Current Pharmaceutical Biotechnology - Volume 12, Issue 10, 2011

Therapeutic proteins produced using recombinant DNA technologies are generally complex, heterogeneous, and subject to a variety of enzymatic or chemical modifications during expression, purification, and long-term storage. Hence the analytical strategies for characterization, quantitation, purity assay and evaluation of the biological activity of recombinant proteins still represent a big challenge and a matter for debate. The aim of the proposed special issue is to point out the main applications as well as future potentialities of the most advanced analytical techniques in the different aspects of the quality assessment of therapeutic proteins and in particular for conformation analysis, aggregates and impurities detection and quantitation, intact protein characterization, post-translational modifications (PTMs) identification and biological activity assessment. The special issue contains six in depth reviews and two original papers. Protein conformation is a key aspect to be assessed, because a specific conformation is essential for the biological function of the protein. The paper by Bertucci et al. points out the growing role of circular dichroism (CD) as a valuable and reliable technique to obtain this information, representing a useful tool for the study of pharmaceutical peptides themselves, in new formulations, after new processes of derivatization, production and storage. The paper also contains examples on the use of CD spectroscopy in the structural characterization of free and formulated recombinant proteins, looking at the prediction of the secondary structure, propensity to conformational changes, stability, and tendency to aggregation. Characterization of protein conformation by high resolution mass spectrometry (direct ESI-MS and hydrogen/ deuterium exchange) is reviewed by Bobst & Kaltashov. The paper provides an overview of the MS techniques and current trends for the characterization of the higher order structure and dynamics of biopharmaceutical products. Recombinant proteins often fail to reach their native conformation and in such cases they form different kinds of aggregates which are unsuitable for the intended applications. This has pressed, on one hand, the exploration of different approaches to favour protein folding, and on the other hand, the development of analytical strategies aimed at detecting soluble and insoluble aggregates. In the review by Garcia- Fruitos et al. the biological aspects of protein folding and unfolding are addressed together with an overview of the most advanced analytical techniques suitable for the fast evaluation of conformational quality and aggregation of recombinant drugs, even if showing apparent solubility......

Most of the protein therapeutics are now produced by recombinant DNA technology. The advantages of recombinant proteins are related to their higher specificity and to their safety as exposure to animal or human diseases. However, several problems are still present in development of recombinant proteins as therapeutics, such as low bioavailability, short serum half-life, and immune response. Their successful application hinges on the protein stereochemical stability, and on the folding and the tendency to aggregate induced by purification steps and storage. All these aspects determine the failure of many potential protein therapies, and limitations in the development of the formulation. The application of multiple analytical techniques is important in order to obtain a detailed product profile and to understand how manufacturing can influence product structure and activity. Surely the protein conformation is a key aspect to be assessed, because a specific conformation is often essential for the biological function of the protein. Thus, there is a growing need to perform structural studies under the conditions in which the proteins operate, and to monitor the structural changes of the protein. Circular dichroism has been increasingly recognised as a valuable and reliable technique to get this information. In particular, examples will be here reported on the use of circular dichroism spectroscopy in the structural characterization of free and formulated recombinant proteins, looking at the prediction of the secondary structure, propensity to conformational changes, stability, and tendency to aggregate.

Mass spectrometry has already become an indispensable tool in the analytical armamentarium of the biopharmaceutical industry, although its current uses are limited to characterization of covalent structure of recombinant protein drugs. However, the scope of applications of mass spectrometry-based methods is beginning to expand to include characterization of the higher order structure and dynamics of biopharmaceutical products, a development which is catalyzed by the recent progress in mass spectrometry-based methods to study higher order protein structure. The two particularly promising methods that are likely to have the most significant and lasting impact in many areas of biopharmaceutical analysis, direct ESI MS and hydrogen/deuterium exchange, are focus of this article.

Production of protein-based pharmaceuticals is a major issue in conventional pharmacology, biomedicine and nanomedicine. Being mostly obtained by genetic engineering, the quality and activity of protein drugs is a steady matter of concern. Although the physiology of the host recombinant cells, mostly mammalian and microbial, is progressively understood, the complexity of the cellular quality control systems escapes rational protein and process engineering, and recombinant proteins are often unstable, aggregate and/or do not reach the fully native conformation compatible with proper biological activity. In this review, we summarize the main biological aspects of protein folding and misfolding, mainly focusing in microbial cells, the newest insights in the biological control of protein quality and the main and analytical approaches that are suitable for the fast evaluation of the conformational quality and aggregation of recombinant drugs, even if showing apparent solubility.

