If one looks at the various areas in which microbial exopolysaccharides are currently employed, it may be possible to make some predictions about future usage. The increase in interest in the physical properties of these polymers, together with a much better understanding of the relationship between physical properties and chemical structure and the continued search for new polysaccharides, will inevitably lead to new discoveries. Relatively few of these are likely to have properties suited to new applications or their use in place of currently used polymers. There are two major constraints: legislative and financial.

In the food industry, xanthan is currently unique in its acceptability. As has been mentioned earlier, gellan from Pseudomonas elodea is currently undergoing safety evaluation. These two polymers can potentially fulfil many of the perceived needs of the food industry for microbial polysaccharides as well as replacing some established plant or algal products. Any new polymer would only have a small market niche and this would probably be insufficient to justify the expense of development and of the safety appraisal needed to obtain legislative approval. It is more likely that new applications will be found for the polysaccharides, such as xanthan, which already are approved. An exceptional situation exists in Japan, where the microbial polysaccharides, being regarded as natural products, are acceptable food ingredients. Perhaps some new polysaccharides will find applications in Japan, which may justify their introduction into other countries. One such might be curdlan.

In non-food applications, xanthan currently holds a commanding situation.

Introduction

While microbial exopolysaccharides, in common with similar polymers from other sources, are the substrates for degradative enzymes, the number of polysaccharases that have been isolated and characterised is relatively small. Only a small number of the polysaccharide-producing microbial species also yield enzymes degrading the same polymers. The exceptions include some of the bacterial species synthesising alginate and hyaluronic acid. A rich source of enzymes degrading bacterial exopolysaccharides has proved to be bacteriophages. These viral particles contain polysaccharases as part of the particle structure, usually in the form of small spikes attached to the base-plate of the phage. After phage infection, the bacterial lysates normally contain further amounts of the same enzyme in soluble form. The advantage of bacteriophages as sources of enzymes degrading polysaccharides is their freedom from other associated glycosidases, which might further degrade any oligosaccharide products. On the other hand, yields of phage-induced enzymes are low and they can only be regarded as laboratory tools of value in structural studies, unless the genes for the enzymes can be cloned and expressed on a large scale in microbial hosts. In addition, not all bacteriophages for exopolysaccharide-producing bacteria yield such enzymes.

There are very few commercially available enzymes acting on microbial polysaccharides. Consequently, the laboratory interested in using enzymes for structural determinations or for quality control must normally isolate its own enzymes. A number of polysaccharases have been obtained from bacterial and fungal sources by using enrichment procedures, with the polysaccharides as substrates.

Introduction

Although some of the earliest studies on bacterial transformation utilised as a model system polysaccharide production in Streptococcus pneumoniae and its relation to virulence, further progress in studying the genetics of polysaccharide synthesis has taken some considerable time. Much effort was applied to studies on the genetics of colanic acid synthesis in E. coli and Salmonella typhimurium, but this has proved to be a very complex system with numerous regulatory mechanisms. The complexity may perhaps be, at least in part, related to the relatively large hexasaccharide repeat unit of this exopolysaccharide and the ability of most bacteria synthesising it to produce more than one extracellular polysaccharide. Recently, however, detailed knowledge of the genetics of exopolysaccharide synthesis has derived from various systems. The interest in xanthan as an industrial product from X. campestris, and in the bacteria per se as plant pathogens, has prompted their study. Rhizobium species, as well as producing at least two different polysaccharides of potential industrial interest, have received attention because of their symbiotic relationship with leguminous plants and the associated bacterial fixation of dinitrogen. Alginate production by Pseudomonas aeruginosa has been studied because of the correlation between polysaccharide secretion and the infection of cystic fibrosis patients. Finally, a range of (mainly pathogenic) Gram-negative bacterial species have been examined, E. coli strains being used for a number of studies. All this information enables us to see some common aspects in the genetic control and regulation of exopolysaccharide synthesis; the concept of a ‘cassette’ of biosynthesis genes unique for each polysaccharide, first conceived in exopolysaccharide-synthesising E. coli by Boulnois and his colleagues, may well be at least partly applicable to many, if not all, exopolysaccharide-synthesising bacteria.

