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1.
《MABS-AUSTIN》2009,1(3):190-209
The Second International Conference on Accelerating Biopharmaceutical Development was held in Coronado, California. The meeting was organized by the Society for Biological Engineering (SBE) and the American Institute of Chemical Engineers (AIChE); SBE is a technological community of the AIChE. Bob Adamson (Wyeth) and Chuck Goochee (Centocor) were co-chairs of the event, which had the theme “Delivering cost-effective, robust processes and methods quickly and efficiently.” The first day focused on emerging disruptive technologies and cutting-edge analytical techniques. Day two featured presentations on accelerated cell culture process development, critical quality attributes, specifications and comparability, and high throughput protein formulation development. The final day was dedicated to discussion of technology options and new analysis methods provided by emerging disruptive technologies; functional interaction, integration and synergy in platform development; and rapid and economic purification process development.MAbs. 2009 May-Jun; 1(3): 190–209.
March 10, 2009 Day 1, Emerging Disruptive Technologies and Cutting-Edge Analytical Techniques
Janice M ReichertAuthor information Article notes Copyright and License information DisclaimerTufts Center for the Study of Drug Development; Boston, MA USACorresponding author.Correspondence to: Janice M. Reichert; Tufts Center for the Study of Drug Development; 75 Kneeland Street; Suite 1100; Boston, MA 02111 USA; Email: ude.stfut@trehcier.ecinajReceived 2009 Mar 20; Accepted 2009 Mar 20.Copyright © 2009 Landes BioscienceThe meeting was opened by Bob Adamson (Wyeth) who remarked that it is the responsibility of biological engineers to develop technologies that will produce drug products rapidly and cost effectively. On average, protein therapeutics cost more than small molecule drugs. However, technological advances can help to drive down the cost of these products. For example, penicillin was scarce in the 1930s and 1940s because of production issues, but this drug is easily and cheaply obtained today.Recent developments in biosimilars, a potentially disrutive group of products, were discussed by Rob Garnick (Lone Mountain Biotechnology). He first noted the various names by which biosimilars are known. While the term biosimilars is favored in Europe, the US Food and Drug Administration (FDA) uses the term ‘follow-on biologics,’ and Health Canada prefers the phrase ‘subsequent entry biologics.’ The term biogenerics is not usually used because this word implies that the products are identical to approved innovator biologics. The European Medicines Agency and Health Canada have issued regulatory guidances for approval of biosimilars, and some of these products have been approved in Europe.1 However, the process has gotten stalled in the US for various reasons, including questions surrounding the reliability of sourcing, magnitude of price reduction, need for clinical trials done against a national comparator and comparability issues. On the other hand, the global economic problems have focused political attention on healthcare reform and unsustainable increases in the cost of healthcare.Dr. Garnick noted that in the US the general consensus is that the Drug Price Competition and Patent Term Restoration Act of 1984, also known as the Hatch-Waxman Act, has been successful in promoting generics while still providing financial incentives for research and development by innovators. In addition, Congress believes that scientific issues surrounding biosimilars are addressable and so a regulatory pathway can be established for approval of biosimilars. Due to the progress in defining a regulatory pathway, some major pharmaceutical firms, including Pfizer, AstraZeneca, Novartis and Merck, have recently indicated that they will develop these products. Biosimilars development is attractive because the success rates should be 100% if the products are developed correctly, the manufacturing processes are well-understood and can be out-sourced, and the markets are potentially large. With a global market over US $5 billion, rituximab will certainly be targeted as a biosimilar. As with the Hatch-Waxman Act, the key to success of any US biosimilars legislation will be the maintenance of incentives to innovate.There are still numerous scientific and legal problems to address,2 including the exact nature of legislation, patent issues, design of clinical trials, substitutability, interchangeability, safety and post-approval surveillance. The challenge lies in the details, e.g., establishing product specifications and test methods, and defining comparability. Dr. Garnick noted that cautionary tales on comparability come from the experience of a number of innovator companies. For example, efalizumab produced by XOMA was found to have differences when compared to efalizumab produced by Genentech. The differences, which included minor changes in acidic forms, galactosylation, charge heterogeneity and an increase in C-terminal processing, were expected to be inconsequential, but translated into different clinical study results. This experience suggests that a combination of written procedures, training, analytical testing and regulatory agency inspections are needed to control the production of biological products. Quality control release tests need to be supported by rigorous product and process characterization and process control.Outside the US, the reality is that biosimilars are being marketed in Europe, India and China as well as other countries. Marketed biosimilars span a broad range of complexities and include monoclonal antibodies. Reditux, a rituximab biosimilar, was approved in India in April 2007 for non-Hodgkin lymphoma and rheumatoid arthritis. However, the clinical trials included relatively few patients and limited analytical data has been made available. In conclusion, Dr. Garnick remarked that the world is gaining experience with biosimilars, and the products will likely become a reality in the US by 2009. FDA will need input to develop effective guidance documents for assessment of biologics. Immunogenicity will be a key concern of regulators. Comparability studies will be required, product differences will need to be investigated and appropriate clinical studies must be done, but biosimilars will come to market.Global trends in antibody development by innovator companies were presented by Janice Reichert (Tufts University). Clinical development of protein therapeutics is on the rise worldwide.3–5 Approximately 120 recombinant proteins and 240 monoclonal antibodies (mAbs) are currently in clinical studies. While recombinant proteins have historically entered the clinic at a rate of fewer than 20 candidates per year, mAbs are now approaching the 40 candidate per year mark. Recombinant proteins have somewhat