DE102007063440B4 - Screening system for the implementation and direct analysis of biological, biochemical and chemical synthesis and reaction reactions - Google Patents

Abstract

screening system
full
an autosampler (2) of an analysis device (1) with computer-assisted evaluation and a reaction vessel arrangement (3) adapted to the size of the functional area of the autosampler (2) and arranged within the functional area,
characterized in that
the reaction vessel arrangement (3) has a reaction vessel carrier (31) with reaction vessel locations (33) and reaction vessels (4) held on the latter,
in that a magnetic stirrer (32) designed as a multi-stirrer system is arranged below the reaction vessel carrier (31) and has a magnetic stirrer drive (34) for each reaction vessel parking space (33),
wherein the reaction vessels (4) have a closure (41) with gas-permeable or gas-impermeable covers (42, 43).

Description

object The patent is a screening system for direct implementation and Analysis of aerobic and anaerobic biological, biochemical and chemical synthesis and reaction reactions. By linking a stirred multiple reaction system with a tempering unit became a novel independent screening system created that directly with a sample name and dosing system an analysis system such as GC or HPLC coupled and by a computer-aided evaluation added can be. This system works without additional robotic and pipetting systems or other refurbishment and evaluation lines in one Microtiter plates based on specially adapted tube system.

The Biotechnology has become more important in recent years in the development of active pharmaceutical ingredients, the implementation of biocatalytic synthesis of substances and degradation of pollutants. For the development These processes require systems in which high levels of Number of samples Experiments performed and can be analyzed and evaluated in a timely manner. It makes sense the possibility the recording of a reaction kinetics by a multiple sampling schedule in the course of the reaction.

at the implementation of biological, biochemical and chemical synthesis and reaction reactions The analysis is usually by gas chromatography (GC) and high performance liquid chromatography (HPLC) performed.

The screening systems, which are currently in development and are available on the market, have reaction tubes which are placed on microtiter plates ( WO 2005 107931 A1 ), Serial cultivation of microorganisms with dosage in shake flask batteries ( US Pat. No. 6,202,713 B1 ) or micro-fertilizer batteries (Dasgip, Sartorius / Braun). In these reactors, the experiments take place, which are measured analytically depending on the structure during the reaction and at the end. The sampling and processing is carried out manually via pumps and hose systems (eg multi-channel sampling and analysis system for the analysis of fluids, DE 197 58 356 A1 ) and via a diffusion membrane and pump (methods and apparatus for determining analyte concentrations WO 01 90718 A1 and WO 01 90720 A2 ), by means of dialysis hoses ( WO 99 02961 A1 ) or via pipetting robots ( DE 102 60 691 A1 ), Some parameters such as pH, O ₂ or CO ₂ can be determined directly (minerizer, shake flask batteries). Direct data acquisition without intermediate stations via substance detection with GC or HPLC is not possible with these systems. There are always intermediate steps for sample preparation and cleaning necessary. This is the reaction systems with shake flasks and fermenters in the dimensions, which can not be approached by a sampling system and in the reaction systems with microtiter plates is because of their low reaction volume (<1 ml) and the high shaking frequency (> 300 U / min) during the Reaction No interruption-free sampling and automated recording of reaction kinetics possible.

In the analytical measuring methods, developments for high-throughput screening and substance analyzes ( WO 2005 108597 A2 ) and for high-throughput chromatography systems ( WO 01 88528 A2 ) made. Alternatively, optical methods have also been carried out with substrates which allow a color reaction (optical sensor, DE 101 01 576 A1 ),

Out WO 01 59429 A1 For example, a screening system is known which represents a flow cytometry arrangement. Between the autosampler and analyzer, a pump is installed, which is used to transport the sample to be analyzed.

In US Pat. No. 6,265,226 B1 an analysis unit for the characterization of polymers by gel permeation chromatography is described. The system includes a measuring unit with sample vessels that are not suitable as reaction vessels.

It Two major variants have been developed in biological screening systems Service. The one variant is usually upgraded by robotics Microtiter plate based screening technique developed by the experimental set up to the sampling and processing completely over Robotic systems led becomes. These usually very costly systems reach through the high degree of automation and the replacement of human labor high throughputs, but only for Major research institutions and large farms are affordable. The second variant is the miniaturized fermenter. In these systems, a fermenter or shake flask is tried with his instrumentalization to a miniature scale, one as possible size Number to use at the same time. The data obtained here serve the fermentation development and optimization. A direct coupling a screening system with an analysis system and thus a direct Analysis in the reaction approach and course is with the previous Screening systems not possible.

