8.1 Improvement of the purification procedure
After the fusion protein MUC1-IgG2a Fc is cleaved into IgG2a Fc and the target protein MUC1, it is of importance to obtain a pure product protein. Several different purification techniques have been applied including ion exchange chromatography and ultrafiltration.
In case of ion exchange chromatography, a strong anion exchange material has been used allowing the elution of the proteins with a specific salt gradient according to the isoelectric point of the different proteins. This method has already been described by Bäckström et al [6] for the purification of MUC1. Here, the fusion protein was incubated with soluble enterokinase until complete cleavage of the fusion protein was detected. The reaction mixture was then applied to ion exchange chromatography separating IgG2a Fc and MUC1.
Although using the same purification procedure and receiving a similar chromatogram for elution some distinct impurities at 30 kDa and 43 kDa were detected with SDS-PAGE followed by Silver staining (Figure 8-1A). This suggests that the undesired proteins possess a similar isoelectric point as the target protein MUC1 being eluted at the same salt concentration. By changing the pH of the used elution buffer the overall charge of the proteins changes resulting in a different elution pattern. Since the amino acid composition of the unwanted proteins was not known, it was investigated whether a change in pH and in the elution gradient may improve product purity. Unfortunately, this did not yield higher purity of MUC1. With this purification procedure, a product purity of approximately 60 % was achieved. For further research or a pharmaceutical application, a purity degree of more than 95 % of the product is at least necessary.
According to SDS-PAGE, the size of the unwanted proteins is smaller than 100 kDa.
Therefore, ultrafiltration using a membrane with a 100 kDa cut-off was applied after ion exchange chromatography. As it can be seen on the SDS-gel (Figure 8-1B), the product solution was more concentrated rather than further purified. When comparing the solution before (B) and after (A) the filtration step, the two major impurities at 30 and 43 kDa are still present in the product solution suggesting these proteins to have sterically large structures that cannot diffuse through the pores of the membrane. This is supported by the fact that no proteins were found in the filtrate (F) solution.
Keeping in mind the size of MUC1 with 140 kDa, membranes with a cut-off higher than 100 kDa may lead to a loss of the product protein, which might diffuse through the pores
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of the membrane. Nevertheless, a membrane with a molecular weight cut-off of 300 kDa (XM300) was used for ultrafiltration. After the cleavage reaction was completed, the protein solution was directly applied to ultrafiltration. To wash out the undesired proteins and to remove residual salt, the retentate was washed several times with ultra-pure water.
According to Figure 8-1C, the main impurities at 30 and 43 kDa are washed out with each washing step. Comparing the product solution before (B) and after filtration (A) the amount of impurities was significantly reduced with regard to the proteins found at 30 and 43 kDa.
For complete removal, the number of washing steps was increased, which resulted in the production of MUC1 with a purity of more than 90 %.
Figure 8-1 Techniques applied for the purification of MUC1.
A) MUC1 was eluted with a gradient of NaCl in IEC: I and II represent different fractions of the eluting process;
B) Solution of cleavage reaction was applied to ultra-filtration with a 100 kDa cut-off:
B – before filtration, F – filtrate, A – after filtration;
C) Reaction solution was applied to a membrane with a 300 kDa cut-off: B – before filtration, A – after filtration, circle – very slight MUC1 band.
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With the finally used membrane having a 300 kDa cut-off, the unwanted proteins were successfully removed, with only a neglectable amount of MUC1 – less than 5 % - being lost in the first washing step.
Figure 8-2 SDS-PAGE of the obtained lyophilisates containing MUC1.
I to V – different lyophilisates (1:100). M – size marker, S – first extracted standard:
MUC1-IgG2a Fc (0.233 mg*mL-1).
The reaction mixtures of the different cleavage reactions – using free enterokinase, using immobilized enterokinase, continuous cleavage, and repetitive cleavage – were purified according to the finally developed purification procedure involving the membrane XM300.
The received retentates were lyophilized and analyzed for their MUC1 content using SDS-PAGE followed by Silver and Alcian staining (Figure 8-2). Afterwards, the MUC1 content was determined using the analytical method described in 10.2.19. With the received results, the purity of the lyophilisates was determined, which are summarized in Table 8-1.
Table 8-1 MUC1 content and purity degree of the obtained lyophilisates.
Lyophilisate /
mg
MUC1 content / mg
Purity / %
I II III IV V
repetitive batch 1 repetitive batch 2
(Mg-dependency) continuous cleavage analytical investigations
soluble EK analytical investigations
immobilized EK
43.0 42.7 30.0 12.0 11.0
40.9 39.3 18.6 11.3 10.3
95.1 92.1 62.0 94.6 93.5
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So far, the purification method for MUC1 involved the application of ion exchange chromatography, which is limited by the amount of protein that can be loaded onto the column and the long process time. Furthermore, a sufficient purity could not be achieved.
Now, ultrafiltration using a membrane with a cut-off of 300 kDa is applied, significantly reducing process time and increasing product purity from 60 to more than 90 %. Although, this is still not sufficient for pharmaceutical applications, the approach, however, gave promising results, with still some room for improvements.
8.2 Summary: Product purification
The findings of this chapter can be summarized as follows:
The application of ion exchange chromatography results in a purity of MUC1 of 60 %, not sufficient for further investigations or pharmaceutical applications.
The purity of MUC1 can be improved by using ultrafiltration. Here, a membrane with a molecular weight cut-off of 300 kDa was most suited increasing the purity of MUC1 to more than 90 % and significantly reducing process time. The entire purification procedure was more simplified.
Finally, 100 mg lyophilized MUC1 with a purity of 94 % could be produced (Figure 8-3).
Figure 8-3 Lyophilized MUC1 with a purity of 94 %.