higher success rates on average (approximately 30% vs. 20% for mAbs) and have been studied in a wider array of therapeutic categories compared to mAbs, but, because of their versatility as therapeutics, mAbs are clearly the focus of the biopharmaceutical industry''s attention. A total of 22 mAbs are approved in the US, and eight of these products have global markets over US$1 billion. Six additional candidates are currently undergoing regulatory review.Dr. Reichert noted that the ascendancy of mAbs is due to technologies that addressed immunogenicity, affinity, specificity, stability and production challenges. While murine versions dominated in the 1980s, the less immunogenic humanized versions comprised 45% of the total mAbs in clinical study in the 1990s. In the 2000s, the human versions have comprised the largest contingent. Historically, mAbs have not been discontinued while in regulatory review. Assuming the six mAbs in review are approved, then cumulative FDA approval success rates for humanized and human mAbs will be nearly identical (19 and 18%, respectively). mAbs are commonly studied as either anticancer6 or immunological treatments. There are currently nine anticancer and ten immunological mAbs approved in the US. These products have taken approximately the same length of time for clinical development (6.5 years). The length of the FDA review period was found to vary depending on whether the product was given priority or standard review (average 6.9 or 20.4 months, respectively).Looking forward, Dr. Reichert suggested that antibody fragments and modified versions (pegylated, alternate glycosylation, Fc engineered) are likely to enter the clinical pipeline in increasing numbers. The focus is likely to remain on human IgG, but designed protein scaffolds and domain antibodies will also be included in company pipelines.The meeting then turned to discussion of potentially disruptive science and technologies. Stefan Wildt (Merck) reviewed the development of glycoengineered yeast, which he described as a versatile glycoprotein expression platform. He first emphasized the importance of glycosylation, which affects circulating half life, tissue distribution, potency and immunogenicity of therapeutic proteins. Any new bioprocesses need to be scalable, portable, and provide analytically comparable protein at all scales. The primary biomanufacturing platforms are bacterial (e.g., E. coli), fungal (e.g., Pichia pastoris), and mammalian cell culture (e.g., CHO cells). Fungal platforms are currently used for production of common industrial enzymes, but have not been used extensively for production of therapeutics because the yeast glycosylation pathway yields products that are potentially immunogenic in humans. GlycoFi Inc., a wholly owned subsidiary of Merck & Co., Inc. has developed Pichia with humanized glycosylation to circumvent this problem.In yeast, the carbohydrate processing occurs sequentially in the secretory pathway, like an assembly line. The enzymes act one after another and their actions are separated in time and space. As a consequence, humanized yeast produce proteins with human glycans that are highly uniform. In contrast, traditional mammalian cell production systems produce functional glycoproteins that are heterogeneous and contain non-human glycoforms. As reported by Hamilton et al.,7 GlycoFi eliminated yeast-specific glycosylation in Pichia pastoris and introduced 14 heterologous genes; this process yielded yeast capable of producing complex glycoproteins with greater than 90% terminal sialylation. Candidate protein can be produced in a bioreactor process that takes three to seven days, which is somewhat shorter than the time for production in mammalian cells.For example, Dr. Wildt discussed MK2578, which is a pegylated erythropoietin that is terminally sialylated with N-glycans. The candidate is currently in Phase 1 studies as a potential treatment for anemia. The glycosylation fidelity from the Pichia platform is retained when protein is produced at laboratory scale to up to 2,000 L. The yeast can also be used to produce antibody as IgG1. Compared to CHO cell produced IgG1, candidates produced in yeast were found to be more potent in inducing ADCC and could bind antigen as well. In preclinical studies, yeast-produced mAb glycovariants demonstrated good results in PK studies in Rhesus monkeys and C57BL mice. In conclusion, Dr. Wildt remarked that the selection process, which includes screening for titer, fermentability, glycosylation and protein quality, can result directly in production strains. Yield is approximately 1.4 grams per liter for antibody candidates, but yields up to 2 grams per liter can be achieved.Annie De Groot (EpiVax) presented information on methods to reduce protein immunogenicity by design through deimmunization and tolerance induction. She noted the parallels between vaccine use, when an immune response is desired, and immunogenic therapeutic proteins, which elicit an immune response that is not desired. In both cases, a payload coupled with a delivery vehicle and an adjuvant determine immunogenic potential. T-cell epitopes are a key contributing factor. Like proteins, antibodies are processed by antigen presenting cells. The activated T-cells in turn activate B-cells; in the absence of T-cells, no antibody formation is observed.EpiVax has developed an array of in silico tools and techniques to predict whether proteins will be immunogenic. These include EpiMatrix, in which overlapping 9-mer peptide frames are evaluated for binding potential to eight common class II HLA alleles. The ClustiMer algorithm can be used to find regions of high immunogenicity. Using these methods, an overall immunogenicity score can be estimated. These in silico results can be validated in vitro and in vivo (e.g., HLA transgenic mice).The approach has been clinically validated. Koren et al.8 reported on use of EpiMatrix analysis of a recombinant fusion protein that predicted promiscuous T-cell epitopes in the C-terminal region. In a phase 1 study of 76 subjects, 37% developed antibodies after one injection of the protein candidate. EpiMatrix correctly predicted the immunogenic region and the likelihood that the protein would be immunogenic in the clinic.These results suggest that the technology might be useful as part of an overall strategy for assessing antibody responses in non-clinical and clinical settings.9, 10 In addition, rational modification of epitopes identified using the technology could effectively ‘deimmunize’ protein candidates. Dr. De Groot also discussed the discovery of ‘Tregitopes’ (highly conserved regulatory T-cell epitopes) that are promiscuous, high affinity HLA binders found in IgG. She noted that there is a correlation of antibody immunogenicity with the presence of Tregitopes. Dr. De Groot and co-workers have demonstrated that co-incubation of peripheral blood mononuclear cells (PBMCs) with the Tregitopes can lead to suppression of immune response to other antigens. This suggests that the engineering of Tregitopes into antibodies or other proteins might lead to the development of less immunogenic candidates.Modular IMmune In vitro Constructs (MIMIC), which is an in vitro biomimetic human immune system developed to accurately model the immunotoxicity and immunogenicity of drug candidates, was reviewed by William Warren (Vaxdesign). The MIMIC system is designed to serve as a ‘clinical trial in a well’ by providing predictive HTP in vitro immunology assessment of drug candidates. Primary human donor cells are used to simulate human responses to agents such as vaccines and drugs. The system consists of three modules: (1) Simulation of innate immunity with a peripheral tissue equivalent (PTE) module; (2) Simulation of adaptive immunity with the lymphoid tissue equivalent (LTE); and (3) a functional assay or disease model. The PTE module comprises one monolayer of endothelial cells grown over a 3D collagen matrix. Human PBMCs from donors are used to seed the module; monocytes extravasate through the endothelial cells and differentiate into antigen presenting cells. The PTE module can be used to assess reactogenicity and immunotoxicity responses. The LTE module functionally reproduces the environment of a human lymph node. Within the module, T-cells, B-cells, antigen-presenting cells or follicular dendritic cells interact, leading to immune stimulation that results in activation of lymphocytes, cytokine generation and antibody production. The activated lymphocytes, cytokine profiles and antibodies are then characterized using various methods.Dr. Warren discussed use of the modules to measure the magnitude and quality of T-cell response to vaccines. He noted that primary CD8 T-cell response, in vitro humoral and B-cell response, antibody titer, and microneutralization can be assessed. A correlation analysis of MIMIC response versus serum titer in hepatitis B and influenza vaccination has been performed. Results suggest that the MIMIC system can be used to predict whether a vaccine would be efficacious before going to the clinic. In addition to immunogenicity, the system acts as a biomimetic for evaluation of immunotoxicity and can be used as an inflammation model or in vitro infectious disease model. Use of the system has the potential to accelerate the entire drug development timeline, and decrease failures by providing better data for evaluation of preclinical candidates.Karyn O''Neil (Centyrex, a Johnson & Johnson Internal Venture) described alternative scaffolds that are being used as new biotherapeutic platforms by Johnson & Johnson. She started by wondering whether mAbs are always the best choice since there are reasons to develop alternatives. For example, desirable epitopes might be immunologically silent, alternatives to injection delivery are a challenge, full-size antibodies penetrate tissue and tumors poorly, and royalties might be due on numerous phases of the mAb discovery, screening, development and production process. However, requirements for a next-generation platform, which include the expansion of the range and possibility of targets, lower cost of development and manufacturing complexity, novel delivery, elimination of cold storage, clear freedom to operate and no intellectual property issues, are difficult to meet. Alternative scaffolds do meet many of the aforementioned requirements, and these molecules have favorable biophysical characteristics. Alternative scaffolds can be readily formatted into multi-specific binders with relevant biological activity. For example, Lu et al.11 combined variable regions of two antagonistic antibodies to produce a human IgG-like bispecific antibody that could strongly inhibit the growth of two different human tumors in HT29 xenografts in vivo.Johnson & Johnson''s strategy toward the use of alternative scaffolds involves development of both Centyrins™ and DARPins™, which are viewed as complimentary molecule types. Both are small (10 to 18 kDa) single domain proteins that have high affinity (low picomolar to femtomolar range) and high selectivity for their targets. They are compatible with technologies that improve serum half-lives and seem to have low immunogenicity and low toxicity. They are also very stable and can be expressed at high levels in soluble form. DARPins™ have flat wide surfaces that are better suited for disrupting protein-protein interactions,12 whereas Centyrins™ have extended loops that can interact in protein clefts, enzyme active sites and protein channels.13DARPins™, which are being developed as part of a collaboration between Molecular Partners and Johnson & Johnson, are selected from in vitro display of very large (1012) libraries. The method uses PCR and affinity maturation, and candidates with slow off-rates can be selected. In this way, high affinity, neutralizing DARPins™ can be selected within weeks. Melting properties can be used for selection, resulting in DARPins™ candidates with good biophysical properties. Small scale (2 mL) expression of DARPins™ can yield approximately 1 mg each for additional characterization.The Centyrins™ scaffolds have loops that are analogous to the CDRs of antibodies. The molecules have excellent biophysical properties (>100mg/mL expression, >170mg/mL solubility, >82°C melting temperature, low predicted immunogenicity, stable in serum for more than one month), and can be engineered for improved stability. An in vitro display system licensed from Isogenica utilizes CIS-display technology for Centyrins™ selection. This is proven technology for peptide display. Libraries are potentially quite large (1013). The CIS-display allows rapid panning and selection of binders with a PCR step that allows for in vitro evolution of binders. Rational design of library diversity can improve scaffold properties.14 A green fluorescent protein solubility/folding reporter assay15 is used to assess library quality. The unique properties of alternative scaffolds can be exploited in numerous areas, such as bispecific molecules, medical device, encapsulation, novel formulation and delivery, drug/toxin or radionuclide conjugation, imaging, biosensor, purification technologies and intracellular expression.Cutting-edge analytical technologies became the focus of the meeting in the afternoon session. Steve Cohen (Waters Corporation) discussed chromatographic analysis for biopharmaceuticals with an emphasis on current trends and future prospects. He first discussed ultra performance liquid chromatography (UPLC) utilizing sub-2 micron particle packing (1.7 µmeter with either 130 angstrom or 300 angstrom pores). Compared to HPLC with 3.5 µmeter particle packing, UPLC gives sharper peaks, and can improve resolution of samples in the same run time or achieve comparable resolution and selectivity in a reduced run time (80 versus 120 