Introduction

When one considers that the number of microorganisms known to produce exopolysaccharides is very large indeed, the number of structures of these polymers which have been exhaustively studied is still relatively small. As with all polysaccharides, the microbial products can be divided into homopolysaccharides and heteropolysaccharides. Most of the former are neutral glucans; the majority of heteropolysaccharides appear to be polyanionic.

Three types of homopolysaccharide structure are found. Several are linear neutral molecules composed of a single linkage type. (Microorganisms do not appear to yield the ‘mixed linkage’ type of glucan found in cereal plants such as oats and barley.) There are also several polyanionic homopolymers and these, unlike the glucans, also contain acyl groups. Slightly more complex structures are the homopolysaccharides of the scleroglucan type, which possess tetrasaccharide repeating units due to the 1,6-α-D-glucosyl side-chains present on every third main chain residue. Finally, branched homopolysaccharide structures are found in dextrans.

Microbial heteropolysaccharides are almost all composed of repeating units varying in size from disaccharides to octasaccharides. These frequently contain one mole of a uronic acid, which is usually D-glucuronic acid. Very occasionally, two uronic acids are present. The uniformity of the repeat units is based on chemical studies and it is possible that some irregularities may be found, especially in the polymers composed of larger and more complex repeat units. The heteropolymers commonly possess short side-chains, which may vary from one to four sugars in length. Very rarely, the side-chains themselves may also be branched.

Introduction

The surface of the microbial cell is a rich source of carbohydrate-containing molecules. Some of these are unique types, confined to a limited range of microorganisms. These are the components of the microbial cell walls such as yeast mannans, bacterial teichoic and teichuronic acids, lipopolysaccharides and peptidoglycan. However, in addition to these wall components, polysaccharides may be found either associated with other surface macromolecules or totally dissociated from the microbial cell. These are exopolysaccharides, extracellular polysaccharides showing considerable diversity in their composition and structure. Some of these polymers may bear a strong chemical similarity to cell-wall components, but the majority are distinct chemical structures totally unrelated to cellular constituents.

Exopolysaccharides occur widely, especially among prokaryotic species, both among those that are free-living saprophytes and among those that are pathogenic to humans, animals and plants. Most microalgae yield some type of exopolysaccharide but they are less common among yeasts and fungi. However, some of those isolated from fungi do possess interesting physical and pharmacological properties.

Definition of exopolysaccharides is more difficult than definition of the carbohydrate-containing polymers found in microbial walls. The term exopolysaccharide has been widely used to describe polysaccharides found external to the structural outer surface of the microbial cell and it can be applied to polymers of very diverse composition and of different physical types. The term glycocalyx, introduced by Costerton, fails to differentiate between the different chemical entities found at the microbial surface. It has been used to represent a complex array of macromolecular species inlcuding components which are truly extracellular, together with wall polysaccharides and many other non-carbohydrate-containing chemical species.

Both microbial and non-microbial polysaccharides find a wide range of nonfood industrial uses. In such usage, the polysaccharides may compete with synthetic organic polymers, but in some applications only natural products are acceptable because of their biodegradability and their lack of toxicity. As far as microbial polymers are concerned, xanthan has the largest share of the market. The non-food usage of polysaccharides directly reflects their various physical attributes. Before their use is contemplated, they must be fully evaluated against other possible synthetic and natural products. If these other products are cheaper, they may be preferred even though the microbial polysaccharide is superior in the application envisaged. They are more expensive than starch or than synthetic products such as polyacrylamides, so use of the exopolysaccharide may incur an unacceptable cost penalty. However, because of changes in prices and availability of plant and algal products, there is considerable opportunity for expansion of the industrial use of microbial polysaccharides, especially as new products with unique physical properties are discovered. The oil industry provides one major scenario in which exopolysaccharides have readily found acceptance for a number of purposes, in competition with plant gums and their derivatives and with synthetic chemicals. If the numerous proposed enhanced oil-recovery developments come to fruition, the use of xanthan and other microbial exopolysaccharides could well increase very significantly indeed. Other industrial applications have secured a wide range of technological uses for exopolysaccharides with different physical properties. Some of these are limited in scale and will require only small amounts of polymer, but others contribute significantly to the overall demand.