The object of the invention is therefore a miniaturized screening system for aerobic and anaerobic biological and chemical reactions, with directly suitable for the analysis sterilizable sample vessels and a temperature for the reaction mixtures that for the direct analysis of the Gas or liquid phase of the reaction mixtures can be used and connected to a computer-controlled data acquisition, offer.

The solution The object is achieved with the features of claim 1.

advantageous Further developments are specified in the subclaims.

So includes the screening system of the invention an autosampler of an analyzer with computer-aided evaluation and one to the size of the functional area adapted to the autosampler and arranged within the functional area Reaction vessel arrangement, the reaction vessel arrangement a reaction vessel carrier with Reaction vessel parking spaces and having on this supported reaction vessels, below the reaction vessel carrier designed as a multi-stirring system magnetic is arranged for each Reaction vessel Pitch a magnetic stirrer drive has and the reaction vessels a Closure with gas-permeable or gas-impermeable covers.

A advantageous development is characterized in that for aerobic Try passive ventilation through a gas-permeable Cover made of sterilizable materials such as porous silicone and for anaerobic experiments the gas-impermeable cover of sterilizable Materials such as nonporous Silicone, Teflon or rubber exist.

In a further embodiment is in the gas-tight cover overpressure capillary, which is in communication with the environment and the reaction space, arranged.

A Another advantageous embodiment is characterized in that the reaction vessel carrier as temperable block or is designed as a water bath.

Base of the screening system are reaction tubes which are individually in the system find use and sterilized and with media, starting materials, microorganisms and enzymes can be. These vessels typically have one body made of glass, a lid and an insert in the lid to the closure of the vessel. According to the invention special in the lid the reaction sequence suitable materials used. That's ever after process flow:

1. Aerobic reactions

Around the reaction tubes for the To be able to use analytics must typically be an insert in the lid, with which the Close the containers airtight. For aerobic Processes, however, a gas exchange is necessary. To a contamination in biological processes and excessive evaporation of the reaction liquid To avoid, a shielding of the vessels is necessary. The typical used in microbiology piston seal with cotton plug was for This system is unsuitable because a sampling by means of sampling syringe through the cotton plug because of the high resistance is not possible. Surprisingly proved tailored to the lid size porous Sterilizable silicone as a suitable closure agent, both allows sufficient gas exchange as well as well from needle the sampling syringe is pierceable. This closure guarantees despite the gas exchange, a stable gas phase equilibrium, whereby reproducible measurements in the gas and liquid phase of the tubes can be performed.

2. Anaerobic reactions

For the use of the screening system for anaerobic processes, a tight closure and avoidance of overpressure by gas formation in processes such as CO ₂ formation in the biological fermentation had to be ensured. A tight seal, which is also easy to pierce with the needles of the sampling syringes, can be ensured by a closure with silicone. However, escape of overpressure during gas formation is not possible. By installing a short Überdruckkapillare at the edge of the septum, a pressure equalization could be created, which avoids the emergence of higher pressures in the reaction vessel but allows only a negligible entry of air through the capillary. Since gas formation usually occurs in anaerobic biological systems, the overpressure capillary is mainly used for pressure equalization from the inside to the outside.

These Reaction vessels are with multiple stirring systems connected, which is a uniform mixing allow a high number of samples in a tight arrangement and ensure ventilation over the surface during aerobic processes.

This Mixing System is provided with a customized temperature control unit, which is connected to the stirring Holes for contains the reaction vessels and means Circulation thermostat or Peltier temperature control in a temperature range be tempered from 0-100 ° C. IENN. The temperature control unit is separated from the stirrer by an insulating layer. The described components have become a compact screening system together.

This screening system can by his Compact design can be mounted directly under autosampler for chromatography and thus be used for a direct measurement in the reaction mixtures. Due to the high flexibility of modern autosamplers, the option of automatic software-controlled metering of fluids through the autosampler into the individual experimental approaches during the course of the experiment. As a result, a substrate or Cosubstratfeeding is possible. For even greater integration of the screening system with the autosampler, it is also possible to use a magnetically tempered tray mounted directly on the sampler. By integrating the screening system via software, the control of the screening system and evaluation of the results is carried out directly via a computer.

The Chromatography system (eg a gas chromatograph), is with a Connected autosampler. The sampler of the autosampler is in three directions movable and freely programmable in its movement. Below the Autosamplers becomes the screening system instead of the usual sample storage Installed. The screening system according to the invention can be used with commercially available multiple stirring systems as well as being equipped with modified special systems. With The sampler and injection needle of the autosampler can be placed in programmable time intervals samples are drawn from the reaction vessels be and abandoned in the injection valve of the gas chromatograph and analyzed.