minutes). Dr. Cohen then presented results of LC/ultraviolet (UV) analysis of a reduced monoclonal antibody, and a murine monoclonal IgG reduced and alkylated standard run at elevated temperature. He noted that the high temperature (80–90°C) is absolutely required for reasonable chromatography. Analytical methods for monitoring glycosylation of mAbs are important because bioprocess conditions can cause variation in high mannose type, truncated forms, reduction of tetra-antennary and increase in tri- and biantennary structures, less sialyated glycans and less glycosylation.Dr. Cohen also reviewed new separations technologies such as monolithic materials and chip based nanoscale separations. He presented an example of the use of a ceramic microfluidic UPLC system and the software tool BiopharmaLynx 1.2 to perform humanized peptide mapping. Three dimentional structure analysis using amide hydrogen exchange was also discussed. In a continuous labeling experiment, labeling occurs at 25°C, pH 7, and aliquots are removed and quenched at 0°C, pH 2.5. The protein sample can be subjected to HPLC/UPCL directly, or subjected to online digestion and then HPLC/UPCL. Electrospray mass spectrometry then provides information about isotope pattern and deuterium content that can be used to determine exchange rates. Use of UPLC will provide sharper peaks and improved spectral quality.16 The technique can be used for quality control or comparability of samples, e.g., differentiation of correctly folded protein from incorrectly folded protein.Tom Laue (University of New Hampshire) discussed advances in analytical ultracentrifugation (AUC) and analytical electrophoresis (AE). Dr. Laue remarked that AUC provides a framework for thinking about concentrated solutions and proximity energies. AUC can be used to characterize proteins in high concentration formulations. Proximity energies at high concentrations may be positive or negative, and are dependent on such factors as distance, orientation, solvent and time. Potential energy is dependent on forces such as charge-charge, charge-dipole, dipole-dipole, hydrogen bonding, dispersion, dipole induced dipole, charge induced dipole and van der Waals interactions. AUC with fluorescence detection can be used to characterize labeled proteins in concentrated samples such as plasma. For example, mAb interactions in plasma can be observed using fluorescence detected sedimentation. Weak electrostatic interactions will dominate molecular behavior in concentrated solutions.Dr. Laue then discussed AE as a technique to determine accurate values of protein charge. Interestingly, monoclonal IgGs have an actual charge that is aberrantly low compared to the calculated value (e.g., 2 versus 24). The low charge leads to problems with poor solubility and high viscosity. Dr Laue speculated that the mAb charge suppression may have some housekeeping function such as weakening charge interactions with anionic plasma proteins, or altering co-operativity for Fc FcR binding or other functions. The low charge may be due to a combination of pKa shifts, anion binding (territorial or site), and carbohydrate involvement. Many of the viscosity and solubility problems encountered during processing may be traced to the low charge on IgGs. He urged attendees to measure the charge and not to rely on calculated charge estimates (e.g., from isoelectric point measurements).Kermit Murray (Louisiana State University) discussed coupling microfluidic chips to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The chips can speed proteomics by serving as a single platform for automated cell culturing, digestion, separation and sample deposition. The system is based on synthetic polymer microfluidic devices, with chip components fabricated on a poly(methyl) methacrylate plate using the hot embossing method and off-line MALDI analysis. A key component of the system is a trypsin microreactor. Assembled chips are processed using a pressure-driven or electrokinetic flow; the system utilizes a Dionex LC and a Probot MALDI plate spotter for the former. Digested peptides are coaxially mixed with a MALDI matrix solution and deposited on a MALDI target. Chips are stable for about one month.Dr. Murray presented experimental results from analyses of cytochrome c under various flow rates. A flow rate of 1 µL/min, with a residence time of approximately 24 seconds within the reaction bed, provided 67% sequence coverage, which increased to 72% when residence time was increased to 48 seconds. Use of the 1 µL/min flow rate resulted in sequence coverages of 35%, 58%, and 47% for 10 µM samples of bovine serum albumin, myoglobin and phosphorylase b, respectively. The digestion efficiency was improved using an electrokinetically driven microreactor using a micro-post structured chip. Using the micro-post system, sequence coverage of 10 µM cytochrome c was 89%; sequence coverage decreased when protein concentration of the sample was lower. Whole bacterial cells can be analyzed using the system. Digestion and deposition of E. coli resulted in identification of the aminoglycoside 3′-phosphotransferase type 1, with 57% sequence coverage.A two-chamber chip developed by Dr. Murray and colleagues can also be used to provide MALDI MS results for bacteria. It has applications for analysis of sepsis, pneumonia, tuberculosis, blood supply QA/QC, and environmental pathogen samples. The cell culture chamber has sample and media inlet, as well as outlet, channels; the culture chamber itself has a 3 mm diameter and 300 µm depth. The system uses a PMMA chip and PDMS cover. The channel surfaces are sterilized with UV. After assembly, the chamber is filled with nutrient broth, approximately 4,000 E. coli cells are added and the reservoirs are closed. The bacteria are cultured for 24 hours at 37°C. One µL of E. coli is then deposited on the MALDI target plate. In an experiment using ATCC#9637, #11303, or #11775, some cellular protein peaks were found. The results suggest that such on-chip culturing could be used for fingerprint analysis. Finally, Dr. Murray discussed preliminary work on a temperature regulated chip with heating and cooling elements.The topic of mass spectrometry (MS)-based strategies to study protein architecture, dynamics and binding was reviewed by Igor Kaltashov (University of Massachusetts, Amherst). He first noted that biopharmaceuticals have higher order structure and conformational heterogeneity, and various perturbations or changes in the production process can result in alterations of the primary or higher order structure that can have deleterious effects on the efficacy, immunogenicity or stability of the protein. MS has been applied to the structural characterization of recombinant protein pharmaceuticals17 and specifically therapeutic antibodies.18 The tertiary and quaternary protein structure can be characterized directly in solution by electrospray ionization (ESI) MS.As an example of the use of MS in structure characterization, Dr. Kaltashov discussed studies done on alkylated interferon β-1a (IFN-β-1a). Alkylation at Cys-17 of the protein results in 50–90% reduction of the antiviral activity. He remarked that ‘classical’ biophysical techniques such as size exclusion chromatography, fluorescence, far-UV circular dichroism (CD) and near-UV CD were not very informative regarding conformational changes between the alkylated and unmodified forms. Two complementary MS-based techniques, analysis of ionic charge state distribution and hydrogen/deuterium exchange (HDX), were used to monitor conformational changes.19 The analysis of the ionic charge distributions indicated a decrease in conformational stability in the alkylated form; the partial unfolding was revealed by the presence of protein ions in the ESI mass spectra with significantly higher charge density compared to the unmodified version. In HDX MS, measurements can be carried out under conditions closely mimicking the formulation buffer, and thermodynamic information is derived from biophysical measurements. Global HDX MS revealed higher flexibility of alkylated IFN. Backbone flexibility was observed to be distributed unevenly across the polypeptide sequence. Structural studies suggest that the loss of antiviral activity of the alkylated form is due to destabilization of a region of IFN that binds with its low affinity receptor (IFNAR1), and disruption of ternary complex formation.Dr. Kaltashov concluded by suggesting that ESI MS can be used to characterize highly heterogeneous systems and presented findings from a study of heparin, which is very heterogeneous, and difficult to characterize by MS. With colleagues, he has developing a mass spectrometry-based strategy for characterization of anti-thrombin interaction with low molecular weight heparin and heparin oligomers.20Genentech''s use of high throughput (HTP) methods in bioprocess development was discussed by Judy Chou (Genentech). She described analytical methods as the ears and eyes of the production process. Use of a high throughput platform is directly related to the need for rapid analysis of bioprocess samples. The need for speedy analysis, which enables new products to get to patients in a timely fashion, has to be balanced with the need for extensive sample characterization that might be time-consuming. The aim is to leverage new technology, especially in HTP purification and automation to increase the number of experiments while reducing resources required and shortening timelines. The HTP approach involves scaling down cell culture, use of protein A well-plate purification (PAWP), purification in plate (PiP), HTP impurity analysis and an at-line reverse phase high performance liquid chromatography (RP-HPLC) method.In the PAWP method, 24- or 96-well plates with protein A are used. The plates can be subjected to centrifugation or vacuum procedures, then samples are directly analyzed by HPLC, liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis (CE), or image capillary isoelectric focusing (icIEF) techniques. The method allows product quality tests to be expedited, and allows the company to address product quality issues early on and reduce resource and time cost later. Use of the PAWP method was directly compared with use of a standard purification procedure (protein A column). Samples subjected to both methods gave similar results in an array of tests (SEC IEC, CZE, icIEF analysis, CE-Glycan assay, peptide mapping).RP-HPLC rapid monitoring can be used as a fast method to monitor mAb fragments in both the purified samples and the cell culture fluids without any sample preparation procedure. It provides a powerful tool to look into antibody reduction issues and helps to monitor as well as to develop bioprocess to mitigate the risk of losing product quantity and quality. Furthermore, the on-line RP-HPLC-Mass Spectroscopy (MS) enabled the understanding of the new peaks identified in the cell culture fluids and increased the process knowledge during the development and operation phases.HTP impurity assays were also developed as a tool to quickly narrow down purification conditions. A CHO protein Meso Scale Discovery (MDS) impurity assay that utilizes electrochemiluminescence was incorporated in an HTP process. Plates can be prepared and stored for up to nine months. The MSD assay was compared to ELISA at various purification steps and the difference was found to be less than 15%, whereas only 10% of resources are used to perform assay and the total assay time was only 2.5 hours for ∼400 samples. In addition, a leached Protein A HTP assay based on MSD technology was also developed. In order to prevent the signal masking introduced by the products, a novel approach of acidification combined with effective blockers was implemented. The new method is generic to all the molecules tested so far and only takes three hours for ∼400 samples with limited amount of resources needed.A novel TEACAN system that puts all the relevant analytical assays as well as the PiP and High throughput Formulation development in an assembly line is currently being used. Dr. Chou mentioned that the details of the methods she described will be published soon. 相似文献2.
3.
《MABS-AUSTIN》2013,5(4):308-317
The conference, which was organized by Visiongain and held at the BSG Conference Center in London, provided an excellent opportunity for participants to exchange views on the development, production, and marketing of therapeutic antibodies, and discuss the current business environment. The conference included numerous interactive panel and group discussions on topics such as isotyping for therapeutic antibodies (panel chair: Nick Pullen, Pfizer), prospects for fully human monoclonal antibodies (chair: Christian Rohlff, Oxford BioTherapeutics), perspectives on antibody manufacturing and development (chair: Bo Kara, Avecia), market impact and post-marketing issues (chair: Keith Rodgers, Bodiam Consulting), and angiogenesis inhibitors (chair: David Blakey, AstraZeneca). 相似文献
4.
《MABS-AUSTIN》2009,1(4):308-Aug;1(4):308
The conference, which was organized by Visiongain and held at the BSG Conference Center in London, provided an excellent opportunity for participants to exchange views on the development, production and marketing of therapeutic antibodies, and discuss the current business environment. The conference included numerous interactive panel and group discussions on topics such as isotyping for therapeutic antibodies (panel chair: Nick Pullen, Pfizer), prospects for fully human monoclonal antibodies (chair: Christian Rohlff, Oxford BioTherapeutics), perspectives on antibody manufacturing and development (chair: Bo Kara, Avecia), market impact and post-marketing issues (chair: Keith Rodgers, Bodiam Consulting) and angiogenesis inhibitors (chair: David Blakey, AstraZeneca). 2009 Jul-Aug; 1(4): 308.
