in the development of biopharmaceuticals.

Comparative functional and pharmacological characterization of Sandoz proposed biosimilar adalimumab (GP2017): rationale for extrapolation across indications.

BACKGROUND: Biosimilars are approved biologics that match reference medicine in quality, safety, and efficacy. The development of Sandoz proposed biosimilar adalimumab (SPBA; GP2017) involved a target-directed, iterative state-of-the-art quality-by-design development program. Here, we describe the functional and pharmacological characterization of SPBA and its proposed mechanism of action in immune-mediated inflammatory diseases. METHODS: Sensitive in vitro binding and functional characterization studies, and nonclinical evaluations (pharmacokinetics, pharmacodynamics, and safety/toxicology) were performed as part of a stepwise approach to confirm the biosimilarity of SPBA with reference adalimumab. RESULTS: Matching values were reported for SPBA and reference adalimumab in binding assays involving tumor necrosis factor (TNF)-α, complement 1q and human immune effector cell Fcγ receptor subtypes in cell-based bioassays for Fc receptor function (complement- and antibody-dependent cytotoxicity), and in apoptosis inhibition. Furthermore, SPBA and reference adalimumab were equivalent in terms of membrane TNF binding and induction of reverse signaling. Pharmacokinetics of SPBA and reference adalimumab were comparable in rabbits, and the two biologics were equally effective in a human TNF transgenic mouse model of polyarthritis. CONCLUSION: SPBA matches reference adalimumab with regards to target binding, functional, pharmacokinetic, and pharmacodynamic properties at the nonclinical level supporting its approval in all indications of the reference adalimumab.

HX575: established biosimilarity in the treatment of renal anemia and 10 years of clinical experience.

Erythropoiesis-stimulating agents, such as recombinant human erythropoietin, are commonly used for the treatment of anemia in patients with chronic kidney disease (CKD). In 2007, HX575 (Binocrit®) became the first biosimilar epoetin alfa to be approved by the European Medicines Agency (EMA). The decision to approve a biosimilar is based on the totality of evidence obtained in a comprehensive comparability exercise that involves extensive analytical characterization, nonclinical studies and clinical studies. The development process for HX575 included extensive analytical characterization and comparison with the reference epoetin alfa. This was followed by a clinical development program, comprising Phase I pharmacokinetic/pharmacodynamic studies to show bioequivalence to the reference medicine and a confirmatory Phase III study to demonstrate therapeutic effectiveness in anemia related to CKD. In addition to the comparability exercises, extensive clinical experience over the last decade also confirms that HX575 provides an effective treatment for CKD-related anemia, with a favorable safety profile. Growing clinical experience with EMA-approved biosimilars, including HX575, should offer additional reassurance to health care professionals and patients that these agents are as effective and well tolerated as others in the therapeutic class.

Development and 10-year history of a biosimilar: the example of Binocrit®.

Patent expirations for several biological products have prompted the development of alternative versions, termed 'biosimilars', which have comparable quality, safety and efficacy to a licensed biological medicine (also referred to as the 'reference' medicine). The first biosimilars developed in oncology were the supportive-care agents filgrastim and epoetin. Binocrit® (HX575) is a biosimilar version of epoetin alfa, indicated in the oncology setting for the treatment of chemotherapy-induced anemia (CIA). The process for development and approval of Binocrit® as a biosimilar included extensive analytical characterization and comparison with the reference epoetin alfa. This was followed by a clinical development program comprising phase I pharmacokinetic/pharmacodynamic studies to show bioequivalence to the reference medicine and a confirmatory phase III study to confirm therapeutic effectiveness in CIA. Since its approval, Binocrit® has been extensively used and studied in real-world clinical practice. The accumulated data confirm that Binocrit® is an effective and well-tolerated option for the treatment of CIA in patients with cancer.

Maintaining consistent quality and clinical performance of biopharmaceuticals.