Innovative in this system, the utility for aerobic and anaerobic reaction approaches is the evolution a temperature-responsive reaction system for temperature ranges from O-100 ° C with direct for the Analytics in gas chromatography usable reaction vessels (sample quantities 1-20 ml). The reaction vessels are sterilizable and therefore for the Cultivation of microorganisms and cell cultures usable. As well important is the development of a special analytics, which is the direct Sampling in the running process from the reaction mixtures allows. Thus, in a miniaturized reaction approach a complete Reaction kinetics are included. Through the continuous analytics the product implementation can With this system faster processes can be developed. Applicable is this system in the areas of the development of biocatalysis, in the study of metabolic processes (metabolomics), in detection of biodegradation processes, in the pursuit of chemical reactions and in the biochemical Diagnostics.

The Invention is based on embodiments and drawings closer explained.

It demonstrate

1 a schematic representation of the screening system with a GC autosampler,

2a in a perspective schematic representation of a reaction vessel arrangement,

2 B a schematic cross section of the reaction vessel arrangement with thermoblock,

2c a schematic cross section of the reaction vessel arrangement with water bath,

2d a schematic plan view of the reaction vessel arrangement,

3a a reaction vessel for aerobic reaction,

3b a reaction vessel for anaerobic reaction,

4 a representation of an SPME and headspace measurement with acetophenone (AP) and phenylethanol (PE),

5 a representation of the growth of E. coli in HS comparison silicone and cotton plugs,

6 a representation of the B. subtilis growth in GC-HS tubes as a function of the stirring frequency,

7 a representation of the reduction of a ketone (5-fold in 20 ml tubes),

8th Screening of various yeasts for the reduction of a ketone and

9 a representation of the optimization of the reduction of a ketone with yeast.

In 1 is a schematic sectional view of an inventive screening system in combination with an autosampler 2 a gas chromatograph 1 shown. Below a guide 22 the autosampler 2 is a reaction vessel arrangement 3 appropriate. In the reaction vessel arrangement 3 There is a large number of reaction vessels 4 , One on the lead 22 movable injection needle 21 can now be software-controlled in the reaction vessels 4 be introduced. One from the injection needle 21 recorded sample is then via an injection valve 11 of the gas chromatograph 1 fed to the analysis. The analysis of the analysis results is done via a computer not shown here.

In 2a is the reaction vessel arrangement 3 shown schematically in perspective. In a reaction vessel carrier 31 the reaction vessel arrangement 3 are a variety of reaction vessel sites 33 incorporated. An underlying magnetic stirrer 32 is designed so that every single reaction vessel 4 to stir is able.

2 B shows a cross section through the reaction vessel assembly 3 with the magnetic stirrer 32 and the usable as a thermoblock reaction vessel carrier 31 , In the reaction vessel carrier 31 incorporated reaction vessel parking spaces 33 are the reaction vessels 4 , Directly below the reaction vessels 4 , integrated in the magnetic stirrer 32 , are magnetic stirrer drives 34 arranged, which in function magnetic stirring bars 35 in the reaction vessels 4 move.

2c shows a cross section through the reaction vessel assembly 3 with the magnetic stirrer 32 and in a water bath 36 temperature-adjustable reaction vessel carrier 31 ,

2d shows the top view of a reaction vessel assembly 3 , In the reaction vessel carrier 31 are 60 reaction vessels 4 arranged at regular intervals, leaving an autosimpler 2 an analyzer 1 fully automatic sampling at any time of a reaction process can make. A power connection 37 provides the required energy supply.

3a shows in schematic section a reaction vessel 4 for carrying out an aerobic reaction. The reaction vessel 4 has a closure 41 on. The closure 41 has a porous cover 42 , As a result, gas exchange with the environment is possible for aerobic reaction systems. The reaction vessels typically have a diameter of 10 to 30 mm and a height of 20 to 100 mm.

The reaction vessel 4 in the 3b is particularly suitable for anaerobic reaction processes. The closure 41 has a gas-tight cover 43 , One in the gas-tight cover 43 arranged overpressure capillary 44 ensures that the pressure conditions in the reaction vessel 4 corresponds to the normal pressure.

As a porous cover 42 Expediently, porous silicone is used. As a gas-tight cover 43 is used pierceable material made of silicone, rubber or Teflon.

Examples

example 1

Headspace and SPME analytics in the screening system

basis for the Screening system is the application of modern analytics such as the SPME technique, a measurement of even low sample concentrations from the gas phase or the liquid Approach allows.

With The headspace and the SPME system were measured for analysis of substances and implemented in biotransformation systems. she showed a good reproducibility and allowed a quantitative Evaluation of the implementation. It also showed the much higher sensitivity of the SPME system.

