Contrastación de las Hipótesis Específicas

The methods for disruption o f microbial cells to release intracellular products both by large scale and laboratory techniques have been compared in reviews by Cbisti and Moo-Young, (1986) and Engler, (1985). At present non-mechanical methods such as osmotic shock, chemical and enzymatic lysis are generally more appropriate for small scale laboratory cell disruption due to the specificity o f these techniques for single organisms or products and difficulties with scale-up. Mechanical methods may involve liquid or solid shear operations and the most commonly used methods at industrial scale are high pressure homogenisation, (Hetherington, et aL, 1971), microfluidisation, (Sauer, et aL, 1989) and bead milling (Schutte, et aL, 1983).

The most widely used device for cell disruption on an industrial scale, is the APV high pressure homogeniser (Chisti and Moo-Young, 1986; Engler, 1985). This consists o f a positive displacement pump which may have one or more plungers, and on the pressure stroke the cell suspension is forced through an adjustable discharge valve with a restricted valve seat. During discharge the suspension passes between the valve, the valve seat and an impact ring, impinging on these surfaces. Disruption in high pressure homogenisers generally has been found to be a first order process with respect to the number o f passes, with the rate constant a function o f temperature and pressure (Hetherington, et aL, 1971). Disruption is also dependent on other factors such as organism, growth history, culture medium and valve design but independent o f concentration up to a limit.

Operation at high pressures and increased number o f passes will result in complete cell disruption nullifying the advantages o f periplasmic expression, and provide very fine particles o f cell debris which can cause a number o f problems. These include enhancing chances for blockage o f process equipment and making centrifugal clarification procedures immediately downstream inefficient when attempting clean recovery o f soluble proteins.

1.5.3.2 Specific periplasmic release

Numerous treatments for specific release o f periplasmic contents from bacteria have been demonstrated at laboratory scale. For E. coli and other organisms enzymatic treatment with lysozyme has been extensively investigated. Lysis o f E. coli. Pseudomonas aeruginosa and Azotobacter vinelandii was achieved at a pH o f 7.5-8.0 with a combination o f lysozyme and EDTA (Repaske, 1956). EDTA has a weakening effect on the outer membrane allowing exposure o f the peptidoglycan layer to lysozyme.

Lysis was shown to be immediate and rapid, and it was observed that for optimum lysis the balance o f Tris buffer, lysozyme and EDTA was important. Spberoplast formation was later refined by resuspension o f washed cells in sucrose-Tris buffer followed by addition o f EDTA and lysozyme (Neu and Heppel, 1964). It was observed that improved yields o f spberoplasts were obtained by this method using cells in exponential growth phase compared with those in the stationary phase o f growth. Osmotic shock by dilution o f cells suspended in sucrose/Tris/EDTA in the presence o f lysozyme increased accessibility o f lysozyme to the peptidoglycan layer further enhancing release o f periplasmic contents (Birdsell and Cota-Robles, 1967; Witholt and Boekhout, 1978). These early demonstrations were at bench scale, however more recently adaption to larger volumes at pilot plant scale has been made (French, et aL, 1995).

Exponentially growing cells were found to be more susceptible to osmotic shock than cells in stationary phase, in agreement with lysozyme treatment, and required reduced concentrations o f EDTA and sucrose in cell resuspension buffers (Nossal and Heppel, 1966). Osmotic shock took effect when the sucrose/EDTA treated cell pellet was dispersed into a solution o f 0.5 mM MgCl2. This allowed for optimal release o f various

enzymes whilst maintaining viability o f cells. This method was later modified to a three step osmotic shock for release of heparinase from the periplasm o f Flavobacterium heparinum (Zimmermann, et aL, 1991). Following resuspension o f cells in sucrose/EDTA buffer, two further resuspensions, firstly in an osmotically non­ stabilizing buffer o f low salt concentration and then in a high salt concentration, resulted in up to 75% o f heparinase released. Although protein release was approximately equal in each o f the two salt solutions, heparinase activity was observed in the second high salt wash achieving 7-15 fold increase in purity. Frequently, osmotic shock treatments are performed using agitation for resuspension followed by centrifugation. The use o f a closed system comprising a tank and pump with tangetntial flow microfiltration for separation o f biomass material offers an alternative for release o f periplasmic enzymes (Biedermann and Jepsen, 1989). This method can be performed with reduced relative volumes when scaled up and does not jeopardise the cytoplasmic membrane by recylcing through the system.