Introduction

The information that needs to be obtained in respect of defining microbial exopolysaccharides can be summarised under the headings of polymer composition and of structural analysis. Composition covers not only the monosaccharide components but also the various possible acyl groups and inorganic substituents. Structural analysis involves various approaches. These range from partial fragmentation by acid or enzymic hydrolysis to produce oligosaccharides, the structures of which must then themselves be determined, to methylation and sequence analysis.

Analysis of polysaccharide composition

Exopolysaccharides are composed of three distinct types of monomer. They are of course predominantly carbohydrate in nature but, in addition to the various sugars, there may be organic and inorganic substituents. The individual sugars can seldom be analysed in the intact polymer although it is often possible to use colorimetric assays to quantitate specific types of sugars such as pentoses, 6-deoxyhexoses, heptoses, uronic acids and amino sugars. If the specific monosaccharides are to be determined quantitatively, they must first be released by hydrolysis.

Determination of the composition of microbial exopolysaccharides is essentially similar to that of any other comparable polymer. The polymer is hydrolysed with acid to yield the component monosaccharides, which are then identified and quantified. During hydrolysis, labile groups such as ester-linked components and ketals are likely to be removed and must be separately recovered and identified. A careful choice of hydrolysis conditions must also be made. Monosaccharides differ in their stability to acid at high temperatures; the glycosidic bonds vary considerably in their resistance to hydrolysis.

Industrial interest in microbial polysaccharides has been stimulated by their unique properties and the opportunity to provide a guaranteed supply of material of constant quality and stable price. One must set against such positive aspects the relatively high costs of the product, of process development, and of downstream processing, especially if the intention is to provide material with approval for food usage.

The aim of this book is to present information relating to microbial exopolysaccharides which have actual or potential industrial or medical importance, rather than to provide comprehensive coverage of the whole field. It indicates the mechanisms by which these polymers are synthesised, as well as techniques used in their chemical and physical characterisation.

There has been a marked upsurge of interest in microbial exopolysaccharides in recent years, from biologists and non-biologists alike. In particular, recent studies on the physicochemical properties of polysaccharide solutions are providing a new insight into the physical structures of these polymers and furnishing the industrialist with a clearer indication of their useful properties. The increased interest in microbial polysaccharides mirrors a growth in the use of water-soluble polymers generally, and also an appreciation of the environmental advantages to be gained from use of water-soluble rather than solvent-based systems.

As many students now receive little instruction in the chemistry of carbohydrates and related molecules, readers may need to refer to a suitable text on that subject. It should, however, be remembered that in many respects the physical properties of polysaccharides are frequently not greatly dissimilar from those of DNA and RNA.

In Chapter 2, the theory of hybridoma generation was described, and the variation in methodology used was discussed. The procedures for the production of hybridomas may differ substantially for different antigens; §2.5.2 showed how the immunisation schedule used for one antigen may not be optimal for another. The use of different myeloma cell lines can also produce different results (see §2.5.8). Thus, the determination of the optimal conditions for generating hybridomas against a particular antigen is central to the success of the technology. Chapter 3 will expand on the discussion of Chapter 2 concerning factors which are influential in the successful generation of hybridomas.

The use of different immunisation protocols for different antigens and how these influence the type of immune response generated was introduced in Chapter 2 (§2.5.2). In this chapter, §3.1 will look at the relative efficacy of different immunisation schedules, and the reasoning behind choosing a particular protocol for a particular antigen. Chapter 2 identified the major influential factors in the fusion event as the myeloma cell, the fusing agent (usually PEG) and the fusion ratio. How the source of myeloma cell, source of PEG and fusion ratio can influence the success of hybridoma production will be discussed in this chapter (§§3.2–3.4), since the physical generation of hybridomas is probably the most important, sensitive and delicate of the in vitro steps.For fusion to occur, cell membrane structure must be drastically altered, and thus slight variations in the toxicity of the fusing agent can be detrimental to the success of generating hybridomas. Likewise, the fusion ratio (splenocytes : myeloma cells) must be such as to guarantee fusion between myeloma cells and some antigen-specific lymphocytes.