INTRODUCTION: Biopharmaceuticals are large protein based drugs which are heterogeneous by nature due to post translational modifications resulting from cellular production, processing and storage. Changes in the abundance of different variants over time are inherent to biopharmaceuticals due to their sensitivity to subtle process differences and the necessity for regular manufacturing changes. Product variability must thus be carefully controlled to ensure that it does not result in changes in safety or efficacy. AREAS COVERED: The focus of this manuscript is to provide improved understanding of the science and strategies used to maintain the quality and clinical performance of biopharmaceuticals, including biosimilars, throughout their lifecycle. This review summarizes rare historical instances where clinically relevant changes have occurred, defined here as clinical drift, and discusses modern tools used to prevent such changes, including improved analytics, quality systems and regulatory frameworks. EXPERT OPINION: Despite their size complexity and heterogeneity, modern analytics, manufacturing quality systems and comparability requirements for the evaluation of manufacturing changes cumulatively help to ensure the consistent quality and clinical performance of biopharmaceuticals throughout their product lifecycle. Physicians and patients can expect the same safety and efficacy from biopharmaceuticals and their respective biosimilars irrespective of batch or production history.

Rational Selection, Criticality Assessment, and Tiering of Quality Attributes and Test Methods for Analytical Similarity Evaluation of Biosimilars.

Leading regulatory agencies recommend biosimilar assessment to proceed in a stepwise fashion, starting with a detailed analytical comparison of the structural and functional properties of the proposed biosimilar and reference product. The degree of analytical similarity determines the degree of residual uncertainty that must be addressed through downstream in vivo studies. Substantive evidence of similarity from comprehensive analytical testing may justify a targeted clinical development plan, and thus enable a shorter path to licensing. The importance of a careful design of the analytical similarity study program therefore should not be underestimated. Designing a state-of-the-art analytical similarity study meeting current regulatory requirements in regions such as the USA and EU requires a methodical approach, consisting of specific steps that far precede the work on the actual analytical study protocol. This white paper discusses scientific and methodological considerations on the process of attribute and test method selection, criticality assessment, and subsequent assignment of analytical measures to US FDA's three tiers of analytical similarity assessment. Case examples of selection of critical quality attributes and analytical methods for similarity exercises are provided to illustrate the practical implementation of the principles discussed.

T-cell assays confirm immunogenicity of tungsten-induced erythropoietin aggregates associated with pure red cell aplasia.

Immunogenicity of biotherapeutics and the elicitation of anti-drug antibodies are a key concern for their efficacy, pharmacokinetics, and safety. A particularly severe consequence of immunogenicity of a biotherapeutic is the rare development of antibody-mediated pure red cell aplasia (PRCA) in anemic patients treated with aggregated forms of recombinant human erythropoietin (rhEPO). Here, we investigated in vitro T-cell responses to experimentally heat-induced rhEPO aggregates, and to tungsten-induced rhEPO aggregates in clinical lots associated with rhEPO-neutralizing antibodies and PRCA. Heat-stressed rhEPO elicited T-cell responses only in blood obtained from healthy individuals identified as responders, whereas nonstressed rhEPO overall did not induce reactions neither in responders nor nonresponders. Tungsten-induced rhEPO aggregates in clinical lots associated with rhEPO-neutralizing antibodies and PRCA could induce in vitro T-cell responses in blood obtained from healthy donors, in contrast to rhEPO from low tungsten syringes. Importantly, ex vivo T-cell recall responses of patients treated with rhEPO without PRCA showed no T-cell responses, whereas T cells of a patient who developed PRCA after treatment with a clinical batch with elevated levels of tungsten and rhEPO aggregates showed a clear response to rhEPO from that clinical batch. To our knowledge, this is the first time that T-cell assays confirm the root cause of increased rhEPO immunogenicity associated with PRCA.

The structure-function relationship of disulfide bonds in etanercept.

