The use of predictive models to develop chromatography-based purification processes.

Chromatography is the workhorse of biopharmaceutical downstream processing because it can selectively enrich a target product while removing impurities from complex feed streams. This is achieved by exploiting differences in molecular properties, such as size, charge and hydrophobicity (alone or in different combinations). Accordingly, many parameters must be tested during process development in order to maximize product purity and recovery, including resin and ligand types, conductivity, pH, gradient profiles, and the sequence of separation operations. The number of possible experimental conditions quickly becomes unmanageable. Although the range of suitable conditions can be narrowed based on experience, the time and cost of the work remain high even when using high-throughput laboratory automation. In contrast, chromatography modeling using inexpensive, parallelized computer hardware can provide expert knowledge, predicting conditions that achieve high purity and efficient recovery. The prediction of suitable conditions in silico reduces the number of empirical tests required and provides in-depth process understanding, which is recommended by regulatory authorities. In this article, we discuss the benefits and specific challenges of chromatography modeling. We describe the experimental characterization of chromatography devices and settings prior to modeling, such as the determination of column porosity. We also consider the challenges that must be overcome when models are set up and calibrated, including the cross-validation and verification of data-driven and hybrid (combined data-driven and mechanistic) models. This review will therefore support researchers intending to establish a chromatography modeling workflow in their laboratory.

Plant-made immunotoxin building blocks: A roadmap for producing therapeutic antibody-toxin fusions.

Molecular farming in plants is an emerging platform for the production of pharmaceutical proteins, and host species such as tobacco are now becoming competitive with commercially established production hosts based on bacteria and mammalian cell lines. The range of recombinant therapeutic proteins produced in plants includes replacement enzymes, vaccines and monoclonal antibodies (mAbs). But plants can also be used to manufacture toxins, such as the mistletoe lectin viscumin, providing an opportunity to express active antibody-toxin fusion proteins, so-called recombinant immunotoxins (RITs). Mammalian production systems are currently used to produce antibody-drug conjugates (ADCs), which require the separate expression and purification of each component followed by a complex and hazardous coupling procedure. In contrast, RITs made in plants are expressed in a single step and could therefore reduce production and purification costs. The costs can be reduced further if subcellular compartments that accumulate large quantities of the stable protein are identified and optimal plant growth conditions are selected. In this review, we first provide an overview of the current state of RIT production in plants before discussing the three key components of RITs in detail. The specificity-defining domain (often an antibody) binds cancer cells, including solid tumors and hematological malignancies. The toxin provides the means to kill target cells. Toxins from different species with different modes of action can be used for this purpose. Finally, the linker spaces the two other components to ensure they adopt a stable, functional conformation, and may also promote toxin release inside the cell. Given the diversity of these components, we extract broad principles that can be used as recommendations for the development of effective RITs. Future research should focus on such proteins to exploit the advantages of plants as efficient production platforms for targeted anti-cancer therapeutics.

[Special situations of preconditioning and prehabilitation in oncological visceral surgery]. / Spezialsituationen der Präkonditionierung und Prähabilitation in der onkologischen Viszeralchirurgie.

BACKGROUND: Prehabilitation prior to complex visceral oncological surgery is playing an increasingly important role. OBJECTIVE: The aim of this review article is to present special situations of preconditioning in visceral oncological patient cohorts. The following conditions were defined as special situations with subsequently increased risk profile: cardiopulmonary comorbidities, geriatric patients, neoadjuvant therapy and simultaneous fatigue. MATERIAL AND METHODS: A selective literature review based on a search in the electronic databases MEDLINE, PubMed, Cochrane Library and the International Standard Randomization Controlled Trial Number (ISRCTN) was performed. RESULTS: The identification of high-risk patients is an essential part of the preoperative evaluation conducted by the anesthesiologist prior to surgery. The cardiovascular and the pulmonary risk profile are determined by means of prediction indices evaluating patient-specific and surgery-related risk factors. The increased use of new oral anticoagulants and dual platelet aggregation inhibition requires individualized treatment strategies. Numerous studies have shown clinically relevant effects of exercise therapy interventions throughout all phases of oncological treatment. In addition to positive effects on therapy-associated side effects, sport can also counteract the effects of sedentary behavior in cancer patients and improve the health-related quality of life. The effectiveness of sport and exercise therapies as well as psychological interventions in oncological patients with fatigue (CRF) is broad, with important components being motivation and compliance. DISCUSSION: In high-risk patients an interdisciplinary approach to planning and conduction of prehabilitation is essential for the early detection and optimization of perioperative risk factors and potential complications. The aim is faster recovery, reduced morbidity and mortality and the possibility to improve long-term survival and quality of life.

