ESTADOS FINANCIEROS

Under appropriate pH conditions these can act as flocculating agents by effecting polymer bridging between particles, and can also cause protein precipitation by the same mechanism. Such interactions involve the adsorption of segments of the polymer onto different molecules or particles, thus creating a bridge between the two, and can be due to direct interactions between the functional groups of the flocculants and the proteins or particles, including hydrogen-bonding, chemical and co-ordination bonding, hydrophobic association through non-ionic hydrocarbon groups and long-range electrostatic attraction through ionisation of the functional groups. It can also be due to mutual interactions of the polymer and the protein or particle with bridging metal cations. Adsorption of polymers (Attia, 1987) is due to;- — Chemical forces : co-ordination bonding, hydrogen- bonding; these act over very short distances.

bonds, hydrophobic bonding, London-Van-der-Waals forces; these act over longer distances.

— A combination of the two.

Care must be taken that the particles do not become completely covered in the polymer, or they will be restabilised, and no flocculation will occur. Flocculation can also be achieved through the use of polyelectrolytes with an opposite charge of much higher density to that of the particles, through charge reversal of 'patches' on the particle surfaces. These patches then interact directly with the normally-charged surfaces of other particles, creating much shorter bonds than are seen in the previously described form of polymer bridging (Gregory, 1987; Gregory, 1989). Extensive studies have been undertaken on the cationic polyelectrolyte polyethyleneimine, and this method is thought to be the mechanism behind the effectiveness of the lower molecular weight.PEIs, where the polymer chain length may be insufficient for bridging between particles (Akers, 1975), while other researchers believe it plays a dominant role in high molecular weight polyelectrolytes also (Treweek & Morgan, 1977a). This dominance is likely to be dependant on other factors; Lindquist and Stratton (1976) state that "... the relative importance between polymer bridging and electrostatic considerations is pH dependant. At pH 9 and greater, polymer bridging is the dominant mechanism of destabilization due to the low cationic charge of the PEI molecule. However, at pH less than 9, charge interaction between PEI and colloidal silica is the dominant consideration". More recent work (Bulmer, 1989) indicates that this explanation may be over­ simplified; colloid titration shows that PEI retains substantial charge at pH 9.

Such methods of flocculation are much favoured because of the low polyelectrolyte concentrations at which flocculation is achieved. Cationic polyelectrolytes such as polyethyleneimine (PEI) are frequently used. The polymer is weakly cationic and is thus affected by the type and concentration of any counterions present, as well as pH. "The PEI's constitute a large family of polyamines of varying molecular weight and

degree of modification. They act as weak bases and exhibit a cationic character depending on the extent of protonation"

(Horn, 1980). They have the following general formula:- HgNfCgH^NHinCgH^NHg

PEIs form compounds with macromolecules containing anionic domains, including nucleic acids, some proteins, and cell debris (Jendrisak, 1987). Such complexing is thought to be due to the formation of 'patches' of charge neutralisation on the species to be flocculated. The effectiveness of this flocculation increases with higher PEI concentration (up to a maximum concentration, after which restabilisation of the colloid occurs) and molecular weight. However, charge density is thought to be the important factor rather than molecular weight, and that is pH-controlled (Eriksson & Hardin, 1987). Protonation of PEI rarely exceeds 75%, due to the proximity of some charged groups on the polymer to several others. Such mutual charge repulsion also leads to expansion of the molecules. The expansion, which is obviously pH-related, can be measured via viscosity or dynamic light scattering. The isoelectric point of PEI is 10.8. A reduction to pH 5.0 will give an expansion of up to 65%. The degree of polymer dispersion and the rate of polymer addition are also important factors in flocculation, as are the shear conditions. It is reported that if the floes are disrupted by high shear conditions, they will reform as soon as the shear stops (Horn, 1980). To avoid viscosity problems with high molecular weight PEI, Klotz and Sloniewsky (1968) suggest using cross-linked or highly branched forms of the polymer, consisting of 25% primary amines, 50% secondary amines and 25% tertiary amines. All commercially available PEIs conform to these specifications. This could result in a lower degree of protonation, however, and would have to be balanced carefully to gain the optimum degree of flocculation while remaining at a manageable viscosity.

To function as a flocculant, a polymer must have an extended and flexible configuration, the former to achieve bridging

between particles and the latter to withstand shear (Attia, 1987) . PEI is unusual in that the molecule is spherical in solution, and must thus have a large enough diameter to achieve polymer bridging. The ideal flocculant dosage is that which causes partial coverage of the particles with polymer, as polymer : polymer or 'patch':'patch' attachment is unlikely. Higher dosages will cause a reversal of the net surface charge of the particles, leading to particle repulsion and restabilisation of the suspension. Other factors affecting the efficiency of the polyelectrolyte at aggregating cells and debris include the release by the cells of proteins, nucleic acids and polysaccharides. PEI has been shown to remove these contaminants along with the cells, however (Milburn et a l ,

1990) .

Polygalacturonic acid (PGA) is an anionic polymer made by deacylating pectin. Surprisingly, despite its overall negative charge, it can flocculate mammalian cells, which are also anionic overall. Furthermore, out of all the anionic polymers (including dextran sulphate, polyglutamic acid and polyvinylsulphate) tested by Celltech (European Patent Application, 25th April, 1985) it was the only one to exhibit this property. It is thought to be due to the matching of the charge distribution on the polymer with positive local regions of charge on the cell surfaces. Further work on PGA by Sirica and Woodman (Aunins, 1989) reported no aggregation of mouse leukaemia cells at neutral pH, nor did it aggregate CRL 1606 cells, which are murine-murine hybridomas.

Dextran sulphate, which is anionic, has been reported to aggregate erythrocytes in the presence of calcium by a postulated method of specific calcium adsorption to the cells, where it aids the coordination of a bridge between the polymer and cell membrane (Aunins, 1989). Further studies by Aunins showed that high molecular weight dextran sulphate failed to aggregate CRL 1606 cells, even in the presence of calcium. Poly-L-histidine is a cationic polymer found to be effective at flocculating a range of cell lines (Aunins, 1989; Aunins & Wang, 1989). At pH 7, PLH is an insoluble precipitate, and is only slightly charged. As its pH drops, its charge and

solubility increase. By using PLH at neutral pH, Aunins suggested that selective flocculation of mammalian cells without membrane disruption would be possible.

Possible drawbacks with all the methods described here are that they all involve the addition of possibly toxic chemicals that may have detrimental effects on the product, or on later processing steps, and which must be proved to have been removed from the final product. This is not always the case; PEG with a molecular weight of 4,000 Daltons is used as a plasma precipitant. Furthermore, a flocculation process that for any reason was incomplete would bring no advantage to the purification system while still adding the problems mentioned above. Process-specific trials to optimise flocculation with minimum effect on product activity or subsequent processes would be vital before a decision could be made on the appropriateness of including a flocculation step in any purification sequence.