The technique of combinatorial peptide ligand libraries (CPLL), for capturing and amplifying low-abundance proteins in r-DNA products as well as in a number of other biological systems, is here analyzed in depth and reviewed. This methodology is based on a creation of several millions of bio-specific ligands composed of hexapeptides produced in a combinatorial way. When acting on an overloading and saturation principle, high-abundance species are captured in limited amounts, whereas low-abundance ones keep being concentrated on their bio-specific ligand till substantial harvesting from solution (the capture process occurring in general from ca. 50% up to 90% efficiency). Examples are given on tracking host-cell impurities present in, e.g., recombinant albumin or monoclonal antibodies. Additionally, other examples of detecting traces of additives and fining agents in such beverages as white and red wines are presented. The unique mechanisms underlying the protein capture in the CPLL methodology, as opposed to capture by homogeneous beads, as represented by ion-exchangers and by hydrophobic resins, are discussed in depth.

Molecular weight determination of intact recombinant therapeutic proteins is a challenging analytical tool which furnishes valuable information not only for protein structure characterization but also to assess purity and heterogeneity. Up to now several mass spectrometric (MS) approaches have been reported for the measurement of monoisotopic or average masses of intact recombinant therapeutic protein. The choice of a suitable approach depends on several factors, among which are the sample complexity and homogeneity and the size of the target protein. MALDI-TOF, since it is rapid, simple and adaptable for automation has been found suitable for in-process monitoring quality of low/middle protein. High resolution MS such as Q-TOF, orbitrap and FT-ICR are excellent analytical tools for assessing protein heterogeneity and for structural characterization, in some cases without the need for proteolytic digestion and analysis of resulting products. High resolution techniques such as capillary electrophoresis (CE) and UPLC have been successfully coupled to high resolution MS analyzers in cases where the sample is complex and highly heterogeneous. The paper is a review of the most used and advanced MS strategies so far reported as well as of their applications in the biopharmaceutical field.

Mass spectrometry is the method of choice for sequencing peptides and proteins and is the preferred choice for characterizing post-translational modifications (PTMs). The most commonly used dissociation method to characterize peptides (i.e. collision-induced dissociation (CID)), however, has some limitations when it comes to analyzing many PTMs. Because CID chemistry is influenced by amino acid side-chains, some modifications can alter or inhibit dissociation along the peptide backbone, thereby limiting sequence information and hindering identification of the modification site. Electron transfer dissociation (ETD) has emerged as an alternate dissociation technique that, in most cases, overcomes these limitations of CID because it is less affected by side chain chemistry. Here, we review recent applications of ETD for characterizing peptide and protein PTMs with a particular emphasis on the advantages of ETD over CID, the ways in which ETD and CID have been used in a complementary manner, and how peptide modifications can still influence ETD dissociation pathways.

Influenza vaccination is recognized as the most effective method for reducing morbidity and mortality due to seasonal influenza. To improve vaccine supply and to increase flexibility in vaccine manufacturing, cell culture-based vaccine production has emerged to overcome limitations of egg-based production. The switch of production system and the need for annual re-evaluation of vaccines for the effectiveness due to frequent viral antigenic changes call for methods for complete characterization of the hemagglutinin (HA) antigens and the final vaccine products. This study describes advanced liquid chromatography-mass spectrometry (LC-MS) methods for simultaneous identification of HA proteins and process-related impurities in a trivalent influenza candidate vaccine, comprised of purified recombinant HA (rHA) antigens produced in an insect cell-baculovirus expression vector system (BEVS). N-linked glycosylation sites and glycoforms of the three rHA proteins (corresponding to influenza A subtypes H1N1 and H3N2 and B virus, respectively) were profiled by peptide mapping using reversed-phase (RP) LC-MSE (data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode). The detected site-specific glycoforms were further confirmed and quantified by hydrophilic interaction LC (HILIC)-multiple reaction monitoring (MRM) assays. LC-MSE was used to characterize the vaccine candidate, providing both protein identities and site-specific information of glycosylation and degradations on each rHA protein. HILIC-MRM methodology was used for rapid confirming and quantifying site-specific glycoforms and potential degradations on each rHA protein. These methods can contribute to the monitoring of vaccine quality especially as it pertains to product comparability studies to evaluate the impact of production process changes.