March 24, 2009 Day 1
Mari HerigstadAuthor information Copyright and License information DisclaimerVisiongain; London, UKCorresponding author.Correspondence to: Mari Herigstad; Visiongain; BSG House; 226-236 City Road; London EC1V 2QU UK; Email: moc.liamg@datsgireh.iramCopyright © 2009 Landes BioscienceThe first day was dedicated to discussion of antibody development and engineering, as well as debate on use of various types of antibodies. The session was chaired by David Blakey (AstraZeneca). The day opened with an overview on global trends in the antibody development and probabilities of approval success for human and humanized monoclonal antibodies (mAbs). The speakers then provided insights into the engineering and development of new therapeutic antibodies. Prospects for novel antibody formats, and assessment of immunogenicity, stability and aggregation risks in the development of therapeutic antibodies through use of in vivo and in silico methods were reviewed.Global trends in antibody development were discussed by Janice Reichert (Tufts Center for the Study of Drug Development and Editor-in-Chief, mAbs). Dr. Reichert emphasized the increased focus on mAbs as therapeutic agents. Of the therapeutic proteins entering clinical study each year, the majority are mAbs. Major pharmaceutical firms are acquiring biotechnology companies to enter this market and new solutions to problems of immunogenicity, stability, affinity, specificity and production are being developed. The research on clinical pipelines undertaken at Tufts CSDD allows calculation of metrics such as clinical development and approval times and probabilities of approval success. Insights gained from these results are important for strategic planning.The cumulative approval success rate for humanized mAbs was 16% for candidates entering clinical study during 1988 and 2008, and 29% for candidates entering clinical study during 1988 and 1997.1 A conservative estimate of the success rate for humanized monoclonal antibodies would be somewhere in between, at approximately 20%. The trend, however, is toward fully human monoclonal antibodies. There are currently two marketed human mAbs, with another four in regulatory review. The cumulative US approval success rate for human antibodies is currently low, but will rise to 18% if the four in regulatory review are approved.In terms of therapeutic categories, oncology mAbs comprises approximately 50% of the total. Of 228 oncology mAbs that have entered clinical study since 1988, 56% are currently in clinical development. By comparison, 125 immunological mAb therapeutics have entered clinical study since 1990, of which 54% are currently in clinical development. The cumulative success rate for humanized oncology and immunological mAbs is 15% and 20%, respectively. Other therapeutic categories are being considered, including infectious disease. Sixteen anti-infective mAbs are currently in clinical study and one anti-infective mAb (palivizumab) has been approved to date. Oncology and immunology mAbs exhibit similar patterns for phase lengths and transition probabilities. The phase transition probability for phase 1 to 2 is high, followed by a lower phase 2–3 transition probability due to a proof-of-concept barrier. The transition probability for phase 3 to approval is comparable to that of phase 1 to 2.1Other interesting trends include an increasing emphasis on antibody fragments.2 Fragments may be easier and less costly to produce, but have shorter circulating half-life compared to full size antibodies and no effector functions unless this is added. Also worth noting is the growing prevalence of modified versions of mAbs (glycosylation and Fc region engineering) and improvements on circulation half-life through PEGylation.3,4 Production methods as well as development and approval pathways for mAbs are well established and marketing approvals are set to increase if success rates are consistent with previous rates. This, together with competitive R&D times and potentially large markets, makes mAbs attractive for development as therapeutics.Julian Burke (Genetix) presented a clinical update on the selection of cell lines for antibody expression and protein production. A hybridoma is a hybrid cell that has been engineered to produce a desired antibody in large amounts. ClonePix FL is an antigen based system for in vitro detection and selection of hybridomas. The system incorporates plating hybridomas into a 3D cell matrix-a method which was first described 25 years ago.5 Whilst this method is not new, the novel aspect of the ClonePix system lies in the screening and collection of only those clones secreting a specific antibody. There are two options for screening hybridomas: immunoglobin G (IgG) secretion assays and antigenspecific assays. Unlike IgG secretion assays, antigen-specific assays isolate only antigen-specific clones with the desired IgG isotype. The system can also optimize production through detection of the highest producing cell lines. This approach allows the production of 10,000 clones in three weeks compared to the conventional approach which produces approximately 1,000 clones in two months. After a few days growth post-selection, isolated clones can be rapidly re-screened for cell-line stability. This stability test can be run in parallel with the scaling up of clones, thus making the process highly time efficient. To summarize, ClonePix minimizes the labor requirement, shortens the process timeline and permits parallel interrogation of multiple antigens.Masa Fujiwara (Chiome Bioscience) described the generation of antibodies using a novel antibody-generation technology called the ADLib (Autonomously Diversifying Library) System. This is a selection technology system based on cell-cell interactions and surface-displayed antigens in their native conformations. The system provides high-affinity antibody generation against difficult antigens such as self/human/homologous antigens, GPCRs, sugars/lipids, haptens and pathogens.Dr. Fujiwara explained how the ADLib system can be used to generate specific monoclonal antibodies using a chicken B-cell line (DT40) that undergoes gene conversion at immunoglobulin loci. This gene conversion is enhanced by treatment of the cells with trichostatin A, a histone deacetylase inhibitor. DT40 cells that are specific to the target antigen are obtained through ‘fishing’ the ADLib library with antigens conjugated with magnetic beads. This selection process and the subsequent screening for specificity can be completed in approximately one week.6,7 As such, one of ADLib''s attractive features is the system''s ability to develop diverse monoclonal antibodies within weeks, not months.Optimization of antibodies, with a focus on structure-function relationships, was discussed by Bryan Edwards (MedImmune). Complementary determining regions (CDRs) are found in the variable domains of antibodies and confer the antigen specificity of the molecule. The CDR regions show high levels of natural sequence variability and are targeted during somatic