4 shows the much higher sensitivity of the SPME analysis compared to the normal headspace analysis. Acetophenone gave an 85-fold stronger signal and phenyethanol a 480-fold stronger signal. In the case of phenylethanol, this high amplification could be achieved by an alcohol-selective SPME material. This shows the importance of adapting the SPME measuring system to the analytes.

Example 2

Growth of microorganisms in the developed screening system

to Evaluation of the applicability of the reaction tubes were various Basic parameters for studied the biotransformation. Solutions were developed for various applications, which allow a stable use of the system.

In the reaction tube various gas-permeable plug were used and their usability tested by growth experiments with microorganisms. Furthermore, various magnetic stirrers were tested and determined by comparative experiments with different levels, the optimum filling level in aerobic experiments ( 5 ), By experiments with different stirring speeds, the optimal stirring frequency for aerobic experiments could be determined ( 6 ),

In the 5 shown test series with assessment of the microorganism growth of E. coli in batches with 5, 10 and 15 ml filling the 20 ml reaction tubes was performed with two different gas-permeable plug (cotton plug, porous silicone plug). It was found, that the best oxygen supply is given at a level of 5 ml. The gas-permeable cotton and porous silicone stoppers were both equally usable.

Example 3

Biotransformations in the developed screening system

at The biotransformation are produced by chemical syntheses Starting materials by means of biochemical reactions with biocatalysts (Enzymes, microorganisms, plant and animal cells) in one Target substance converted. In the evaluation of these biotransformations is a specific analysis for the detection of the implementation important. The screening of substance conversions with a quick result detection is always a bottleneck in the development of biotransformations.

An example of biotransformation is the reduction of prochiral keto compounds to chiral alcohols (see 7 ),

in the The scope of work to date has become an application-specific one Screening system developed, which allows in small test approaches a large number to investigate different reactions and get fast results over the reaction courses to deliver. The reaction vessels are sterilizable and therefore also for biological processes usable.

Example 4

execution screening series on biotransformation

In the developed and constructed screening system, series of biotransformations can be performed, analyzed and evaluated. Both screening tests for the reaction of certain substances with different biocatalysts ( 8th ), as well as optimization series for certain biocatalysts with certain substances ( 9 ) be performed. By using the miniaturized system, a large number of approaches could be prepared and investigated from a small amount of microorganisms and small amounts of test substances.

through Gas chromatography with chiral separation column can measure the experiments directly and be evaluated. Thus, a quick evaluation of the experiments possible.

The 8th and 9 show typical screening and optimization series. In 8th the comparison of 11 yeasts for the reduction of a ketone is shown. In 9 are the results of the variation of parameters (yeast concentration and feeding) for the reduction of a ketone with yeast 1 shown.

LIST OF REFERENCE NUMBERS

1

analyzer

11

injection valve

2

autosampler

21

injection needle

22

guide

3

Reaction vessel arrangement

31

Reaction vessel carrier

32

magnetic

33

Reaction vessel Pitch

34

Magnetic drive

35

magnetic

36

water bath

37

power connection

4

reaction vessel

41

shutter

42

porous cover

43

density cover

44

Überdruckkapillare

Claims (4)

Screening system comprising an autosampler ( 2 ) of an analyzer ( 1 ) with computer-aided evaluation and a size of the functional area of the autosampler ( 2 ) and arranged within the functional range reaction vessel arrangement ( 3 ), characterized in that the reaction vessel arrangement ( 3 ) a reaction vessel carrier ( 31 ) with reaction vessel locations ( 33 ) and on these supported reaction vessels ( 4 ), that below the reaction vessel carrier ( 31 ) a magnetic stirring device designed as a multiple stirring system ( 32 ) arranged for each reaction vessel ( 33 ) a magnetic stirrer drive ( 34 ), wherein the reaction vessels ( 4 ) a closure ( 41 ) with gas-permeable or gas-impermeable covers ( 42 . 43 ) exhibit. Screening system according to claim 1, characterized in that for aerobic experiments a passive ventilation by a gas-permeable cover ( 42 ) made of sterilizable materials such as porous silicone and for anaerobic experiments the gas-impermeable cover ( 43 ) made of sterilizable materials such as nonporous silicone, Teflon or rubber. Screening system according to claim 1 or 2, characterized in that in the gas-tight cover ( 43 ) an overpressure capillary ( 44 ), which with the Environment and the reaction space communicates, is arranged. Screening system according to one of claims 1 to 3, characterized in that the reaction vessel carrier ( 31 ) as a temperable block or as a water bath ( 36 ) is trained.

Images