Interleukin (IL)-7 is a cytokine inducing the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway. As a consequence of IL-7 activating this pathway, STAT5 is phosphorylated. In pharmaceutical quality control, the potency of biopharmaceuticals is commonly assessed by proliferation assays. This is also possible for IL-7 conjugates. However, the disadvantage of these classical “endpoint-assays” is that they require very long incubation times, up to several days, since they measure the downstream events of a cellular response. As an alternative to this, we developed a rapid intracellular phosphorylation assay, measuring IL-7 induced STAT5 phosphorylation in Kit 225 cells. The Kit 225 human T cell line expresses the IL-7 receptor and is responsive to IL-7, therefore making it a good candidate cell line for assay development. Like the Kinase receptor activation (KIRA) assay, developed by Sadick et al. [1], the STAT5 phosphorylation assay was performed using two separate microtiter plates: the first one for cell stimulation and lysis, the second one for enzyme-linked immuno sorbent assay (ELISA). The assay showed a high accuracy and precision with a mean recovery of 102% and a mean coefficient of variation of 9%. In comparison to the classical proliferation assay, the phosphorylation assay is much faster. Thus, the assay procedure time can at least be reduced from six to three days by using STAT5 phosphorylation instead of proliferation as an endpoint due to the shorter incubation time with IL-7. Moreover, the phosphorylation assay shows a wider dynamic range and higher signal to noise ratios and is thus more robust than the proliferation assay.mAs a consequence, this assay could serve as reliable, accurate, precise and fast alternative to the classical proliferation assay for IL-7. This study also serves as an example for the typical steps during development and qualification / validation of a potency assay for quality control testing.

Chronic pain is an unmet medical need. Millions of people suffer from some form of chronic pain that often is co-morbid with cancer, diabetes, arthritis, anxiety and depression: a growing list of diseases that contribute to short/long-term disability and affect quality of life. According to American Pain Foundation, more Americans suffer from some form of chronic pain than diabetes, heart disease, stroke and cancer. Chronic pain encompasses back/neck pain, headache (migraine), visceral pain, fibromyalgia, cancer pain, neuropathic pain and pain associated with osteoarthritis or rheumatoid arthritis. Pain therapy is primarily driven by modestly effective, side-effect limiting opioid therapy although pregabalin and duloxetine with new mechanisms have gained momentum, especially for neuropathic pain and fibromyalgia. Efficacy remains the largest unmet need in the area of chronic pain. Several pharmaceutical companies have been engaged in analgesic drug discovery programs prosecuting novel targets with differentiated mechanism of actions: while the effort has greatly contributed to our overall understanding of the role of different targets (GPCRs, ion channels, enzymes) in pain physiology, clinical success has been limited. A good example of such a target is TRPV1: while the pain community has benefited immensely from selective TRPV1 antagonists, many of which have contributed in further elucidation of the role of the target in nociception, several companies have suspended clinical development of TRPV1 antagonists due to target limiting adverse events. Continued clinical failures results in short term panic by the sponsoring organizations that has precipitated in many notable pharmaceutical/biotechnology companies moving out of pain area or significantly reducing workforce in analgesic drug discovery and development. The question that I like to ask is: is the future of pain drug discovery and development really that bleak? This issue is being put forward to demonstrate that the future of pain drug discovery is not that bleak after all. One of the major challenges in the field has been lack of proper translation of preclinical pain data to the clinic. Several novel mechanisms seem to pass the test in various models of pain (acute, spontaneous, neuropathic, inflammatory, cancer, arthritis) in rodents but then fall short in early proof of concept human trials. In this issue, we have review on animal models of pain followed by more focused articles on animal models of osteoarthritic pain and fibromyalgia. Cancer pain is an area of neglect and there is a huge unmet medical need; the group from Eli Lilly focuses on that aspect in their contribution. Biologics has become an important part and parcel in our arsenal to target novel and ‘ undruggable’ targets (Druggability defined by small molecule success) and there is chapter that reviews that approach as well. The rest of the issue deals with analgesic drug targets across various target families with a view to highlight state of the research in the field. In closing, I like to thank all the contributors who have worked tirelessly with me to bring this issue to fruition.

The increasing debate regarding the predictiveness of rodent persistent pain models for clinical efficacy has spurred rapidly evolving numbers and types of novel models from which to choose. While several excellent reviews of these models have been published in recent years, few focus on their specific applications and particular challenges with the use of these models in the setting of drug discovery. Thus, in this review, how models of persistent pain may be used to: 1) screen molecules for in vivo efficacy, 2) advance lead compounds and 3) guide decision making for clinical trial design is discussed. Relative to other disease areas for potential drug discovery and development, chronic pain appears to be well-poised for drug discovery and development. This is in large part due to the advanced understanding of pain mechanisms and the upsurge in the development of novel, specialized rodent models of persistent pain and improvements in methods of pain assessment in animals.

