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Chapter 1Upstream and downstream stepsin biochromatography

Mookambeswaran A.Vijayalakshmi

Introduction

The Biochromatography or chromatography of biological molecules is a multidisciplinary

involving the expertise development in:

1 Polymer chemistry, for the development of column packing materials/adsorbents with dif

composition, different derivatization and different particle sizes etc.

2 Mechanical designing of the columns with fluid mechanics knowledge.

3 Development of software for data handling, operation controls and even simulations.

4 Detection systems with optics, lasers, amperometrics etc.

5 Sophisticated physico-chemical analytical methods on-line or off-line such as ESI-MS.

The striking development of highly efficient and sophisticated microanalytical and prepa

chromatography has led to tremendous progress in the study of proteins and peptides. Neverth

optimisation of upstream and downstream operations, in chromatography steps, such as findin

right conditions of extraction, sample preparation and suitable methods for monitoring the proteibiological activities remain crucial. Several manuals are available which cover the detailed oper

of protein separation and purification covering relevant details of the extraction methods (Sc

1987; Ladish et al., 1990; Janson and Ryden, 1989; Methods in Enzymology, 22, 34 and 204).

We can divide protein separation and purification into four broad steps: extraction;frac

precipitation; purification and final polishing(Fig. 1.1). Before these four steps the s

identification and choice of raw material for the targetted protein is an important issue which in turn

determine/orientate the choice of extraction methods.

Choice of sources of raw materials

In the case, particularly, of biochromatographic operations of proteins, the following questions h

be answered before designing any experimental set up (Fig. 1.2). What is the final targetted use

protein? Is the protein for purification meant for basic studies on the protein, such as crystal struct

is it meant for large scale production to be used in agro food industry or pharmaceutical indu

If the final use is pharmaceutical, then one has to take into consideration the high demand on the

of the final product: 99.999.99%. In terms of choice of raw material, these questions have a

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relevance. If we use animal sources e.g. albumin or IgG from animal/human sera, we have to ser

consider alternative sources to avoid the potential HIV, Hepatitis B, etc. contamination, from th

material.

These alternative sources are mainly based on recombinant technology; human/animal protei

expressed in micro-organisms or humanised cells or plant cells or whole plants, seeds, fruits etc

use of the latter sources, also termed as Molecular Farming, is more recent and is quite promis

terms of cost effective and facile production of the raw material. But, here the purification, partic

the extraction steps prior to the chromatographic steps needs special care, both in terms o

enormous quantity of the raw material to be treated (leaves, seeds, fruits etc.) and also the compoof buffers (e.g. addition of polyvinylpyrolidone) for preventing the complexation of the target pr

with polyphenols.

Extraction methods

The extraction step is meant for obtaining the target molecule (protein) in solution. It is obviou

certain raw materials of biological origin constitute an almost clear solution of the protein. Some

Figure 1.1 General purification scheme with the four major steps, in downstream processing.


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examples are: blood sera, urine, milk, extracellular protein in the animal, bacterial, yeast, mamm

and plant cells culture media. The main upstream step in these cases is just the cell separatio

concentration step by membrane (ultrafiltration) processes or by precipitation techniques.

In the case of insoluble raw materials such as animal or vegetal tissues and in the ca

intracellular proteins produced in the cell culture systems, not only adequate extraction methods s

be used to obtain the protein in its soluble form, but also specific additives such as pro tease inhib

reducing agents, polyphenol blockers (e.g. polyvinylpyrolidone) should be added to the extraction mA classification of extraction methods and their applicability to different raw materials is desc

inTable 1.1.