Recently osmotic shock was compared to treatment with guanidine for release o f periplasmic cystatin C from E. coli (Chaib, et aL, 1995). Both methods were considered options for scale-up by these researchers. A product stream at high dilution resulted from osmotic shock which may not be attractive for product pruification. Guanidine treatment however, resulted in a highly purified product in a small liquid volume. The latter method was adapted from the protocol devised by Naglak and Wang (1990) who investigated the effects o f both guanidine and the non-ionic detergent Triton X-100. Guanidine is a chaotropic agent which has the ability to denature proteins at high concentration, and can solubilise proteins from the outer membrane o f E. coli effecting

release o f periplasmic contents. Studies with recombinant E. coli producing p-lactamase revealed that 94% recovery o f this periplasmic enzyme was achieved using 0.2 M guanidine over a 2-5 h period (Naglak and Wang, 1990). Due to the low levels o f protein released by this method specific activity was high. Introduction o f 0.5 % Triton X-100 solubilised proteins from the cytoplasmic membrane effecting further release o f overall protein reducing purity o f the periplasmic protein o f interest. Guanidine/EDTA treatment was the most favourable method when compared with a number o f other treatments for release o f periplasmic penicillin acylase from recombinant E. coli

(Novella, et aL, 1994), recovering 95 % o f the enzyme in 10 h with purification factor o f 25 when compared with disruption by sonication.

Other chemical permeabilisation methods developed for periplasmic release have included treatments with chloroform, where the mechanism o f release appeared to partially solubilise the outer membrane with effects similar to those o f osmotic shock (Ames, et aL, 1984). Selective release o f periplasmic p-lactamase was possible by treatment with the amphiphilic quaternary ammonium compound tetradecyl betainate under certain conditions (Ahlstrom and Edebo, 1994). Alkaline pH improved release as did increased concentration o f tetradecyl betainate, however the latter also caused p- lactamase inactivation. This was alleviated by modification o f the protocol to utilise high salt concentrations. Alkaline pH also affects the permeability o f the outer membrane during fermentation resulting in an increase in excretion o f enzymes located in the periplasmic space and also lipopolysaccharide into the culture medium (Georgiou,

et aL, 1988). Investigations with E. coli producing recombinant p-lactamase did however show reduced enzyme activity at alkaline pH.

Addition o f low concentrations of glycine (1 % w/v) to fermentation medium influenced the release o f recombinant periplasmic a-am ylase from E. coli over a 3 h period (Ariga,

et aL, 1989). 70-80 % o f a-am ylase was released under such conditions and increases in both time and glycine concentration were o f no additional benefit. Co-expression o f bacteriocin release protein from a second plasmid in the same E. coli cell and induced by an appropriate concentration o f mitomycin C had a synergistic effect (Yu, et aL,

1991). It was possible to release 78 % o f a-am ylase without release o f cytoplasmic contents when a combination o f mitomycin C and glycine were employed.

Application o f various physical methods to specific release o f periplasmic enzymes has been demostrated on laboratory scale. Genentech (1987) have patented a technique whereby cells are contacted with ethanol or another lower alkanol to kill cells without damage to the inner membrane, followed by freezing and thawing. Resuspension o f cells into buffer relases periplasmic proteins. Repeated cycles o f freezing and thawing in the absence o f other additives was recently used to recover a number o f recombinant proteins located in both the periplasm and cytoplasm selectively without release o f bulk endogenous E. coli proteins (Johnson and Hecht, 1994). This offers a gentle method

which yields recombinant protein in a relatively pure form. Finally, disruption using critical fluids was reported to specifically release periplasmic heparinase from

Flavobacterium heparinum without significant damage to the cytoplasmic membrane (Castor, 1991). Cells are inflated with a critical fluid such as carbon dioxide and nitrous oxide followed by rapid decompression. Although details o f this technique remain unreported Castor suggests that it is gentle and scaleable.

Methods such as osmotic shock and lysozyme treatment have the ability to release antibody fragments at bench scale, however, no pilot scale recovery methods have been reported. Application o f some o f the diverse techniques reported in the literature to the recovery o f periplasmic antibody fragments may prove to be successful. O f these methods few have been demonstrated at scale. Those requiring addition o f enzymes such as lysozyme will be uneconomical at increased scale due to the cost o f the enzyme, and addition o f chemicals such as chloroform at large scale will have environmental implications. Factors such as these must be taken into consideration when developing a scalable process for specific release o f periplasmic antibody fragments.

1.5.4 Biomass separation