Osteoarthritis (OA) is a complex disease plagued by a significant unmet need for treatment. To date, no disease- modifying OA drugs (DMOADs) exist and the available symptom-modifying OA drugs (SMOADs) have limitations. Although a complete understanding of the mechanisms of OA pain in humans is lacking, animal models have helped provide insight into the multifaceted origin and manifestation of OA pain. Success in discovering new therapeutics will likely require reliance on good animal models. This review summarizes the animal models available for studying pain associated with OA.

Fibromyalgia syndrome (FMS) is a chronic pain syndrome characterized by diffuse musculoskeletal pain. In quantitative sensory testing studies, FMS patients display alterations in heat, cold, and mechanical sensitivity. Genetic studies support a key role for the biogenic amine system, and single nucleotide polymorphisms have been identified in serotonin and dopamine transporter and receptor genes of FMS patients. The pathophysiology of fibromyalgia includes contributions from both the ascending and descending somatosensory systems, and decreased central nervous system inhibition of peripheral nociceptive signalling. Three drugs have been approved for the treatment of FMS: Lyrica® (pregablin), Cymbalta® (duloxetine), and Savella® (milnacipran). These drugs were originally developed for indications other than FMS, and were later approved for FMS after successful clinical trials. One hurdle in the development of drugs specifically for FMS is the availability of preclinical animal models of the disease. Recently, several rodent models have been described with potential for translation to the human pain syndrome. In this review, we discuss recent developments toward understanding the pathophysiology of FMS, currently available pharmacologic therapy, ongoing clinical trials, and potential animal models of FMS.

Most cancer patients will experience moderate to severe pain and/or neuropathy during the course of their disease. Recent improvements in the primary treatment of cancer have increased the life span of cancer patients, but not necessarily their quality of life (QoL). The pain and suffering cancer patients experience may be the result of the tumor itself, or the treatments required to arrest tumor growth and progression. In contrast to the rapid, highly mechanistic, tailored medicine approach used to target and treat the primary tumor burden, the evolution of pain and other supportive treatment approaches for cancer patients have been slow to non-existent. A movement is emerging to use more rational mechanistic approaches to the treatment of pain created by cancer and chemotherapeutics. This review briefly describes the most severe and debilitating symptoms (endophenotypes) from the cancer patient's perspective, the biochemical/neurobiological sequalae associated with tumor growth and therapies designed to arrest tumor progression, and highlights some promising pharmacologic mechanisms that may be used to treat cancer-related pain, sensory neuropathies, and associated endophenotypes. Delivering improved broader spectrum supportive care medicines to cancer patients will fill a significant unmet need and enable them to live productive, fulfilling lives that preserve their overall QoL.

The need for new pain therapies that provide greater relief without unwanted side-effects drives the search for new drug targets. The identification of endogenous lipid ligands for the two known cannabinoid receptors (CB1 and CB2) has led to numerous studies investigating the role of these endocannabinoids in pain processes. The two most widely studied endocannabinoids are anandamide (AEA; arachidonoyl ethanolamide) and 2-arachidonoylglycerol (2-AG), but there are also a number of structurally related endogenous lipid signaling molecules that are agonists at cannabinoid and noncannabinoid receptors. These lipid signaling molecules are not stored in synaptic vesicles, but are synthesized and released on-demand and act locally, as they are rapidly inactivated. This suggests that there may be therapeutic potential in modulating levels of these ligands to only have effects in active neural pathways, thereby reducing the potential for side-effects that result from widespread systemic cannabinoid receptor activation. One approach to modulate the levels and duration of action of these lipid signaling molecules is to target the enzymes responsible for their hydrolysis. The two main enzymes responsible for hydrolysis of these lipid signaling molecules are fatty acid amide hydrolase (FAAH) and monoacylglyceride lipase (MGL). This article will discuss the role of the endocannabinoid system in the modulation of pain and will review the current understanding of the properties of the hydrolytic enzymes and the recent advances in developing inhibitors for these targets, with particular relevance to the treatment of pain.

Chronic pain conditions present a huge burden on modern society. Both inflammatory and neuropathic pain are poorly treated in man; the majority of patients do not benefit from adequate pain relief, and side effects of currently used treatments are common. Discovery and development of novel therapies remains an imperative, but the ability to genuinely test the efficacy of novel therapies is often limited by effects at targets other than intended, particularly with novel small molecule approaches. Approaches which limit these off-target activities, and thus avoid the commonest cause of terminating development of new therapeutics may provide a greater ability to genuinely test targets of choice clinically, and here, biologic therapeutics provide such an opportunity; in the major class of biologic therapies, monoclonal antibodies, inherent exquisite selectivity is intrinsic to their nature. Antibody therapeutics have been developed successfully in the immunology and cancer fields, and recent progress in analgesia suggests that these therapeutics may transform treatment paradigms in a similar manner to that observed within, for example, the rheumatoid arthritis space. In addition, opportunities with other biologic approaches, such as peptides, further broadens the potential to bring forward genuinely novel approaches to pain. In this review, the current status of biologic therapies, as well as future opportunities are reviewed.