The use of detergents in the extraction step needs some special comments. The additi

detergents is used mainly for the extraction of membrane bound proteins, by reducing the hydrop

interactions involved in the protein binding to the membranes. Some chaotopic agents can re

detergents for the same purpose. Some detergents may denature the proteins; hence attention has

paid both for the choice of the detergents for extraction, their concentration and also whether to

the presence of detergent in all the steps including the final chromatographic steps. This last

evokes the question of compatibility of the specific detergent added to the buffer an

chromatographic adsorption media chosen. Non-ionic detergents such as Triton x-100 are usually

compatible with the different affinity adsorbants than the ionic detergents such as Sodium Do

Sulfate (SDS). Moreover SDS can denature the protein and interfere with its biological activity

ionic, non-ionic and zwitterionic nature of the specific detergent may have an impact both on p

surface as well as on the chromatographic adsorbent properties, thus influencing both adsorptio

the elution of the protein in the chromatographic steps. Thus a fairly good knowledge of the che

Figure 1.2 The multifaceted nature of protein purification process development. (From HO 1990permission.)


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structure of the detergent to be chosen is very useful. The reader can get more information o

detergents and their physicochemical properties, in Hjelmelands chapter in Methods in Enzym

vol. 124, 1986.

The optimum concentration of the detergent to be used, should invariably be below the cr

micelle concentration, in order to avoid the protein being entrapped into the micelle and thus no

to interact with chromatographic adsorbent surfaces. However, in the electrokinetic chromatogr

mode, the protein-detergent micelles are exploited for separating/studying the proteins as a funct

their size (Landers, 1993).

The other additives to be used, including the buffers, during the extraction step and up tchromatographic steps are enumerated in Table 1.2.

Fractionation techniques

This step is intended mainly for reducing the volume of the solution to be handled in further ste

also reduces the total number of components present in the medium. The fractionation method

Table 1.1 Initial fractionation (corresponding to step 1 ofFig. 1.1)


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mainly based on precipitation of the target protein by decreasing its solubility by modifying th

concentration (salting in and salting out approaches); or by the addition of organic solve

organic polymers. Temperature and/or pH variations leading to targetted denaturation o

contaminating proteins and precipitation of the desired protein may also be used as a fraction

step.

In the salting out approach the property of a particular salt (e.g. (NH4)2SO4) as an eff

precipitation agent is deduced from the so called Hofmeister series

anions:

i.e. and are the most effective precipitating agents.

Cations:

i.e. is the most efficient agent.

Table 1.2 Extraction medium


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Hence it follows from the above that (NH4)2 SO4 is a very good precipitation agent. Moreove

salt ensures minimisation of any denaturation as it stabilises the native conformation of the prot

be precipitated.

Organic solvents promote the precipitation of proteins by disturbing the organised water mole

around the protein. Even though, it may, at first sight look like organic solvents may denature the pro

two organic solvents namely, ethanol and acetone have been very successfully used for the sel

precipitation of serum proteins, enzymes or hormones.

The ethanol precipitation of serum proteins known as the COHN fractionation method intro

in 1946 (Cohn, 1946) is used even today at all levels, may be with some modification (Taylor, 1

This method, by a proper control of pH and temperature, enables one to prepare fractions partic

enriched in albumin, globulins and globulins. In addition, the use of ethanol and su

temperatures ensures certain safety against microbial and other contamination during the process

The organic polymers such as polyethylene glycol (PEG) in the molecular weight range of 60

20000 are able to precipitate the proteins by sequestrating the water molecules in a rather simila

to the organic solvents (Curling, 1980). PEG is the most widely used polymer due to its inocuity

toxicity, low immunogeneicity etc.

In some cases inorganic polymers are used to concentrate the protein solutions. Her

phenomenon exploited is not a precipitation, but a physical adsorption. A case in study is the silica or cesium, Kieselger etc. to concentrate proteins from culture broth or even from urine

adsorbed proteins are then stripped off using minimum quantity of buffers to be used in further st

Sample preparation from the precipitates

In the fractional precipitation step where the sample is concentrated, the precipitates are reco

either by simple centrifugation most often in laboratories or by liquid-liquid two phase partition

Scopes, 1987 for more details of the technique) at larger scales. Then the precipitated protein fra

is solubilized in a minimum quantity of buffer and either used as such (if hydrophobic intera

chromatography is used as the first step) or desalted using either membrane process (ultrafiltratiby desalting chromatography using a sephadex G25 gel filtration mode.