Chapter 1 described the interactions and communications which occur during an immune response against non-self material (antigen). From this, it can be seen how hybrid cell lines could be produced which would secrete antibody reactive against a particular antigen. In this chapter, the requirements for the production of such hybrid cell lines–hybridomas – will be explained. The two major factors involved are the use of a tissue-culture-adapted lymphoblastoid cell line (myeloma cell line) and antigen-stimulated (in vivo or in vitro)lymphocytes. The characteristics and production of myeloma cells will be described; details of how these cells play a major role both in the production of hybridomas, through chemical-induced fusion with the antigen-stimulated lymphocytes, and in subsequent stabilisation of the hybrid cells in vitro will be presented. Procedural requirements for generating antigen-stimulated lymphocytes which are to be used in hybridoma production will be explained; and §2.6 will present a practical guide to the preparation of the myeloma cells and lymphocytes, fusion to produce hybridomas and selection of myeloma cell-lymphocyte hybrid cells. Since this book is concerned with antibody-secreting hybridomas, most of the details will be concerned with these; but reference will be made to other types of lymphocyte derived hybrid cell lines, in particular in §2.7.

Basic requirements

The basic constituents of any hybridoma or hybrid cell line are the parent cells required for its formation.With the immune B cell hybridomas (which secrete antibody) these parents are splenocytes from the appropriate animal immunised with the antigen of choice, and tissue-culture adapted myeloma cells (1–6).

The production of monoclonal antibodies can be divided into a number of phases. (i) Immune splenocytes are fused with myeloma cells, and the resultant hybridomas are selected and adapted to growth in tissue culture. (ii)The hybridoma media (culture supernatants) are tested for the presence of antibody. (iii) Positive cultures are ‘weaned off’ feeder cells and, when fully adapted to growth in vitro,are cloned. (iv) Growing colonies of hybridoma cells are tested for secretion of antibody specific for the appropriate antigen, and positive cultures expanded (by growth and passage) and recloned. (v) Of these ‘re-clones’, those secreting the highest relative quantities of antibody (relative to the cell concentration) are selected for expansion and eventual storage. Chapters 2 and 3 dealt in detail with the preparation and passage of hybridoma cultures, and the conditions required for successful hybridoma production. This chapter will describe the methods for detecting antibody in hybridoma cultures, and the procedures of isolating monoclonal hybridomas (cloning).

The antibody-detecting assays will be described in approximate order of frequency of use in reports of hybridoma technology, and only the major assays will be discussed in detail. Other procedures will be mentioned, but the assays described in detail are those which should prove most applicable to hybridoma technology.

The three most commonly reported methods of cloning hybridoma cells will be described; the theoretical and practical advantages and disadvantages will be discussed.These procedures are cloning established hybridomas by limit dilution, isolation of colonies from the ‘masterplate’ (plate used for seeding the fusion mixture) – micromanipulation – and isolation of colonies in semi-solid ('sloppy') agar. A more recent adaptation, that of cloning hybridoma cells immediately after fusion, will also be described.

Introduction

There are two pressing reasons for producing large quantities of monoclonal antibodies by in vitro technologies. The first is associated with the problems of the presently popular alternative route: that of using mice and rats to produce ascites fluids from the inherently carcinogenic effects of inoculating them with a monoclonal antibody-producing hybridoma. This route has five major problems:

1. (i) it is costly in manpower and facilities;

2. (ii) the materials produced, although present in high concentration (2–10 mg/ml) are contaminated with other immunoglobulins, plasma proteins (70–80%) and other possibly infectious adventitious agents;

3. (iii) there is a growing movement in modern societies to cause the minimum discomfort to those animals which are of value nutritionally, scientifically and technically;

4. (iv) there is a lack of reproducibility; and

5. (v) it is not possible to produce human antibodies in rodent bodies.

The more positive reasons for looking towards in vitro technologies are:

1. (i) the ability to scale up such cultures and thus to be able to satisfy requirements for kilogram or hundred to thousand kilogram (1,2) quantities of pure antibody for a variety of therapeutic and preparative purposes; and

2. (ii) the exploitation of the potential of animal cell systems for the more efficient production of antibodies by using very dense suspensions of animals cells.

The difference between the rodent and tank methods can be illustrated by presenting the alternative systems necessary to produce 1 kg of antibody (Table 5.1). The size of the tank culture for such a production (5000 1) is well within current practice for animal cell cultures; lymphoblastoid interferon is presently produced commercially from mammalian cells in 8000 1 volumes (3, 4).