Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin.

Three macromolecular crystallography (MX) beamlines at the Helmholtz-Zentrum Berlin (HZB) are available for the regional, national and international structural biology user community. The state-of-the-art synchrotron beamlines for MX BL14.1, BL14.2 and BL14.3 are located within the low-ß section of the BESSY II electron storage ring. All beamlines are fed from a superconducting 7 T wavelength-shifter insertion device. BL14.1 and BL14.2 are energy tunable in the range 5-16 keV, while BL14.3 is a fixed-energy side station operated at 13.8 keV. All three beamlines are equipped with CCD detectors. BL14.1 and BL14.2 are in regular user operation providing about 200 beam days per year and about 600 user shifts to approximately 50 research groups across Europe. BL14.3 has initially been used as a test facility and was brought into regular user mode operation during the year 2010. BL14.1 has recently been upgraded with a microdiffractometer including a mini-κ goniometer and an automated sample changer. Additional user facilities include office space adjacent to the beamlines, a sample preparation laboratory, a biology laboratory (safety level 1) and high-end computing resources. In this article the instrumentation of the beamlines is described, and a summary of the experimental possibilities of the beamlines and the provided ancillary equipment for the user community is given.

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin.

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results. Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY  II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input. Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Vacuum decay container/closure integrity testing technology. Part 1. ASTM F2338-09 precision and bias studies.

ASTM F2338-09 Standard Test Method for Nondestructive Detection of Leaks in Packages by Vacuum Decay Method is applicable for leak-testing rigid and semi-rigid non-lidded trays; trays or cups sealed with porous barrier lidding materials; rigid, nonporous packages; and flexible, nonporous packages. Part 1 of this series describes the precision and bias studies performed in 2008 to expand this method's scope to include rigid, nonporous packages completely or partially filled with liquid. Round robin tests using three VeriPac 325/LV vacuum decay leak testers (Packaging Technologies & Inspection, LLC, Tuckahoe, NY) were performed at three test sites. Test packages were 1-mL glass syringes. Positive controls had laser-drilled holes in the barrel ranging from about 5 to 15 microm in nominal diameter. Two different leak tests methods were performed at each site: a "gas leak test" performed at 250 mbar (absolute) and a "liquid leak test" performed at about 1 mbar (absolute). The gas leak test was used to test empty, air-filled syringes. All defects with holes > or = 5.0 microm and all no-defect controls were correctly identified. The only false negative result was attributed to a single syringe with a < 5.0-microm hole. Tests performed using a calibrated air leak supported a 0.10-cm3 x min(-1) (ccm) sensitivity limit (99/99 lower tolerance limit). The liquid leak test was used to test both empty, air-filled syringes and water-filled syringes. Test results were 100% accurate for all empty and water-filled syringes, both without holes and with holes (5, 10, and 15 microm). Tests performed using calibrated air flow leaks of 0, 0.05, and 0.10 ccm were also 100% accurate; data supported a 0.10-ccm sensitivity limit (99/99 lower tolerance limit). Quantitative differential pressure results strongly correlated to hole size using either liquid or gas vacuum decay leak tests. The higher vacuum liquid leak test gave noticeably higher pressure readings when water was present in the defect. Both the ASTM F2338-09 test method and the precision and bias study report are available by contacting ASTM International in West Conshohocken, PA, USA (www.astm.org).

Vacuum decay container/closure integrity testing technology. Part 2. Comparison to dye ingress tests.

Part 1 of this series demonstrated that a container closure integrity test performed according to ASTM F2338-09 Standard Test Method for Nondestructive Detection of Leaks in Packages by Vacuum Decay Method using a VeriPac 325/LV vacuum decay leak tester by Packaging Technologies & Inspection, LLC (PTI) is capable of detecting leaks > or = 5.0 microm (nominal diameter) in rigid, nonporous package systems, such as prefilled glass syringes. The current study compared USP, Ph.Eur. and ISO dye ingress integrity test methods to PTI's vacuum decay technology for the detection of these same 5-, 10-, and 15-microm laser-drilled hole defects in 1-mL glass prefilled syringes. The study was performed at three test sites using several inspectors and a variety of inspection conditions. No standard dye ingress method was found to reliably identify all holed syringes. Modifications to these standard dye tests' challenge conditions increased the potential for dye ingress, and adjustments to the visual inspection environment improved dye ingress detection. However, the risk of false positive test results with dye ingress tests remained. In contrast, the nondestructive vacuum decay leak test method reliably identified syringes with holes > or = 5.0 microm.

