Interference chromatography: a novel approach to optimizing chromatographic selectivity and separation performance for virus purification

Phase Ib Trial of Oncolytic Herpes Virus G207 Shows Safety of Multiple Injections and Documents Viral Replication

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria during growth, mainly under stress conditions. OMV-based vaccines have played an important role in vaccination against Neisseria meningitidis serogroup B (MenB), stimulating research into novel approaches for developing more effective vaccines. OMVs released by the bacterium Neisseria lactamica have emerged as a promising platform for new vaccine development, especially as carriers in subunit vaccines. Despite their importance, some challenges remain in obtaining and purifying OMVs. The most commonly employed methods for OMV isolation and purification are ultracentrifugation (UC) and size exclusion chromatography (SEC). However, these techniques could present limitations for large-scale production and often result in low yields. This study investigated techniques such as tangential flow filtration (TFF), membrane chromatography, and mixed-mode (multimodal) chromatography as potential replacements for UC and SEC. Among the TFF methods evaluated, the sample obtained on the membrane with a 300-kDa cutoff showed a profile more similar to UC but with more than double the total protein recovery. Sartobind® Q membrane chromatography was ineffective for OMV purification, in the conditions evaluated, with a recovery of 8.7%. Conversely, multimodal Capto™ Adhere chromatography recovered 59.0%, while Capto™ Core 400 yielded a recovery of 72.0%, proving to be more effective for purification when analyzed by high-performance liquid chromatography (HPLC). Thus, combining TFF with a 300-kDa membrane followed by Capto™ Core 400 chromatography can be applied as strategy for large-scale applications offering high recovery and purity.Key points• Evaluation of TFF, membrane and multimodal chromatography techniques for OMV purification.• Improved Neisseria lactamica OMV yields combining TFF and multimodal chromatography.• A process for OMV purification from a non-pathogenic organism feasible to scale up.

Antibody products are promising therapeutic candidates for various diseases, such as cancers and autoimmune disorders. Owing to the improvement of cell culture technology in recent years, the production titers of antibodies can reach up to 10 g/L. With such high titers in upstream production, advancements in the development of downstream separation and purification techniques are highly desired. To ensure a balance between handling the increased product supply and maintaining the high purity required for biopharmaceutical applications, membrane chromatography is an efficient and cost-effective technique that can be used to separate and purify proteins and other biological components. In this study, we developed a full-membrane platform that uses affinity membrane chromatography to capture antibodies, which is followed by a two-step flow-through polishing process involving anion exchange membrane chromatography and hydrophobic interaction membrane chromatography. We tested five molecules using this platform, and the outcomes of membrane chromatography were comparable to those of column chromatography in terms of yield and product quality. Moreover, full-membrane chromatography might lead to a cost reduction of > 70% in chromatography media. Altogether, the full-membrane platform developed in this has potential applications in downstream processes of antibody production.

Oncolytic Viruses: Now Interviewing for the All-Star Game

Evaluation of two-species binding model with anion-exchange membrane chromatography to predict pressure buildup during recovery of virus

Engineered oncolytic herpes simplex virus expressing interleukin 12 suppresses tumorigenicity of hepatocellular carcinoma.

Several organizations have raised concerns about the excessive secrecy maintained by regulatory authorities around the world, in particular in the European Union, France, UK, Canada and Australia. However, limited research has assessed the provision of information by regulatory authorities. This study aimed to assess the type and availability of information provided on the regulatory authorities' websites. Regulatory authorities' websites in six countries (USA, Canada, UK, France, Australia and New Zealand) and at the European level (European Medicines Evaluation Agency) were surveyed by two reviewers between October 2005 and March 2006. The survey instrument included 16 criteria organized in 3 domains: information on marketed drugs, information on assessment of drugs and information on drug safety. There was a great variability in the level of information provided. Several medicine agencies did not provide basic information on marketed drugs, such as the summary of products' characteristics. Information on registration dossiers was scant on most websites except that of the US Food and Drug Administration. The European Medicines Evaluation Agency, the French agency and the Canadian agency released public assessment reports that contained only summarized information of registration data. Only one country, Canada, provided full access to pharmacovigilance data. The periodic safety update reports that companies have to provide regularly to regulatory authorities were not available in any country. Information on which regulatory authorities base their decisions for licensing new drugs and the rationales behind these decisions were often not publicly available.