hypermutation to generate higher affinity antibodies to the antigen. When considering strategies for in vitro optimization of antibodies, the CDR regions are typically targeted for mutagenesis. Three CDRs (CDR1, CDR2 and CDR3) are found on both the heavy and light chain regions of an antibody, and the highest level of natural sequence variability is found in the heavy chain CDR3 domain.Dr. Edwards described the optimization of two lead antibody candidates, where the heavy and light chain CDR3 domains were randomized at all amino acid positions and higher affinity variants isolated by both phage and ribosome display. Further gains in antibody potency were then obtained by combining the beneficial amino changes introduced into the heavy and light chain CDR3 domains. Additional sequence space was also explored outside of the CDR3 regions by the generation of error-prone libraries, and subsequent selection by ribosome display. Lead antibody candidates were improved several thousand-fold in potency through a combination of these approaches.By studying the solved crystal structures of Fab:antigen complexes, Dr. Edwards explained that not all CDR regions make direct contact with the target antigen and that amino acids in the framework regions can also contribute to antibody specificity. Moreover, beneficial changes introduced during optimization are not always due to the introduction of new contacts with the target antigen. Several amino acid changes can indirectly improve the potency of the antibody despite being some distance from the antigen binding site. It has been postulated that these amino acid changes improved affinity by reducing the free energy of the antibody:antigen complex, for example by stabilizing CDR loop conformations or by improving the stability of the VH-VL interface.Pavel Bondarenko (Amgen) presented data on the structure and function of disulfide isoforms of the human IgG2 subclass. There are five different known human antibody isotypes; IgA, IgD, IgE, IgM and IgG, of which IgG is the isotype that provides the majority of immune responses against pathogens. IgG antibodies have predictable properties, controlled function and long circulation half-life. Due to these properties, IgG antibodies are the most common therapeutic modalities. There are four human IgG subclasses, IgG1, IgG2, IgG3 and IgG4, of which IgG1 is the most abundant.One therapeutic function of monoclonal IgG antibodies involves binding Fab regions to target receptors, which blocks ligand-receptor interaction. Additional functions include initiating cell destruction through the attraction of immune complexes by the Fc and hinge region on the antibody. Due to their lower affinity for Fc receptors than IgG1s, IgG2s show reduced propensity for activating immune responses. This may be beneficial in some therapeutic aspects.Scientists at Amgen have recently discovered that structural heterogeneity is a naturally occurring feature of human IgG2 antibodies.8,9 These distinct IgG2 forms are due to differences in the disulfide connectivity at the hinge region. There are three human IgG2 isoforms; IgG2-A, IgG2-B and IgG2-A/B. IgG2-A is defined by structurally independent Fab domains and hinge region. In IgG2-B, on the other hand, both Fab regions are covalently linked to the hinge. IgG2-A/B is an arrangement in which only one Fab arm is covalently linked to the hinge through disulfide bonds.The disulfide isoforms may show differences in potency. Against a cell-surface receptor, IgG1 and IgG2-A forms of a mAb were shown to have approximately similar potency and both had greater potency that IgG2-B. The difference between the IgG2 isotopes was attributed to the greater flexibility of IgG2-A and its ability to bind with both Fab regions. IgG2 disulfide exchange is facilitated by the close proximity of cysteine residues at the hinge region of IgG2. The mutation of a single cysteine residue in the IgG2 hinge region resulted in a loss of disulfide heterogeneity.10 Also, redox treatments with a cysteine/cystamine mixture have been shown to cause enrichment of both IgG2-A and IgG2-B.9 Human IgG2 isoforms are dynamic and exhibit disulfide rearrangement in both blood and cell culture.11 Initially, IgG2 exists as IgG2-A, and is then rapidly converted to the asymmetric IgG2-A/B, followed by a slower conversion to IgG2-B. The biological relevance of IgG2 isoforms and in vivo conversion is currently being studied.9Andrew Popplewell (UCB New Medicines) provided an introduction to the prospects for therapeutic antibody fragments. Out of the 22 currently FDA approved monoclonal antibody therapeutics, three are antibody fragment therapeutics. Antibody fragment formats include Fab regions, single chain variable domains (scFv) and variable loops on the heavy and light chain (dAbs).Antibody fragments are highly flexible formats that may be combined to form new multivalent or multi-specific structures. Compared to full-length IgGs, fragments may have improved biodistribution, tissue penetration,12,13 target access, or potentially better safety profiles due to the lack of Fc regions. Antibody fragments can also be expressed in microbial systems. They also show shorter serum persistence, which, depending on use, can be either disadvantageous or advantageous. However, antibody fragment circulation time can be modified either through PEGylation,3,4,14 or through use of serum proteins as carriers.15 Either strategy may alter the pharmacokinetic properties of fragments, allowing the infrequent therapeutic dosing commonly used for full-length IgG therapy.Stability is a major factor for the successful commercialization of antibody-based drugs. Fabs are generally more stable to thermal or physical stress compared to IgGs, and this stability is not affected by PEGylation or by antibody species origin (e.g. mouse, rat, human). ScFvs and dAbs typically exhibit reduced stability compared to Fabs, however advances in technology may contribute to improve stability in these fragments.Dr. Popplewell emphasized that a more complete understanding of biophysical properties and improved stability engineering are required before antibody fragments can reach their full potential. Full-length IgG1s offer active immune cell recruitment, and may thus be better suited for certain therapeutic uses, such as oncology treatments. The development of products with enhanced Fc functionality, the option of using inactive Fcs, and improvements in yields from mammalian cell expression systems are providing further options for the full length IgG format. Dr. Popplewell summarized by pointing out that the intended mechanism of action should guide the choice of format.The challenge of predicting immunogenicity in a potential drug was discussed by Phillipe Stas (Algonomics). This is a difficult, yet important, process because early and precise immunogenicity assessments can reduce the number of drugs that fail to demonstrate efficacy in clinical trials. In order to generate an accurate risk profile of the potential immunogenicity for a given drug, two