Induction chemotherapy (IC) followed by radiotherapy (RT) versus cetuximab plus IC and RT in advanced laryngeal/hypopharyngeal cancer resectable only by total laryngectomy-final results of the larynx organ preservation trial DeLOS-II.

Background: The German multicenter randomized phase II larynx organ preservation (LOP) trial DeLOS-II was carried out to prove the hypothesis that cetuximab (E) added to induction chemotherapy (IC) and radiotherapy improves laryngectomy-free survival (LFS; survival with preserved larynx) in locally advanced laryngeal/hypopharyngeal cancer (LHSCC). Patients and methods: Treatment-naïve patients with stage III/IV LHSCC amenable to total laryngectomy (TL) were randomized to three cycles IC with TPF [docetaxel (T) and cisplatin (P) 75 mg/m2/day 1, 5-FU (F) 750 mg/m2/day days 1-5] followed by radiotherapy (69.6 Gy) without (A) or with (B) standard dose cetuximab for 16 weeks throughout IC and radiotherapy (TPFE). Response to first IC-cycle (IC-1) with ≥30% endoscopically estimated tumor surface shrinkage (ETSS) was used to define early responders; early salvage TL was recommended to non-responders. The primary objective was 24 months LFS above 35% in arm B. Results: Of 180 patients randomized (July 2007 to September 2012), 173 fulfilled eligibility criteria (A/B: larynx 44/42, hypopharynx 41/46). Because of 4 therapy-related deaths among the first 64 randomized patients, 5-FU was omitted from IC in the subsequent 112 patients reducing further fatal toxicities. Thus, IC was TPF in 61 patients and TP in 112 patients, respectively. The primary objective (24 months LFS above 35%) was equally met by arms A (40/85, 47.1%) as well as B (41/88, 46.6%). One hundred and twenty-three early responders completed IC+RT; their overall response rates (TPF/TP) were 94.7%/87.2% in A versus 80%/86.0% in B. The 24 months overall survival (OS) rates were 68.2% and 69.3%. Conclusions: Despite being accompanied by an elevated frequency in adverse events, the IC with TPF/TP plus cetuximab was feasible but showed no superiority to IC with TPF/TP regarding LFS and OS at 24 months. Both early response and 24 months LFS compare very well to previous LOP trials and recommend effective treatment selection and stratification by ETSS. Clinical trial information: NCT00508664.

[Preconditioning prior to visceral oncological surgery : A paradigm shift in visceral surgery?] / Präkonditionierung vor viszeralonkologischen Operationen : Ein Paradigmenwechsel in der Viszeralchirurgie?

BACKGROUND: Postoperative complications after complex visceral oncological surgery can lead to substantial impairment of patients. In addition, preoperative physical performance and the severity of postoperative complications determine the long-term recovery process of physical function. Therefore, preconditioning in the preoperative period should be an important part of the preoperative/neoadjuvant treatment. OBJECTIVE: The aim of this article is a critical appraisal of current concepts of prehabilitation as well as their development potential and applicability in visceral surgery. MATERIAL AND METHODS: Based on a selective literature review, current studies and implemented concepts are presented and therapy algorithms are provided. RESULTS: This study differs in primary outcome, design and temporal framework of the intervention. The study results showed positive effects of an active increase in physical fitness in the preoperative period with respect to the quality of life, convalescence and postoperative pulmonary complication rate. DISCUSSION: In addition to the assessment of the individual risk of complications by means of spiroergometry, a targeted nutrition and exercise program can increase the individual performance level prior to visceral surgery and, thus, influence the postoperative risk of complications. The performance should be understood as a modifiable risk factor, which can also be positively influenced in the preoperative phase, even in a short time period. Individual preoperative care optimizes the physical and psychological situation of patients. To ensure the required individual care, approaches must be created and pursued, which can be implemented in a decentralized way.

Findings on the heavy metal content in herbal drugs and essential oils - an update.

In this contribution, data for 7 elemental impurities originating from quality control analysis of manufacturers of herbal products is evaluated in light of the current requirements of the European Pharmacopoeia (Ph. Eur.) and the European legislative framework. The data shows that the Ph. Eur. limits set for cadmium, lead and mercury in herbal drugs are in principle still appropriate. The probability of herbal drugs exceeding the limits for arsenic, cobalt, nickel and vanadium (based on the ICH Q3D guideline for elemental impurities) appears to be very low, and consequently, it is proposed that general limits for these elements in herbal drugs in the Ph. Eur. are not required. For essential oils, there does not appear to be a risk of heavy metal contamination and a general test on heavy metals is not considered necessary.

A linear epitope coupled to DsRed provides an affinity ligand for the capture of monoclonal antibodies.