Kinetic Modeling of the Release of Ethylene Oxide from Sterilized Plastic Containers and its Interaction with Monoclonal Antibodies.

Ethylene oxide (ETO) is commonly used to sterilize plastic containers, but the effects of residual amounts left after sterilization on protein therapeutics are still not well understood. Here we focus primarily on the factors that influence concentrations of ETO migrating from ETO-treated plastic containers into aqueous solution. A study was designed to investigate the kinetics of this process at various temperatures, and the kinetic data could be fit with a model based on a combination of Fickean diffusion and first-order chemical reaction (to account for observed hydrolysis of ETO). The diffusion and reaction rate constants thus obtained obey Arrhenius-like temperature dependence. These results indicate that for analytical methods involving extraction into water, measurements of residual ETO in a container must account for the effects of ETO hydrolysis. Further, the effects of salt concentration and pH of the fluid in the container on accumulated ETO levels were explored. Finally, interactions of ETO with anti-streptavidin (AntiSA) Immunoglobulin G1 (IgG1) and IgG2 antibodies were studied, with ETO adducts found on all methionine residues when incubated in solutions spiked with ETO at concentrations that could be reached (based on the kinetic studies) in ETO-treated plastic vials. Overall, the likelihood of observable ETO-protein modifications upon storage in ETO-sterilized containers will depend on a complex interplay of protein properties, formulation details, storage conditions, and amount of residual ETO initially in the container. LAY ABSTRACT: Ethylene oxide (ETO) is commonly used to sterilize plastic containers, but the effects of residual amounts left after sterilization on protein therapeutics are still not well understood. Here we describe experiments exploring the factors that influence concentrations of ETO migrating from ETO-treated plastic containers into aqueous solution over time. Additionally, interactions of ETO with model antibodies were studied, with ETO adducts found on all methionine residues when incubated in solutions spiked with ETO at concentrations that could potentially be reached in ETO-treated plastic vials. Overall, the likelihood of observable ETO-protein modifications upon storage in ETO-sterilized containers will depend on a complex interplay of protein properties, formulation details, storage conditions, and amount of residual ETO initially in the container.

Creating a Holistic Extractables and Leachables (E&L) Program for Biotechnology Products.

UNLABELLED: The risk mitigation of extractables and leachables presents significant challenges to regulators and drug manufacturers with respect to the development, as well as the lifecycle management, of drug products. A holistic program is proposed, using a science- and risk-based strategy for testing extractables and leachables from primary containers, drug delivery devices, and single-use systems for the manufacture of biotechnology products. The strategy adopts the principles and concepts from ICH Q9 and ICH Q8(R2). The strategy is phase-appropriate, progressing from extractables testing for material screening/selection/qualification through leachables testing of final products. The strategy is designed primarily to ensure patient safety and product quality of biotechnology products. The holistic program requires robust extraction studies using model solvents, with careful consideration of solvation effect, pH, ionic strength, temperature, and product-contact surface and duration. From a wide variety of process- and product-contact materials, such extraction studies have identified and quantified over 200 organic extractable compounds. The most commonly observed compounds were siloxanes, fatty acid amides, and methacrylates. Toxicology assessments were conducted on these compounds using risk-based decision analysis. Parenteral permitted daily exposure limits were derived, as appropriate, for the majority of these compounds. Analysis of the derived parenteral permitted daily exposure limits helped to establish action thresholds to target high-risk leachables in drug products on stability until expiry. Action thresholds serve to trigger quality investigations to determine potential product impact. The holistic program also evaluates the potential risk for immunogenicity. This approach for primary drug containers and delivery devices is also applicable to single-use systems when justified with a historical knowledge base and understanding of the manufacturing processes of biotechnology products. LAY ABSTRACT: In the development of a drug product, careful consideration is given to impurities that may originate from manufacturing equipment, process components, and packaging materials. The majority of such impurities are common chemical additives used to improve the physicochemical properties of a wide range of plastic materials. Suppliers and drug manufacturers conduct studies to extract chemical additives from the plastic materials in order to screen and predict those that may leach into a drug product. In this context, the term extractables refers to a profile of extracted compounds observed in studies under harsh conditions. In contrast, the term leachables refers to those impurities that leach from the materials under real-use conditions and may be present in final drug products. The purpose of this article is to present a holistic approach that effectively minimizes the risk of leachables to patient safety and product quality.