questions in particular must be addressed. First, the probability of observing an immunogenic response must be analyzed. Second, the severity of the observed immunogenicity needs to be considered.Neutralizing antibody responses can neutralize not only the therapeutic protein, but also its endogenous counterpart; the latter may induce severe side-effects in the patient.16 Risk factors for immunogenicity include the degree of ‘non-self’ of the antibody, the dosing of the drug (acute versus repeated), route of administration (intravenous versus subcutaneous) and other drug characteristics such as clearance rate of the drug. The patient''s immune status and the properties of the disease (i.e., severity and availability of concomitant immunosuppressants) should be included in a risk analysis.The different drug development stages offer opportunities to use different strategies for immunogenicity assessment. In the clinical phase, the general approach is to conduct antidrug antibodies (ADA) screening on individuals exposed to the drug. However, efforts are now geared toward assessing immunogenicity at earlier stages. The generation of ADA is dependent on the presence of T-cell epitopes. These can be measured in the preclinical setting using in-vitro T-cell assays. Prior to this stage, in silico methods may be used to identify T-cell epitopes. One such T-cell epitope screening tool is Algonomics'' platform Epibase®, which can rapidly analyze and predict the potential immunogenicity of therapeutic protein leads. An in silico approach to T-cell identification can offer relatively inexpensive mapping of epitopes from a wide genetic background.17 Also, perhaps more importantly, the combined use of in vitro and in silico tools allows for a much more accurate and less time-consuming assessment of expected immunogenicity in a drug.In silico methods in the development of therapeutic antibodies were reviewed by Jesús Zurdo (Lonza Biologics). Dr. Zurdo focused on assessment of stability and aggregation risks. In addition to increased production costs, aggregation reduces product stability, increases immunogenicity and may also elevate the toxicity. AggreSolve is an in silico protein analysis platform that can be applied to predict and overcome protein stability and aggregation issues. The Aggresolve platform assesses protein aggregation propensity and identifies aggregation ‘hot-spots.’ The platform can also predict sequence changes that are likely to reduce aggregation propensity. This library of potential substitutions can then be used to re-engineer antibodies with elevated stability and fewer aggregation problems. Compared to wild-type, selected re-engineered molecules achieve significantly reduced aggregation levels whilst retaining biological activity. Furthermore, protein stabilisation using re-engineering methods can also translate into elevated antibody productivity. 相似文献5.
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Samantha O Arnett Jean-Luc Teillaud Thierry Wurch Janice M Reichert Cameron Dunlop Michael Huber 《MABS-AUSTIN》2011,3(2):133-152
The 21st Annual Antibody Engineering and 8th Annual Antibody Therapeutics international conferences, and the 2010 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, was held December 5–9, 2010 in San Diego, CA. The conferences featured over 100 presentations and 100 posters, and included a pre-conference workshop on deep-sequencing of antibody genes. The total number of delegates exceeded 800, which set a new attendance record for the conference.The conferences were organized with a focus on antibody engineering only on the first day and a joint engineering/therapeutics session on the last day. Delegates could select from presentations that occurred in two simultaneous sessions on days 2 and 3. Day 1 included presentations on neutralizing antibodies and the identification of vaccine targets, as well as a historical overview of 20 years of phage display utilization. Topics presented in the Antibody Engineering sessions on day 2 and 3 included antibody biosynthesis, structure and stability; antibodies in a complex environment; antibody half-life; and targeted nanoparticle therapeutics. In the Antibody Therapeutics sessions on days 2 and 3, preclinical and early stage development and clinical updates of antibody therapeutics, including TRX518, SYM004, MM111, PRO140, CVX-241, ASG-5ME, U3-1287 (AMG888), R1507 and trastuzumab emtansine, were discussed and perspectives were provided on the development of biosimilar and biobetter antibodies, including coverage of regulatory and intellectual property issues. The joint engineering/therapeutics session on the last day focused on bispecific and next-generation antibodies. Summaries of most of the presentations are included here, but, due to the large number of speakers, it was not possible to include summaries for every presentation.Delegates enjoyed the splendid views of the San Diego Bay and proximity to the Gaslamp Quarter provided by the venue. The 22nd Annual Antibody Engineering and 9th Annual Antibody Therapeutics conferences, and the 2011 Annual Meeting of The Antibody Society, are planned for December 5–8, 2011 at the same location in San Diego, and will include two two-day short courses on Introduction to Antibody Engineering and Protein Characterization for Biotechnology Product Development.Key words: antibody engineering, antibody therapeutics, phage display, biosimilar antibodies 2011 Mar-Apr; 3(2): 133–152. Published online 2011 Mar 1. doi: 10.4161/mabs.3.2.14939
Day 1: December 6, 2010 Antibody Engineering
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The 2nd Annual Antibodies for Cancer Therapy symposium, organized again by Cambridge Healthtech Institute as part of the Protein Engineering Summit, was held in Boston, USA from April 30th to May 1st, 2012. Since the approval of the first cancer antibody therapeutic, rituximab, fifteen years ago, eleven have been approved for cancer therapy, although one, gemtuzumab ozogamicin, was withdrawn from the market. The first day of the symposium started with a historical review of early work for lymphomas and leukemias and the evolution from murine to human antibodies. The symposium discussed the current status and future perspectives of therapeutic antibodies in the biology of immunoglobulin, emerging research on biosimilars and biobetters, and engineering bispecific antibodies and antibody-drug conjugates. The tumor penetration session was focused on the understanding of antibody therapy using ex vivo tumor spheroids and the development of novel agents targeting epithelial junctions in solid tumors. The second day of the symposium discussed the development of new generation recombinant immunotoxins with low immunogenicity, construction of chimeric antigen receptors, and the proof-of-concept of ‘photoimmunotherapy’. The preclinical and clinical session presented antibodies targeting Notch signaling and chemokine receptors. Finally, the symposium discussed emerging technologies and platforms for therapeutic antibody discovery. 相似文献