Monoclonal antibodies (mAbs) dominate the market for biopharmaceutical proteins because they provide active and passive immunotherapies for many different diseases. However, for most mAbs, two expensive manufacturing platforms are required. These are mammalian cell cultures for upstream production and Protein A chromatography for product capture during downstream processing. Here we describe a novel affinity ligand based on the fluorescent protein DsRed as a carrier for the linear epitope ELDKWA, which can capture the HIV-neutralizing antibody 2F5. We produced the DsRed-2F5-Epitope (DFE) in transgenic tobacco (Nicotiana tabacum) plants and purified it using a combination of heat treatment and immobilized metal-ion affinity chromatography, resulting in a yield of 24 mg kg-1 at 90% purity. Using a design-of-experiments approach, we coupled up to 15 mg DFE per mL Sepharose. The resulting affinity resin was able to capture 2F5 from the clarified extract of N. benthamiana plants, achieving a purity of 97%, a recovery of >95% and an initial dynamic binding capacity at 10% product breakthrough of 4 mg mL-1 after a contact time of 2 min. The resin capacity declined to 15% of the starting value within 25 cycles when 1.25 M magnesium chloride was used for elution. We confirmed the binding activity of the 2F5 product by surface plasmon resonance spectroscopy. DFE is not yet optimized, and a cost analysis revealed that boosting DFE expression and increasing its capacity by fourfold will make the resin cost-competitive with some Protein A counterparts. The affinity resin can also be exploited to purify idiotype-specific mAbs.

[Gene expression analyses and their possible clinical benefit in head and neck cancer]. / Genexpressionsanalysen und ihrmöglicher klinischer Nutzen inder Betrachtung der Kopf-Hals-Karzinome

Systems biology approaches for mutational (exome analysis and targeted sequencing) and gene expression analysis (transcriptome-wide gene expression profiling) represent a new and growing scientific field in head and neck oncology. In addition to medical biological expertise, bioinformatic assistance is increasingly required. For squamous cell head and neck cancer (HNSCC), the recent molecular genetic single-gene and signal pathway observations represent basic research. Important aspects of this have now been significantly enhanced by systems biology approaches, which have grown into relevant areas of translational clinical research. It is now known that HPV16 is associated with genetic alterations at various locations, but also that it functionally affects genes not altered in their base sequence at the level of methylation. In transcriptome analyses, various consortia found matching clusters of gene expression and HPV16 association with the spectrum of somatic mutations. The differential methylation of gene promoters discovered in HPV16-driven HNSCC proved predictive for survival­even in HNSCC patients without HPV detection. The authors present an overview of some translationally relevant findings and venture an outlook on possible future clinical developments.

Pesticide residues in essential oils: evaluation of a database.

In the context of a revision of the European Pharmacopoeia (Ph. Eur.) general monograph Essential oils (2098), the need to include a test for pesticides is being discussed. According to published literature, some oils, mainly those produced by cold pressing (e.g. citrus oils), can contain relevant amounts of pesticide residues, whereas distilled oils showed positive findings in only a few cases. Recent evaluation of a database containing 127 517 sets of data compiled over 8 years, showed positive results in 1 150 cases (0.90 per cent), and the limits of Ph. Eur. general chapter 2.8.13 Pesticide residues or Regulation (EC) 396/2005, both applicable to herbal drugs, were exceeded in 392 cases (0.31 per cent, equivalent to 34.1 per cent of the positive results), particularly in cases of oils produced by cold pressing. From these results, it can be concluded that a general test on pesticides in the Ph. Eur. general monograph on essential oils is not required for most oils used in medicinal products. Therefore, it is proposed to limit the testing of essential oils for pesticide residues to those cases where potential residues are more of a concern, either due to the type of production process or to those processes where pesticides are actively used during cultivation of the plant (e.g. as documented according to Good Agricultural and Collection Practice (GACP)). Furthermore, in order to assess any potential risk, an approach using the Acceptable Daily Intake (ADI) can be made.

The heavy metals cadmium, lead and mercury in raw materials of animal origin: evaluation of data from practice.

Raw materials from animal origin are widely used in homoeopathy. Due to the lack of dedicated limits, the quality requirements for herbal drugs of the European Pharmacopoeia (Ph. Eur.) and/or the German Homoeopathic Pharmacopoeia (Homöopathisches Arzneibuch, HAB), including limits for heavy metals such as cadmium, lead and mercury, have been applied. A recent database evaluation shows that for some raw materials of animal origin the Ph. Eur. limits for herbal drugs cannot be met in practice. For this reason proposals for new limits for cadmium, lead and mercury are made based on recent experiences from the companies' daily practice. These specific limits are suggested to be included in the individual monographs of the Ph. Eur. or at least the German HAB, respectively, for Ambra grisea, Euspongia officinalis, Formica rufa and Sepia officinalis.