Oncolytic virus immunotherapy is rapidly gaining interest in the field of immunotherapy against cancer. The minimal toxicity upon treatment and the dual activity of direct oncolysis and immune activation make therapy with oncolytic viruses (OVs) an interesting treatment modality. The safety and efficacy of several OVs have been assessed in clinical trials and, so far, the Food and Drug Administration (FDA) has approved one OV. Unfortunately, most treatments with OVs have shown suboptimal responses in clinical trials, while they appeared more promising in preclinical studies, with tumours reducing after immune cell influx. In several clinical trials with OVs, parameters such as virus replication, virus-specific antibodies, systemic immune responses, immune cell influx into tumours and tumour-specific antibodies have been studied as predictors or correlates of therapy efficacy. In this review, these studies are summarized to improve our understanding of the determinants of the efficacy of OV therapies in humans and to provide insights for future developments in the viro-immunotherapy treatment field.

Pathophysiological hypoxia, which fosters the glioma stem-like cell (GSC) phenotype, is present in high-grade gliomas and has been linked to tumor development, invasiveness, and resistance to chemotherapy and radiation. Oncolytic virotherapy with engineered herpes simplex virus-1 (HSV-1) is a promising therapy for glioblastoma; however efficacy of γ134.5-deleted HSVs, which have been used in clinical trials, was diminished in hypoxia. We investigated the ability of a chimeric HCMV/HSV-1 virus, which expresses the human CMV PKR evasion gene IRS1 and is in preparation for clinical trials, to infect and kill adult and pediatric patient-derived glioblastoma xenografts in hypoxia and normoxia. Infectivity, cytotoxicity and viral recovery were significantly greater with the chimeric virus compared to the γ134.5-deleted virus, regardless of oxygen tension. The chimeric virus infected and killed CD133+ GSCs similarly to wild-type HSV-1. Increased activation of mitogen-activated protein kinase (MAPK) p38 and its substrate heat shock protein 27 (Hsp27) was seen after viral infection in normoxia compared to hypoxia. Hsp27 knockdown or p38 inhibition reduced virus recovery indicating that the p38 pathway plays a role in the reduced efficacy of the γ134.5-deleted virus in hypoxia. Together these findings demonstrate chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

Oncolytic virus therapy (OVT) represents a new class of therapeutic agents in cancer treatment. The molecular and cellular mechanisms of action of OVTs have been evaluated in nonclinical/clinical phase trials. Various genetically modified viruses have been developed as oncolytic agents, and the first approval of an OVT for clinical use was issued by the US Food and Drug Administration in 2015. In this context, more and more clinical development of OVTs is anticipated in the future. This article provides a risk assessment of OVT based on the safety data obtained from all clinical trials to date using a publicly available database. The most common adverse events (AEs) observed in clinical trials have been infection-related symptoms such as fatigue, chills, fever, and nausea; few serious AEs have been observed, regardless of the kind of virus or transfected genes. In vivo systemic infusion of OVTs demonstrated a high percentage of AEs, but most AEs were manageable using common drugs. This paper describes OVTs' specific safety/toxicity profiles and encourages the performance of further clinical trials of OVTs to address the most serious challenges anticipated in the development of OVTs as a new class of drugs for the treatment of cancer.

Cancer has an increasing incidence worldwide in humans and animals. In addition to traditional treatments such as surgery, radiotherapy, and chemotherapy, there is a search for new treatment strategies for cancer treatment. Oncolytic virotherapy arouses great interest in human medicine with the development of biotechnology and increasing knowledge about virus-cell interactions in recent years. Many in-vivo and in-vitro studies have led to the development of a United States Food and Drug Administration (FDA)-approved, genetically modified oncolytic viral therapy. Based on the studies in human medicine, some clinical trials have also been carried out with oncolytic virotherapy in veterinary medicine. But the studies in cats and dogs are very limited. This review aims to compare the development of oncolytic virotherapy in human and veterinary medicine with current studies and to draw attention to the fact that virotherapy can be used as a treatment option for various tumoral diseases in veterinary medicine in the future.

