Consideraciones Éticas:

Fig.5. 8 (a) Elution portion only from the forward wash and elution

chromatogram in Fig.5.7(b).

(b) Purification o f each eluted fraction relative to column feed and ADH yield o f each fraction relative to the total ADH eluted.

(c) Cumulative affect on pool purity and yield on pooling fractions, starting at fraction #50 up to #16.

Chromatography stage Volume (mL) Total ADH (U) Total Protein (mg) PF* (-) Yield (%) Load 50 5,395 493 1 100 Breakthrough 50 0 0 - 0 Wash 250 1,284 402 - 24 Eluate 500 4,002 114 3.22 74

Table 5.6a. Purification table and mass balance fo r the XK 16/20 column loaded with batch PEI clarified supematant. Washing and elution performed in the reverse

direction to flow to loading. "^Purification factors are given relative to column feed

Total Total Chromatography stage Volume (mL) ADH (U) Protein (mg) PF* (-) Yield (%) Load 50 5,395 493 1 100 Breakthrough 50 0 0 - - Wash 250 1.8 302.6 - - Eluate 500 5,542 199.2 2.54 >100

Table 5.6b. Purification table and mass balance fo r the XK16/20 column loaded with batch PEI clarified supernatant. Washing and elution performed in the same direction to flow as loading. ^Purification factors are given relative to column feed.

In summary, reversing the direction o f wash and elution following loading considerably reduced both the time and buffer volumes required for elution in comparison to washing and elution in the forward direction. For recovery of all eluted fractions, reverse elution resulted in a higher purification for all pooled fractions due to the removal o f a portion of the contaminating protein. However this was at the expense o f a drop in the overall yield. Reversing the direction of elution was chosen as the preferred method for future packed bed chromatographic operations.

5.3.3 Case study 3 - Pilot-scale clarification of yeast homogenate by continuous in-line PEI flocculation.

The use of batch PEI flocculation was found to result in several operational problems, not the least o f which was a low overall yield. The pH o f the PEI was subsequently found to play an important role in the flocculation process with pH6.5 producing a more specific flocculation o f cell debris and protein. The use of larger volumes of lower concentration PEI solutions were also found to yield better clarification in small-scale laboratory experiments. As previously mentioned, the use of a more dilute PEI solution might also be expected to increase centrifugal performance through a slight dilution o f the centrifuge feed stream. The use o f concentrated stock solutions in the off-line determination of the PEI to homogenate mix ratio, was later found to yield differing results depending on the concentration o f the PEI stock employed. Finally the use o f a batch PEI flocculation resulted in a less flexible operating procedure. The homogenate feed stream always needed off-line analysis to check on the correct mix ratio, a process taking approximately 0.5h.

The method of addition of PEI to the homogenate was another cause for concern. Despite using the lowest possible stirrer setting on the tank agitator, a large amount o f air was entrained in the flocculant stream. Studies have shown that the density of streams with entrapped air are lower than the same liquid without air resulting in poorer centrifugal separation of the flocculant stream (Hoare, 1982). Thus mixing in the absence o f an air-liquid interface at low shear would help to increase centrifugal performance.

A number o f these observations were used to develop further the actual flocculation process. A 1% (w/v) PEI solution at pH6.5 was subsequently employed for the flocculation o f cell debris as opposed to the higher concentration previously used. Off-line determination o f the appropriate mix ratio was performed using a sample of the pilot-plant stock solution of l%(w/v) PEI. Thus any errors incorporated due to differences between the two PEI stock solutions were eliminated. '

The method o f mixing the homogenate and PEI was changed from a batch to a continuous addition process. Two lobe pumps were used to mix the two streams together at a T-piece junction eliminating the problem o f air entrapment and a

subsequent decrease in the clarification efficiency. The use o f the two pumps meant that the mix ratio could be altered during processing to reflect any variations in the quality o f the homogenate. In theory, this eliminated the need for an off-line determination o f the correct mixing ratio. Different ratios could be investigated in-si tu

through altering the relative flowrates on both pumps. However an off-line check on the PEI to homogenate ratio was still carried out as a precautionary measure.

5.3.3.1 Off-line determination of PEI to homogenate mix ratio.

Using the method described in section 5.2.4.2, the optimum mix ratio for PEI to homogenate was found to be approximately 0.3%(w/v) PEI.

5.3.3.2 Continuous PEI flocculation and disk stack centrifugation.

The overall mass balance o f the continuous PEI flocculation process is illustrated in Fig.5.9. To achieve the above PEI concentration through in-line mixing, the two feed pumps were set to different flowrates; 9L/h for the PEI pump and 21L/h for the homogenate pump thus resulting in a final flowrate to the centrifuge of 30L/h. A mass balance for the continuous flocculation process is given in Fig.5.9 and the purification table for the pilot-plant clarification route is provided in Table 5.7.

The CSA-1 centrifuge was again operated to sacrifice yield for clarification with full discharges performed every 150s. The yield for ADH over the CSA-1 clarification stage was 61% with a purification factor of 2.03 both o f which exceed the relevant figures obtained using the batch PEI flocculation route described earlier (section 5.3.2). Two lOmL samples were removed from the CSA-1 supematant pool and clarified at a relative centrifugal field of 49,000g in the J2-M1 centrifuge. In comparison to the same test performed from the batch PEI route, an extremely small amount of solids was recovered at the bottom of the OM salt centrifuge tube. In the tube to which ammonium sulphate had been added, no separated solids were recovered at all, indicating that the solids recovered where solubilised at 0.78M ammonium sulphate. Therefore no additional clarification would be obtained by passing the CSA-1 supematant pool through the IP centrifuge. The CSA-1 supematant was immediately adjusted to 0.78M ammonium sulphate through the addition of a 1 0 0% saturated solution and this dilution step alone was sufficient to

60L 280g/L Bakers Yeast inO.IMKHzPO^at pH6.5 5 passes 500 barg Q=280L/h 3 volumes PEI to 7 volumes Homog Semi-hermetic feed zone Q=30L/h A D H = 282 U/mL TP = 29.2 mg/mL 60L Yeast homogenate 1%(w/v)PEI pH6.5, 25.7L 41.1 L Slurry ADH= 128 U/mL TP = 24.5 mg/mL A D H = 2 4 4 U/mL TP = 13.3 mg/mL 42.3 L Supematant _ 10.6L, 100% sat. (NH,)2SO. A D H = 210 U/mL TP = 10.5 mg/mL 52.9 L Supematant Homogenisation K3

In line PEI flocculation

CSA-1 Disk stack Clarification

Adjust Ionic Strenth of Supematant to 0.78M (NH4)2S04pH6.5