[Neoadjuvant chemotherapy or primary surgery for colorectal liver metastases. Pro adjuvant chemotherapy]. / Neoadjuvante Chemotherapie oder primäre Chirurgie bei kolorektalen Lebermetastasen. Pro neoadjuvante Chemotherapie.

Medicinal treatment of colorectal cancer liver metastases has undergone significant developments in the past two decades. Cytotoxic treatment regimens have demonstrated greater efficacy and contributed to a significant improvement in survival. Perioperative therapy with FOLFOX has demonstrated progression-free survival benefits in resectable colorectal liver metastases in a prospective randomized controlled trial. The safety of perioperative chemotherapy was found to be acceptable and only a few patients showed initial progression. In general, preoperative chemotherapy should not extend beyond 3-4 months to avoid chemotherapy-associated liver damage. For patients with borderline resectable metastases, a response to chemotherapy can establish secondary resectability. Combinations of cytotoxic drugs are adequate in this situation. The addition of bevacizumab or anti-epidermal growth factor receptor (EGFR) antibodies for tumors without RAS mutations are reasonable choices for a better response and improved survival outcome. It is crucial that all treatment decisions for systemic colorectal liver metastases include correct definitions of treatment goals and endpoints and are derived based on appropriate multidisciplinary considerations.

The effects of focused attention training on the duration of novice drivers' glances inside the vehicle.

Several studies have documented that the failure of drivers to attend to the forward roadway for a period lasting longer than 2-3 s is a major cause of highway crashes. Moreover, several studies have demonstrated that novice drivers are more likely to glance away from the roadway than the experienced drivers for extended periods when attempting to do a task inside the vehicle. The present study examines the efficacy of a PC-based training programme (FOrward Concentration and Attention Learning, FOCAL) designed to teach novice drivers not to glance away forthese extended periods of time. A FOCAL-trained group was compared with a placebo-trained group in an on-road test, and the FOCAL-trained group made significantly fewer glances away from the roadway that were more than 2 s than the placebo-trained group. Other measures indicated an advantage for the FOCAL-trained group as well. Statement of relevance: Distracted driving is increasingly a problem, as cell phones, navigation systems, and other in-vehicle devices are introduced into the cabin of the automobile. A training programme is described that has beentested on the open road and can reduce the behaviours that lead to crashes caused by the distracted driving.

Evaluation of different speech and touch interfaces to in-vehicle music retrieval systems.

In-vehicle music retrieval systems are becoming more and more popular. Previous studies have shown that they pose a real hazard to drivers when the interface is a tactile one which requires multiple entries and a combination of manual control and visual feedback. Voice interfaces exist as an alternative. Such interfaces can require either multiple or single conversational turns. In this study, each of 17 participants between the ages of 18 and 30 years old was asked to use three different music retrieval systems (one with a multiple entry touch interface, the iPod, one with a multiple turn voice interface, interface B, and one with a single turn voice interface, interface C) while driving through a virtual world. Measures of secondary task performance, eye behavior, vehicle control, and workload were recorded. When compared with the touch interface, the voice interfaces reduced the total time drivers spent with their eyes off the forward roadway, especially in prolonged glances, as well as both the total number of glances away from the forward roadway and the perceived workload. Furthermore, when compared with driving without a secondary task, both voice interfaces did not significantly impact hazard anticipation, the frequency of long glances away from the forward roadway, or vehicle control. The multiple turn voice interface (B) significantly increased both the time it took drivers to complete the task and the workload. The implications for interface design and safety are discussed.

Can younger drivers be trained to scan for information that will reduce their risk in roadway traffic scenarios that are hard to identify as hazardous?

Younger drivers (18-21 years) are over-involved in crashes. Research suggests that one of the reasons for this over-involvement is their failure to scan areas of the roadway for information about potential risks in situations that are hazardous, but not obviously so. The primary objective of the present study is to develop and evaluate a training program that addresses this failure. It was hypothesised that PC-based hazard anticipation training would increase the likelihood that younger drivers would scan for potential hazards on the open road. In order to test this hypothesis, 12 trained and 12 untrained drivers' eye movements were measured as they drove a vehicle on local residential, feeder and arterial roads. Overall, the trained drivers were significantly more likely to gaze at areas of the roadway that contained information relevant to the reduction of risks (64.4%) than were the untrained drivers (37.4%). Significant training effects were observed even in situations on the road that were quite different from those shown in training. These findings have clear implications for the type of training of teen drivers that is necessary in order to increase their anticipation of hazards.