Laboratory work: A foundation in discovery

For any drug discovery project the initial steps are born and nurtured in laboratory at the lowest possible scale. Dr Girish Mahajan, Assistant Director, Prokaryote and Marine Technology, Department of Natural Products, Piramal Enterprises gives an insight In the past decade, drug discovery and development science has evolved into a discrete twig of life-sciences. It […]

For any drug discovery project the initial steps are born and nurtured in laboratory at the lowest possible scale. Dr Girish Mahajan, Assistant Director, Prokaryote and Marine Technology, Department of Natural Products, Piramal Enterprises gives an insight

Dr Girish Mahajan

In the past decade, drug discovery and development science has evolved into a discrete twig of life-sciences. It is highly multidisciplinary including the disciplines of chemistry, highly networked branches of biology, computation technologies and engineering. It is unique twig among its tangential major sciences as its focus is to deliver a drug to address the unwell strata on the globe.

Scientists and managers in a drug discovery environment must, therefore, be able to work in multidisciplinary teams and must be able to communicate their specialist knowledge to scientists in other authorities. Generally the major stages involved in drug discovery are, to freeze disease area followed by Target Identification and ‘Validation’, Assay Development and Lead Generation-lead optimisation. At this stage generally one has a lead which has crossed all the hurdles at in-vitro stages. It is ready for entering into developmental mode. The fate of several bioactive leads is decided at this stage by careful analysis of the data, as further stages are demanding more investment in terms of money and time.

For all these preclinical stages laboratory work is very critical. For any drug discovery project the initial steps are born and nurtured in laboratory at the lowest possible scale. Laboratory work is like childhood to adolescence stage of any project. This work sets foundation for any new proposals. It decides the direction of work.

For general purpose the laboratory work is explained in different ways. E.g. at http://encyclopedia2.thefreedictionary. com/Laboratory+Work it is explained as “One of the forms of independent practical work for students in higher, specialised secondary, and general schools.

The aim of laboratory work is to deepen and fix theoretical knowledge and to develop the skills of independent experimentation. The work includes preparing the apparatus, equipment, and reagents necessary for an experiment, diagramming and planning the experiment, carrying out the experiment itself and writing a laboratory report. Laboratory work is widely used in teaching the natural sciences and technical disciplines, with the appropriate ratio of theoretical to laboratory work established for each particular field.

Laboratory work is accorded 10–30 per cent of the teaching time in the higher educational institutions and technical schools of the USSR. In certain disciplines, such as chemistry, physics, biology, and general electrical engineering, 60–70 per cent of the time is spent on laboratory work. As a rule, laboratory work ends with a final report on the whole cycle of experiments performed.

There are several such definitions. For every branch and sub-branch of science the position of experimental work in laboratories has always believed a high profile at all levels of studies. The term laboratory work is very wide and encompasses several aspects depending on subject and coverage. However, as representative here, we are considering a tiny representative aspect in drug discovery area. In drug discovery from microbial resources the lab work has additional roles. To understand it more, let us consider the flow of the work.

Resource sample collection

The resource samples (viz. soil, barks, leaves, compost, stone pieces, mud samples, water sample etc.) collected from pre-decided geographical locations are brought into labs and stored under conditions based on type and ecology sample. They are processed using selective methods (chemical /physical) so as to enrich /select desired group of micro-organisms and to suppress the unwanted group of microbes (e.g. use of antifungal compounds to suppress growth of fungi while selecting growth of bacteria). The sample processing step is a semi skilled lab-work. The critical part during this is to avoid addition of external lab microbes in the resource sample. Hence, this step is carried out under aseptic condition. The processed samples are kept under low temperature (2-4ºC) in laboratory domestic refrigerators till used for isolation of microbes.

Isolation of microbes and purification

The media designed to isolate selected group/sub-group of microbes is prepared using dehydrated media ingredients. Primarily this involves use of fine and ultrafine balance, laboratory autoclave, laminar airflow units, micro-ovens etc. The selected nutrient media are poured in sterile disposable petriplates (with dimensions of: 9cm/15cm outer diameter). Glass petriplates with more depth are preferred for isolation of microbes which needs longer period of incubations (=1 month). The processed resource samples are spread on these selective media surface and plates are incubated in inverted conditions at temperatures based on origin of temperatures based on origin of the samples or desired microbial group to be isolated. The incubation may be three days to two months based on type of microbes we are looking.

During all these steps maintenance of hygienic lab condition is must as to keep away native microbes from media. The well isolated colonies of microbes were observed with naked eyes as well as stereo microscope and transferred to purification petriplate (containing same media on which it has grown). They are again incubated under the same conditions and time period. The well grown and pure microbe is maintained as master culture is glycerol containing nutrient media in microvials. These microvials are maintained at -70ºC. The working cultures are maintained on nutrient medium slants.

Shake-flask fermentation

The fermentation is often carried out in conical flasks with capacities ranging from 50 ml to 1000 ml. Using multiple flasks a higher volume shaker batches can be cultivated which could be as high as 40L. All these need a table top shaker water bath, ground fixed gyratory or stroke shakers, multi-stacked shakers, temperature controlled closed shakers or for some micro-algal work light controlled chambers/shakers. Cell mass in any case is separated by table top centrifuges or cartridged filtration units. Based on need either cell mass or cell free-broth is used for further isolation of active principles.

Natural product chemistry

The later part is to isolate bioactive compounds from cell mass or cell free broth using lab analytical tools or advance platforms, which may consist of various HPLC, GC, HPTLC (both analytical and preparative grade) and many column chromatography techniques. Prior to that enrichment of desired compound can be done by simple liquid-liquid partition or extraction of cell mass or cell free broth using solvents of desired polarity. Characterisation of API involves use of various spectroscopic equipments such as UV, MS, IR, NMR (basically proton and carbon). With generation of all such data chemist elucidate the structure of active principle. Through data bases available on web or licensed data bases we decide if the compound is novel or known.

Bioactivity screening of compounds

Based on the disease area and target decided various assays are available in the literature. They need to re-establish to suit the set up and intended aim in our own labs. This demands a range of assay tools including as simple as UV-visible spectrophotometer to as high end as fully automised high through-put screening (HTS) system. All the data generated in this is stored in equipment connected computers and also on servers.

Analysis and documentation

The critical part of laboratory work is analysis of data using various software’s available on web or licensed versions on server. It could be NCBI blast for phylogeny study of microbes to graphical representation of the huge screening data generated. All these data are to be stored in various formats suitable to report modes. There is need to take back-up of all from IPR point of view.

One of the important part in documentation is to keep the personal laboratory journals [or Laboratory Note books (LNB)] updated with the data entry in particular predefined formats. These are to be reviewed, signed by both experimenter and supervisor. In some labs there is e-journal facility which automises the system and has very powerful reporting and secured editing modes. Well organised LNB management ensures well organised science and helps to maintain IPR value of the generated assets in terms of new drugs, new processes and novel applications.

Final words

The outcome of all the laboratory work should be such that it should ease post-laboratory work and represent that data which could be highly reproducible and could be extrapolated to desired extent. Finally in drug discovery second wider phase begins on strong foundation of this first phase in our labs.


2. Reid%20paper%20final_tcm18-85040.pdf
3. v10_issue2/dikmenli/index.htm#con

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First published on: 06-11-2014 at 13:29 IST