Cybersecurity vulnerabilities of cardiac implantable electronic devices: Communication strategies for clinicians—Proceedings of the Heart Rhythm Society's Leadership Summit

In the pharmaceutical industry, process validation tasks are based on the raw data and its derived analytical results generated from the process. Process validation failure affects both patient safety and the economic success of the manufacturing company. Hence, data integrity is highly critical in this area. Regulatory agencies, such as the Food and Drug Administration (FDA), reacted to past data integrity breaches by publishing new guidelines on data integrity for the correct handling of data in the pharmaceutical context.In this contribution, we want to show how data integrity can be improved on a technological level, removing the need for trusted third parties and centralized systems for this task. Therefore, we implemented an approach that uses existing tools, currently mostly used by software developers, and combined them with a new smart contract built on top of the Ethereum blockchain. In a case study, we test how data manipulation or backdating of results can be easily detected and how regulatory agencies can audit the complete data flow from the regulatory report back to the original raw data.The results of this contribution outline a possible road map for the development of production-ready tools, such as versioned database systems that natively interoperate with distributed ledgers. This will improve the trustworthiness of pharmaceutical manufacturing data by both protecting the intellectual property of the industrial company and improving the safety of the patients.LAY ABSTRACT: In the pharmaceutical industry, economically driven manufacturing companies are regulated and controlled by regulatory agencies. The pharmaceutical manufacturing companies need to produce large amounts of process and analytical data to show that their products are safe for patients. As the decisions of the regulatory agencies rely on this data, manufacturing companies need to prove how their generated data can be protected from technical breaches or data manipulation.As of today, the available technical solutions to provide data integrity are not working well enough. Regulatory agencies have published multiple documents highlighting the current data integrity issues.In this contribution, we show how blockchain, a technology that multiple cryptocurrencies like Bitcoin rely on, can help to improve the integrity of manufacturing data and data science analysis procedures. Therefore, we combined a smart contract on the Ethereum blockchain with tools currently mostly used by software developers. The presented workflow shows how data integrity can be guaranteed on a technological level without the need for trusted third parties.

