Generierung und Charakterisierung eines immunoaffinitätsbasierten Reinigungssystems für rekombinante Proteine

摘要

The aim of this work was the invention of a purification and detection system for recombinant proteins based on the specific interactions of the twelve amino acids containing epitopetag 54 and the corresponding monoclonal antibody 54. As opposed to already established systems the mAb54/tag54 system should provide a cost-effective and robust immunosorbent and the possibility to elute proteins under native conditions. To achieve this, all system-relevant parameters were analyzed whereas the costs for allocation of mAb54 and the necessity to use low pH for elution of bound proteins were determined to be the most crucial points. To minimize the affiliation costs of mAb54 the cultivation of the secreting mAb54 cell-line was analyzed in two different disposable bioreactor systems whereas the product accumulation, handling of reactors and general costs of both systems was compared. Under optimal conditions the amount of 1 mg mAb54 could be produced for ca. 1280 € within 45 days. For the subsequent necessary purification of mAb54 the three sorbents Protein A, Protein G and MEP HyperCel were compared. Best results could be obtained with the MEP HyperCel sorbent, whereas yields of >95 % and purities of >90 % were achieved. With due regard to the costs of the antibody purification the total affiliation costs for 1 g of mAb54 amounted to ca. 1700 €. To enable the use of elution buffers while immunoaffinity chromatographie (IAC) which would not lead to protein denaturing of neither the target protein nor the immunosorbent, the affinity between mAb54 and tag54 was reduced via specific modifications of the tag54. Thereto the essential amino acids within the tag54-sequence for the binding to the mAb54 were scanned via alaninscan (K4A, D5A, W6A) and substituted against structural similar amino acids. Overall there were ten rational designed tag54-variants produced and analyzed with regard to the kinetic parameters of interactions between tag54 and mAb54. The effect of different buffers together with temperature on the elution efficacy of well-chosen tag54-variants was analyzed via Surface Plasmon Resonance Spectroscopy. Thereby the variant P16 – an 18 amino acids containing trimer of the six amino acids core-epitope sequence – could be proven to be most promising. At pH 4,0 with addition of 0,5 M NaCl the elution efficacy could be determined to 70 % while with the unmodified tag-variant under the same conditions only 30 % could be achieved. Because of the addition of different salts the elution efficacy of the P16-variant could be raised to up to 90 %. For production of the immunosorbent the mAb54 was undirected linked NHS-activated sepharose. A linking efficacy of over 99 % in 60 min was reached and the optimal ligand density could be determined to 4 mg mAb54/mL. The specific capacity of the sorbent at this density was 6 nmol/mL. For description and characterization of the mAb54 immunoaffinity system two model proteins, tdTomato and Protein L, were C-terminal fused with the P16-variant of tag54 and recombinant expressed in E. coli. Both proteins could be eluted with high purity and mostly in native form (>80 %). Next to the suitability as terminal fusiontag it was analyzed if the tag54-sequenz could be used as linker between the variable domain of heavy and light chain of scFv-antibodies. The against „Grapevine leafroll-associated virus 3“ generated scFvLR3cp-1 was used as model. The existing Whitlow 218 linker was replaced with three different variants of the tag54-sequence. After expression in E. coli and purification via IMAC and IAC the scFv-antibody was analyzed in regard to purity, the kinetic parameter of interaction between scFv and antigen together with the comparison of functional amounts of scFv. It was ascertained that the attachment of the scFV to the antigen GLRaV3-CP was not affected by the use of tag54 as linker, the purity of IAC-purified scFv was as with Protein L-tag54 and tdTomato-tag54 very high whilst the IMAC-purified scFv showed notably contamination with host-cell proteins. Loss of activity due to the IAC could not be observed. The tag54/mAb54 system could successfully be optimized and used for detection and purification of different model proteins. The purification efficacy was very high, the particular target proteins could be eluted without unspecific contaminations through host cell proteins and for the major part (>80 %) the protein integrity and activity could be obtained. The aim of this work, the development of a cost-effective and robust immunosorbent, which could be used for purification of proteins under mainly native conditions, could be achieved.