Migraine remains one of the most prevalent and disabling neurological disorders that often affects a person during their most productive years. Migraine afflicts approximately 11% of the adult population globally, causes substantial disability, which translates into lost productivity both at home and at work. Clearly there remains a need for new approaches to treat migraine and calcitonin gene-related peptide (CGRP) receptor antagonists have the potential to be a major advance in antimigraine therapy. CGRP was first proposed to play a role in migraine pathophysiology a little over 20 years ago and today there is considerable evidence that CGRP plays a key role in the pathogenesis of migraine. CGRP is a 37 amino acid vasoactive neuropeptide largely expressed in sensory neurons. It was observed that plasma levels of CGRP were elevated during the headache phase of migraine and the levels were normalized concomitantly with pain relief. This observation, along with other evidence, suggested that CGRP receptor antagonists might represent a novel approach to migraine treatment. The advent of small molecule CGRP receptor antagonists has clearly demonstrated a clinical link between blocking the CGRP receptor and effectiveness in treating migraine. This review will highlight the biology of CGRP as it pertains to migraine; discuss the CGRP receptor; spotlight the development of CGRP receptor antagonists; and examine site of action.

The metabotropic glutamate receptors (mGluRs) are expressed pre- and post-synaptically throughout the nervous system where they serve as modulators of synaptic transmission and neuronal excitability. Activation of mGluRs can be pro- or anti-nociceptive, depending on their anatomic location and the signaling cascades to which they couple. Antagonists of Group I mGluRs and agonists of Group II and III mGluRs have shown therapeutic promise in animal pain models. This article reviews the potential therapeutic utility of several agents that act predominantly via mGluRs, specifically focusing on their analgesic efficacy and discussing possible off-target effects. Glutamate, the primary excitatory neurotransmitter in the vertebrate nervous system, mediates its effects via activation of two main classes of receptors: ligand-gated ion channels known as ionotropic receptors and G-protein coupled metabotropic receptors. Antagonists of ionotropic glutamate receptors, such as ketamine, have robust analgesic properties; however, their analgesic utility is limited to monitored clinical settings due to the potential for psychomimetic effects.

Acute pain detection is vital to navigate and survive in one's environment. Protection and preservation occur because primary afferent nociceptors transduce adverse environmental stimuli into electrical impulses that are transmitted to and interpreted within high levels of the central nervous system. Therefore, it is critical that the molecular mechanisms that convert noxious information into neural signals be identified, and their specific functional roles delineated in both acute and chronic pain settings. The Transient Receptor Potential (TRP) channel family member TRP ankyrin 1 (TRPA1) is an excellent candidate molecule to explore and intricately understand how single channel properties can tailor behavioral nociceptive responses. TRPA1 appears to dynamically respond to an amazingly wide range of diverse stimuli that include apparently unrelated modalities such as mechanical, chemical and thermal stimuli that activate somatosensory neurons. How such dissimilar stimuli activate TRPA1, yet result in modality-specific signals to the CNS is unclear. Furthermore, TRPA1 is also involved in persistent to chronic painful states such as inflammation, neuropathic pain, diabetes, fibromyalgia, bronchitis and emphysema. Yet how TRPA1's role changes from an acute sensor of physical stimuli to its contribution to these diseases that are concomitant with implacable, chronic pain is unknown. TRPA1's involvement in the nociceptive machinery that relays the adverse stimuli during painful disease states is of considerable interest for drug delivery and design by many pharmaceutical entities. In this review, we will assess the current knowledge base of TRPA1 in acute nociception and persistent inflammatory pain states, and explore its potential as a therapeutic pharmacological target in chronic pervasive conditions such neuropathic pain, persistent inflammation and diabetes.

P2X7 is an ATP-gated non-selective cation channel expressed primarily on cells of hematopoietic origin, such as macrophages and microglia. Since the initial cloning of this channel, enormous progress has been made in the understanding of the physiology, pharmacology and therapeutic utility of P2X7. This article attempts to review the biology of P2X7 with a focus on the complex pharmacology of this channel. Finally, the authors discuss the role of P2X7 as an analgesic drug target and raise some of the challenges and issues that face the P2X7 research community.