In their paper, Correll et al1 present proposals for strategies to “de-risk” trials of nov­el psychotropic agents. However, several of their suggestions may inadvertently increase the risk that clinical trials be uninformative, especially when considering requirements for drug approval. Here we provide our perspective on their advice. The authors begin their “de-risking” advice with some foundational concepts related to validity, power, and a priori hypothesis generation. They go on to discuss the importance of “clinical equipoise” in randomized controlled trials. This emphasis is reasonable. Without clinical equipoise, trials are vulnerable to bias and are more difficult to interpret. For example, the enthusiasm for psychedelic drug development from both the lay press and investigators may contribute to difficulties separating drug effect from expectation bias. Correll et al subsequently offer sugges­tions for modifying trial designs in an at­tempt to avoid failed studies. One recom­mendation is to consider adaptive trial de­signs whereby the beginning of the trial informs its later stages. The US Food and Drug Administration (FDA) has published a guidance for in­dus­try on the use of adaptive trials2. Compared with a traditional clinical trial, patients enrolling at the start of an adaptive clinical trial may not have the same experience as those enrolling later in the trial (e.g., possible doses). This may lead to challenges in interpreting the trial results. Further, although adaptive trials may be designed to maximize the possibility of quickly detecting efficacy with limited enrollment, more subjects may still be needed to characterize safety. A positive adaptive trial may not translate into approval if there are safety signals that must be explored in larger or longer studies. Sponsors considering adaptive studies in phase 3 should discuss their plans with regulatory authorities before implementation. Phase 2 is an important part of dose ex­plo­ration. Correll et al suggest using adap­tive trials to determine the maximum tolerated dose (MTD) and prevent “expensive and underpowered multi-armed studies”. However, as they later acknowledge, there are chal­lenges to using an MTD when a dose range may be required. The MTD as determined early in a study may not translate to the op­timum dose, considering benefit-risk, as the study progresses. A Phase 2 program examining several doses based on phase 1 data (e.g., receptor binding, tolerability) need not be adequate­ly powered to demonstrate safety and effi­cacy for each arm. It is meant to inform a phase 3 program. Sponsors sometimes de­sign phase 2 studies with characteristics of adequate and well-controlled investigations in hopes that a positive trial may be used to support a marketing application. However, if there are dosing, endpoint, population or safety is­sues, this approach may ultimately prove more costly. The paper's discussion of precision medicine versus generalizability is important. We acknowledge that particular mechanisms of action may have benefits particularly ap­plicable to subpopulations, and that en­riched trials may improve the chance of de­tecting an efficacy signal. However, deve­l­op­ment programs should focus not on ar­ti­ficially narrowed populations, but on a pop­ulation widely inclusive of those likely to re­ceive benefit. A reasonable starting point for separat­ing promising subgroup effects from post-hoc artifact is biological plausibility. Although a collection of clinical characteristics could be representative of a biological construct, there is a public health interest in determining what that underlying construct is. The authors suggest that positive studies from an enriched population could lead to an approval for use of the drug in a subpopulation, with studies of a broader population deferred to post-approval. However, in the absence of a biologically plausible sub­group definition supported by strong scientific understanding, we do not support this approach. Sponsors should explore scientifically justified potential subgroups in phase 2, refer to the appropriate guidance3, and discuss plans with regulatory authorities. Placebo lead-in studies have often not met expectations in psychiatric disorders. Sequential parallel design remains an unproven alternative to traditional placebo lead-in strategies. As with adaptive trials in general, there are significant challenges in interpreting the results of such studies. There is not a standard method for analyzing the results of sequential parallel design studies, and employing such a design in phase 3 entails risk on the part of a sponsor. Sponsors considering sequential parallel design should discuss this with regulatory authorities. Correll et al state that “FDA…[has] taken the position that to assess the efficacy of a new treatment for many mental disorders is not possible without a placebo-controll­ed design”. This is not accurate4, 5. The Code of Federal Regulations, Title 21 (section 314.126)6 describes the characteristics of an adequate and well-controlled clinical investigation, and specifically mentions other types of controls – such as active treatment concurrent control and no treatment concurrent control – in addition to placebo concurrent control. Placebo-controlled trials are often favored and chosen by sponsors because they typically produce the most readily interpretable results. Regarding generalizability of clinical tri­al results, Correll et al note that many “real world” patients would not qualify for pharmaceutical trials because of comorbidities. Sponsors should be prepared to justify their exclusion criteria, focusing on comorbidities that are expected to complicate interpretation of the study or decrease the likelihood of detecting an effect (e.g., active substance use disorders). The paper suggests requiring post-marketing studies to examine drug efficacy in “real world” patients; however, the FDA does not have the statutory authority to require such studies7. Correll et al describe scenarios in which rapid recruitment may impact study quality. Baseline symptom inflation and diagnostic imprecision may speed recruitment but will also make demonstrating efficacy more difficult. Although small sites may be a source of heterogeneity, they may simply be recruiting judiciously. Therefore, we recommend caution regarding the suggestion to drop poorly recruiting sites early in the study. We agree that some new technologies might have the potential to improve assessments; however, before incorporating novel assessments (e.g., digital endpoints), we recommend that sponsors submit supportive evidence that the technology is fit-for-purpose. For example, a computerized system for assessing patient speech may seem to be an improvement on established subjective clinician ratings. However, it is the subjective clinical ratings which would have been tied to dysfunction and prognosis. Unless the computerized system also reflects dysfunction and prognosis, it may not be fit-for-purpose. Additionally, sponsors should ensure that including technology does not discourage or prevent certain groups from enrollment or introduce unanticipated biases. Sponsors should discuss novel statistical approaches with regulatory authorities prior to starting clinical trials. Regarding the suggestion to use an endpoint that reflects symptom course over time (rather than at discrete time points), this may or may not be acceptable for a given trial. Such averaged endpoints may reflect improvement at the start of a trial that is lost as the trial progresses, leading to questions about the durability of effect. Before attempting something novel in a development program, sponsors should meet with regulatory authorities, which can often refer companies to pertinent publi­sh­ed guidances, help think through regulato­ry requirements, and use experience from oth­er programs to offer recommendations.

Since the US Food and Drug Administration first approved talimogene laherparepvec for the treatment of melanoma in 2015, the field of oncolytic immunotherapy (OI) has rapidly evolved. There are numerous ongoing clinical studies assessing the clinical activity of OIs across a wide range of tumor types. Further understanding of the mechanisms underlying the anti-tumor immune response has led to the development of OIs with improved immune-mediated preclinical efficacy. In this review, we discuss the key approaches for developing the next generation of herpes simplex virus-based OIs. Modifications to the viral genome and incorporation of transgenes to promote safety, tumor-selective replication, and immune stimulation are reviewed. We also review the advantages and disadvantages of intratumoral versus intravenous administration, summarize clinical evidence supporting the use of OIs as a strategy to overcome resistance to immune checkpoint blockade, and consider emerging opportunities to improve OI efficacy in the combination setting.