Organisation of different chromatographicsteps in the protein fine purification

In this book more than ten different chromatographic approaches, based mainly on diffusio

adsorption principles are discussed in detail. A clear understanding of their underlying principle

enable one to set up the purification train as described in Fig. 1.1, schematising the differen

operations. The unit no. III englobes the chromatographic steps.

The sequential order in which these chromatographic operations should be set up, must tak

account the minimisation of the total number of steps in a purification scheme. Increasing the nu

of steps not only results in increased investment and operational costs, but, also entails loss of th

protein. In fact, at each step due to denaturation and product loss by strong adsorption etc. the yi

the pure product decreases (Hu et al., 1985).


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Monitoring the fractionation and purification

An important prerequisite for setting up a purification scheme is to have adequate assay procedu

follow the biological activity and protein content. The protein content can be monitored bo

calorimetric assays, such as Lowry method (Lowry, 1951), Bradford method (Bradford, 1976), B

method (Smith et al., 1985) or by bisinconinic acid (Sorenson and Brodbeck, 1986), and b

monitoring at 280 nm or eventually with multiple wavelength detection.

The biological activity and protein contents should be recorded at each step, in order to follo

efficiency of purification and yield. A typical example of a purification table is shown in (Table 1Other methods of control such as in Fig. 1.3 should be performed, mainly at the final ste

purification.

Table 1.3 A typical table presenting the protein purification from a crude extract

Figure 1.3 General process for the quality control of purified enzyme/protein.

Conventionally, the SDS-PAGE and Molecular Sieving by Gel filtration chromatography

using Sepharose, Superose at low pressure or HPLC-SEC approach using TSK range of ge

used. But with the advent of mass spectrometry techniques, particularly the electrospray ionic

spectrometry (ESI-MS) the molecular heterogeneicity and molecular masses can be determined

high precision. In our lab, in the last few years, in the chromatographic steps, meant fo


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purification, the fractions are routinely analysed by ESI-MS. This enabled us to identify the sepa

of subspecies/isoforms of the enzymes (Berna et al., 1997).

References

Berna, P.P., Mrabet, N.T., Van Beeumen, J., Devreese, B., Porath, J. and Vijayalakshmi, M.A. (1997) Bio36 (51), 1635516356.

Bradford, M.M. (1976)Anal. Biochem.,72, 248254.

Cohn, E.J., Strong, L.E., Hughes, W.L., Mulford, D.J., Asworth, J.N., Melin, M. and Taylor, H.L. (19

Amer. Chem. Soc.,68, 459475.

Curling, J.M. (1980) Methods of plasma protein fractionation, Academic Press Ed.

Hjelmeland, L.M. (1986) In: Methods in Enzymology,124, 135164.

Ho, S.V. (1990) Strategies for large scale protein purification. In: Protein purification: from mo

mechanisms to large-scale processes. (Ladish, Willson, Painton and Builder, eds), American Ch

Society, Washington, USA, 1434.

Hu, W.S. and Wang, D.I.C. (1985) In: Mammalian cell technology (Thilly, W.G. and Addison-Wesley,

Menlo Park, CA.

Jansen, J.C. and Ryden, L. (1989) Protein purification: principles, high resolution methods and applications.

publishers Inc.Ladish, M.R., Willson, R.C., Painton, C.D. and Builder, S.E. (1990) Protein purification: from mol

mechanisms to large scale processes. American Chemical Society, pp. 2022.

Landers, J.P. (1993) TIBS,18, 409414.

Lowry, O.H. (1951)J. Biol. Chem.,193, 265267

Scopes, R.K. (1987) Protein purification principles and pratice, Springer-Verlag.

Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto,

Goeke, N.M., Olson, B.J. and Klenk, D.C. (1985)Anal. Biochem.,150, 7685.

Sorenson, L. and Brodbeck, H.M. (1986)Experientia,42, 161162.Taylor, H.L., Bloom, F.C., Mc Call, K.B. and Hyndman, L.A. (1956)J. Am. Chem. Soc.,78, 13561358.

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