Etanercept is a TNFα receptor Fc fusion protein used for the treatment of rheumatic disease and psoriasis. Physicochemical and functional investigation of process fractions during development of the etanercept biosimilar GP2015 (Erelzi®) revealed a correlation between reduced potency and incorrect disulfide bridging between specific cysteines in the receptor domain. This novel structure-function relationship was found to be the molecular basis for reduced potency in recent Enbrel® batches, which exhibit higher levels of incorrect disulfide bridging. Interestingly, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring potency to normal levels. This redox dependent reversibility suggests that these variants are likely not relevant for clinical efficacy once the drug enters the bloodstream. Nonetheless, incorrect disulfide bridging in etanercept represents a new quality attribute that is critical for biopharmaceutical functionality and should thus be carefully monitored and controlled to guarantee patient safety.

N-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins.

Monoclonal antibody and Fc fusion protein drugs are complex heterogeneous mixtures of numerous different protein variants and modifications. N-glycosylation as one of the most complex post-translational modification influences the structural characteristics of the antibodies Fc part thereby potentially modulating effector function and pharmacokinetics. Several investigations on the relationship between N-glycosylation and pharmacokinetics have been published. However, this structure-function relationship is not fully understood. In this review potential alterations with focus on N-glycosylation of mAbs and Fc fusion proteins and the possible effects on the pharmacokinetics are reviewed and the current understandings of the underlying mechanisms are described.

Identification of Low-Level Product-Related Variants in Filgrastim Products Presently Available in Highly Regulated Markets.

BACKGROUND: Filgrastim is a recombinant, non-glycosylated form of human granulocyte colony-stimulating factor, used to stimulate leukocyte proliferation in patients suffering from neutropenia. Since the expiration of patents associated with Amgen's filgrastim biopharmaceutical, Neupogen(®), in 2006, a number of filgrastim products have been marketed; however, a detailed characterization and comparison of variants associated with these products have not been publically reported. OBJECTIVE: The objective of this study was to identify and quantify product-related variants in filgrastim reference products and biosimilars thereof that are presently available in highly regulated markets. METHODS: In this study, we used intact and top-down mass spectrometry to identify and quantify product-related variants in filgrastim products. Mass spectrometry has become the method of choice for physicochemical characterization of biopharmaceuticals, allowing accurate and sensitive characterization of product-related variants. RESULTS: In addition to modifications ubiquitously present in biopharmaceuticals, such as methionine oxidation and asparagine/glutamine deamidation, we identified six different low-level, product-related variants present in some, but not all, of the tested products. Two variants, an acetylated filgrastim variant and a filgrastim variant containing an additional C-terminal tryptophan extension, are newly identified variants. CONCLUSION: This study demonstrates that filgrastim products already in widespread clinical use in highly regulated markets differ in low-level, product-related variants present at levels mostly below 1 % relative abundance. This study provides a comprehensive catalog of minor differences between filgrastim products and suggests that the filgrastim product-related variants described here are not clinically relevant when present at low abundance.

Characterization and non-clinical assessment of the proposed etanercept biosimilar GP2015 with originator etanercept (Enbrel(®)).

BACKGROUND AND OBJECTIVE: Biosimilars are approved biologics that are comparable to an originator product with respect to quality, safety and efficacy. Herein, the authors describe the functional and non-clinical studies designed to determine the biosimilarity of GP2015 and originator etanercept (Enbrel®). METHODS: The development of an Enbrel biosimilar (GP2015) involved extensive characterization of the originator. A step-wise target-directed and iterative technical development program involving state-of-the-art functional characterization studies and non-clinical evaluations (pharmacokinetics, pharmacodynamics and safety/toxicology) was applied with the aim of confirming that GP2015 is comparable to originator (Enbrel) at the non-clinical level. RESULTS: In in vitro tests, GP2015 and Enbrel had comparable binding affinities to TNF-α, C1q complement and a complete panel of Fc-Receptors. Comprehensive functional characterization testing confirmed the comparability of GP2015 with Enbrel in terms of its ability to bind to and neutralize TNF-α, which reflects the primary mechanism of action of etanercept. Non-clinical data confirmed that the proposed biosimilar to Enbrel, GP2015, is comparable with regards to its pharmacokinetic properties and pharmacodynamic activity, and efficacy as well as safety/toxicity. CONCLUSION: The proposed Enbrel biosimilar, GP2015, was shown to be comparable to its originator product in studies designed to confirm biosimilarity.