Detergent-Mediated Virus Inactivation in Biotechnological Matrices: More than Just CMC.

For decades, the ability of detergents to solubilize biological membranes has been utilized in biotechnological manufacturing to disrupt the lipid envelope of potentially contaminating viruses and thus enhance the safety margins of plasma- and cell-derived drugs. This ability has been linked to detergent micelles, which are formed if the concentration of detergent molecules exceeds the critical micelle concentration (CMC). Traditionally, the CMC of detergents is determined in deionized water (ddH2O), i.e., a situation considerably different from the actual situation of biotechnological manufacturing. This study compared, for five distinct detergents, the CMC in ddH2O side-by-side with two biopharmaceutical process intermediates relevant to plasma-derived (Immunoglobulin) and cell-derived (monoclonal antibody) products, respectively. Depending on the matrix, the CMC of detergents changed by a factor of up to ~4-fold. Further, the CMC in biotechnological matrices did not correlate with antiviral potency, as Triton X-100 (TX-100) and similar detergents had comparatively higher CMCs than polysorbate-based detergents, which are known to be less potent in terms of virus inactivation. Finally, it was demonstrated that TX-100 and similar detergents also have virus-inactivating properties if applied below the CMC. Thus, the presence of detergent micelles might not be an absolute prerequisite for the disruption of virus envelopes.

Synthesis of "Nereid," a new phenol-free detergent to replace Triton X-100 in virus inactivation.

In the 1980s, virus inactivation steps were implemented into the manufacturing of biopharmaceuticals in response to earlier unforeseen virus transmissions. The most effective inactivation process for lipid-enveloped viruses is the treatment by a combination of detergents, often including Triton X-100 (TX-100). Based on recent environmental concerns, the use of TX-100 in Europe will be ultimately banned, which forces the pharmaceutical industry, among others, to switch to an environmentally friendly alternative detergent with fully equivalent virus inactivation performance such as TX-100. In this study, a structure-activity relationship study was conducted that ultimately led to the synthesis of several new detergents. One of them, named "Nereid," displayed inactivation activity fully equivalent to TX-100. The synthesis of this replacement candidate has been optimized to allow for the production of several kg of detergent at lab scale, to enable the required feasibility and comparison virus inactivation studies needed to support a potential future transition. The 3-step, chromatography-free synthesis process described herein uses inexpensive starting materials, has a robust and simple work-up, and allows production in a standard organic laboratory to deliver batches of several hundred grams with >99% purity.

Synthesis of a Pentasaccharide Fragment Related to the Inner Core Region of Rhizobial and Agrobacterial Lipopolysaccharides.

The pentasaccharide fragment α-d-Man-(1 → 5)-[α-d-Kdo-(2 → 4)-]α-d-Kdo-(2 → 6)-ß-d-GlcNAc-(1 → 6)-α-d-GlcNAc equipped with a 3-aminopropyl spacer moiety was prepared by a sequential assembly of monosaccharide building blocks. The glucosamine disaccharide-as a backbone surrogate of the bacterial lipid A region-was synthesized using an 1,3-oxazoline donor, which was followed by coupling with an isopropylidene-protected Kdo-fluoride donor to afford a protected tetrasaccharide intermediate. Eventually, an orthogonally protected manno-configured trichloroacetimidate donor was used to achieve the sterically demanding glycosylation of the 5-OH group of Kdo in good yield. The resulting pentasaccharide is suitably protected for further chain elongation at positions 3, 4, and 6 of the terminal mannose. Global deprotection afforded the target pentasaccharide to be used for the conversion into neoglycoconjugates and "clickable" ligands.

"Hypermethylation" of anthranilic acid-labeled sugars confers the selectivity required for liquid chromatography-mass spectrometry.

Analysis of the monosaccharides of complex carbohydrates is often performed by liquid chromatography with fluorescence detection. Unfortunately, methylated sugars, unusual amino- or deoxysugars and incomplete hydrolysis can lead to erroneous assignments of peaks. Here, we demonstrate that a volatile buffer system is suitable for the separation of anthranilic acid labeled sugars. It allows off-line examination of peaks by electrospray mass spectrometry. Approaches towards on-line mass spectrometric detection using reversed-phase or porous graphitic carbon columns fell short of achieving sufficient separation of the relevant isobaric sugars. Adequate chromatographic performance for isomeric sugars was achieved with reversed-phase chromatography of "hyper"-methylated anthranilic acid-labeled monosaccharides. Deuteromethyl iodide facilitates the discovery of naturally methylated sugars and identification of their parent monosaccharide as demonstrated with N-glycans of the snail Achatina fulica, where two thirds of the galactoses and a quarter of the mannoses were methylated.

Towards robotic laboratory automation Plug & Play: The "LAPP" framework.

Increasing the level of automation in pharmaceutical laboratories and production facilities plays a crucial role in delivering medicine to patients. However, the particular requirements of this field make it challenging to adapt cutting-edge technologies present in other industries. This article provides an overview of relevant approaches and how they can be utilized in the pharmaceutical industry, especially in development laboratories. Recent advancements include the application of flexible mobile manipulators capable of handling complex tasks. However, integrating devices from many different vendors into an end-to-end automation system is complicated due to the diversity of interfaces. Therefore, various approaches for standardization are considered in this article, and a concept is proposed for taking them a step further. This concept enables a mobile manipulator with a vision system to "learn" the pose of each device and - utilizing a barcode - fetch interface information from a universal cloud database. This information includes control and communication protocol definitions and a representation of robot actions needed to operate the device. In order to define the movements in relation to the device, devices have to feature - besides the barcode - a fiducial marker as standard. The concept will be elaborated following appropriate research activities in follow-up papers.

Determination of modification degree of polysialylated therapeutic proteins using 1H-NMR spectroscopy.

Water soluble polymers and their derivatives bound to proteins can dramatically favor the biological activity of new drugs and vaccines. Quantification of the modification degree of the protein is crucial during the development and licensing phase and later in order to monitor the industrial production process and to match product specification. In this work, we describe an innovative way to measure directly the modification degree of polysialylated proteins using proton NMR (Nuclear Magnetic Resonance) spectroscopy. Following a calibration step, the modification degree can be easily deduced by the integration ratio of a separate signal from the polymer and selected signals from the protein. In fact, the upfield-shifted signals of methyl groups from Valine, Leucine and Isoleucine can be used as an internal calibration reference for the integration. In this paper recombinant factor VIII (rFVIII) and recombinant factor IX (rFIX) proteins modified by polysialic acid (PSA) are used to illustrate the accuracy, reproducibility and ease of the method that may replace or complement wet-chemistry approaches.

A palladium-catalyzed carbo-oxygenation: the bielschowskysin case.

An asymmetric synthesis of an advanced tetracyclic intermediate toward the synthesis of bielschowskysin (1) is described. A biomimetic [2 + 2]-photocyclization was used to establish the cyclobutane core of bielschowskysin. Macrocyclization under Heck conditions led to an unprecedented carbo-oxygenation of a 1,1-disubstituted double bond.

A non-photochemical approach to the bicyclo[3.2.0]heptane core of bielschowskysin.

An asymmetric synthesis of the tricyclic core (-)-1 of the marine diterpene bielschowskysin is described. In particular, a methodology was developed to introduce the crucial quaternary center at C-12.