This fraction likely consists of host cell proteins, not fully processed VPs, non-capsid viral proteins. Since the ratio of the peak heights of the VP4 and VP1 peaks ...
Appendix 35 A novel proteomics-based approach for the characterization of FMDV antigens M.M. Harmsen, J. Jansen, D. Westra and J.M Coco-Martin Research and Development Department, Products Division, Animal Sciences Group-WUR, Edelhertweg 15 8219 PH Lelystad, The Netherlands. Abstract: During vaccine manufacturing, the golden standard for quantifying the antigen contents are the 146S levels as measured by sucrose density gradient. This method gives information whether the antigen is intact and whether it contains RNA. It does not give any information regarding the proteolytic integrity of the antigen and presence of possible contaminants. In this paper, a novel proteomics-based approach is described for the characterization of antigen preparations. For the analysis of the antigen samples, a Surface Enhanced Laser Diffraction Ionization- Time of Flight- Mass Spectrometer (SELDI-TOF-MS) was used. The antigen samples were applied to protein chip arrays with different surface chemistries, including hydrophobic, cationic, anion and activated protein chips. Analysis took place after addition of the ionization matrix. The VP0 to VP4 capsid proteins could be detected in the antigen preparation with the NP20 and other array types. These findings were confirmed using activated biochips labelled with llama single-domain antibody fragments specific for FMDV of the different serotypes included. All the observed molecular weights were comparable to the theoretically calculated MWs of the different analysed serotypes. Interestingly, more peaks were observed in the expected MW range of the VP proteins. These minor differences could possibly be attributed to modification of the VP proteins caused by post-translational processing of the VP proteins. ProteinChip-assisted analysis using SELDI-TOF-MS is a promising tool for the characterization of antigens during vaccine production. In addition this tool might be valuable to measure product quality at an early stage during manufacturing. Introduction: During vaccine manufacturing, the golden standard for quantifying the antigen contents are the 146S levels as measured by sucrose density gradient (Van Maanen and Terpstra, 1990). This method gives information whether the antigen is intact, containing the RNA or not. It does not give any information regarding the quality of the antigen and possible contaminants in the antigen preparations. In this paper, a novel proteomics-based approach is described for the characterization of antigen preparations. The following structural characteristics of FMDV are relevant for interpretation of SELDI data. The 146S particle consists of the viral RNA molecule and 60 copies each of four viral proteins (VP1-4) that are excised from a polyprotein (Sobrino et al., 2001). VP1-3 are about 24 kDa in size whereas VP4 is about 9 kDa. VP4 is myristoylated at its N-terminus after cleavage from the polyprotein (Chow et al., 1987). VP4 is located internally and is released from the virion upon dissociation into 12S particles (Burroughs et al., 1971). The cleavage between VP2 and 4 occurs quite late in maturation. The uncleaved precursor is termed VP0. This precursor appears to be more often found in 75S particles (Doel and Chong, 1982). Type O viruses often contain a disulfide bridge linking VP1 and 2 (Logan et al., 1993). A disulfide bridge may also occur between VP3 subunits around the five-fold axis (Fry et al., 2005). Materials and Methods: For the analysis of the FMDV antigen samples we employed the ProteinChip technology of Ciphergen Biosystems which is a commercialized form of Surface Enhanced Laser Dissociation and Ionization- Time of Flight- Mass Spectometry (SELDI-TOF-MS). This technique basically combines two well known technologies for the analysis of protein samples. By means of retentate chromatography proteins are a bound to spot surfaces of ProteinChip arrays and subsequently after processing of the arrays for mass spectrometry analyzed in a time of flight mass spectrometer (PCS4000, Ciphergen Biosystems). The arrays are equipped with different chromatographic flavours, including hydrophobic, cationic, anionic surfaces and so called preactivated surfaces to which antibodies can be coupled covalenty. FMDV was cultured using BHK-21 cells grown in suspension. Following cell death and subsequent release of virus in the medium, the suspension was clarified and inactivated with binary
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ethyleneimine. Aliquots were stored at -80 until further use. Llama anti-FMDV mAb fragments were generated as described previously (Harmsen et al., 2006). Briefly, Llamas were immunized with FMDV. B-cells were isolated, mRNA was isolated and llama VHH antibody fragment coding sequences were amplified in order to construct a phage library that expresses llama VHH antigen binding antibody fragments. Phages expressing VHH antibody fragments that recognize FMDV in ELISA format were selected. Selected FMDV binding VHHs were expressed in bakers yeast, purified, and used as baits after immobilization on pre-activated proteinchip arrays. Results: Although the FMDV is a far to big a protein complex (MW~8 x 106 Da) to be detected by the PCS4000, we can detect the individual proteins that make up the viral capsid since the process of laser desorption and ionization (LDI) breaks up the non-covalent protein interactions in the complex. Thus in the acquired mass spectrometry spectra one is able to discern peaks that can be linked to the monomeric viral proteins. Since the resolution of the PCS4000 is much higher than gel electrophoresis or HPLC also minor changes (e.g. post translational modifications or modifications induced during down stream processing) in the molecular weight of proteins can be observed. In our antigen preparations we can detect the readily resolved VP1, 2 and P4 as peaks at 23.2 kDa (doublet), 24.4 kDa and 8.90 kDa (myristoylated) respectively. In contrast, the VP3 protein is not as easy to desorb and ionize as VP1, 2 and 4. The VP3 peak is resolved partly as a 23.9 kDa peak in highly purified virion preprations or after limited trypsin digestion of the antigen preparation. Comparative analyses of antigen preparations that had been submitted to different purification strategies, i.e. sucrose gradient centrifugation, size exclusion filtration and immune affinity purification revealed that a substantial fraction of the proteins that is present in the crude antigen preparations is not incorporated in viral capsids. This fraction likely consists of host cell proteins, not fully processed VPs, non-capsid viral proteins. Since the ratio of the peak heights of the VP4 and VP1 peaks (VP4/VP1) increased in parallel with increasing purification stage we postulate that the VP1, 2 and 3 proteins reside probably in 12S complexes. Also known process related reagents such as PEG6000 and bovine serum albumin could be detected in routinely produced crude viral antigen preparations. To further explore the analytical potential of the SELDI technology we studied the effects of controlled limited tryptic digestion of FMDV antigen preparations on the molecular integrity of the capsid proteins. By employing anti-FMDV single domain llama monoclonal antibody fragments (Harmsen et al., 2006) to study immune affinity purified FMDV protein complexes we were able to visualize the effect of controlled trypsinization in VP1. After limited tryptic digestion of the antigen the peaks representing the intact VP1 protein disappeared from the spectra. Concomitantly, abundant peaks corresponding to the VP1 aa1-145 N-terminal fragment and the near carboxy terminal VP1 aa146-200 and VP1 aa-150-200 fragments can be easily discerned in the spectra. Interestingly, data obtained with a llama-mAb fragment that specifically recognizes the GH-loop strongly suggest that the steric configuration of VP1 embedded in the capsid complex is rather stable. Although the capturing antibody is directed specifically at the intact aa136-aa160 segment of VP1 this monoclonal bait still was able to capture protein complexes containing cleaved GH-loops. Preliminary data indicate that the SELDI-ToF method also can be used to analyse FMDV proteins in vaccine in double oil emulsion. Further experiments are required though to optimize the protein capture conditions. Discussion: Here we showed the use of the ProteinChip technology of Ciphergen Biosystems for the monitoring of constituents of FMDV antigen preparations. Especially in the MW range in which conventional PAGE lacks resolution and detection sensitivity the SELDI-TOF MS approach detected FMDV relevant protein signals i.e. the VP4 capsid protein. Beside the peaks that represent the structural proteins VP1-4 peaks representing other proteins are observed as well as well. So far the nature of these proteins is not clear. These peaks might represent host cell proteins, not fully expressed VPs, non-structural or degraded viral proteins and non-viral proteins. Purification of these particular proteins followed by peptide map mass fingerprinting could reveal the identity of these proteins. The resolution and mass accuracy of the SELDI technology allowed us to identify fragments of VP1 that had been generated by limited tryptic digestion of crude antigen preparations. Clearly under these conditions the peptide bond at 145R-146G of VP1 is very sensitive to enzymatic degradation. Identification of capsid protein related fragments in routinely produced antigen preparations might give us a clue whether proteolytic degradation of proteins is one of the mechanisms that underlies the decline of immunogenic potency of formulated vaccines upon prolonged storage.
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The finding that strategies based on size exlusion for 146S particle purification enriched the antigen preparation for VP4 strongly suggest that crude viral antigen preparations contain VP1, 2 and 3 proteins that are not complexed in 146S particles but are present in 12S complexes or not complexed at all. This observation needs to be confirmed by SELDI-ToF MS analyses of immune affinity captures employing 12S, 12S/146S and 146S specific baits. Preliminary data obtained by employing an anti-146S Llama antibody fragment which only recently became available suggest that, indeed, most of the capsid proteins of a viral antigen preparation are not complexed in 146S particles. Thus , the development of production and DSP strategies focussed on the generation of high yield of 146S particles in antigen preparations might increase the specific immunogenic properties of FMDV antigen and prolong the shelf life of FMDV vaccine potency after formulation. Whereas the ProteinChip technology acquires sensitively high mass accuracy data at the lower MW range PAGE is the method of choice in the higher MW range. Indeed slab gels stained with sensitive protein staining dyes show more peaks in the higher MW range (>30 kDa) as compared to SELDI analyses. In conclusion, full characterization of viral antigen preparations by employing a combination of the two approaches would yield a maximum of information regarding the protein constituents of a crude viral antigen preparation in a production environment. Conclusions: • SELDI—ToF-MS could be used for product/process characterization during process optimization and manufacturing, leading to ultimately faster process development times and improved product properties. • Characterization of FMDV antigen preparations by SELDI-ToF MS measurements could possibly replace the costly FMDV vaccine potency testing in animals. Recommendations: • None at this stage. References: Burroughs, J.N., Rowlands, D.J., Sangar, D.V., Talbot, P., Brown, F. 1971. Further evidence for multiple proteins in the foot-and-mouth disease virus particle. J Gen Virol 13(1): 73-84. Chow, M., Newman, J.F., Filman, D., Hogle, J.M., Rowlands, D.J., Brown, F. 1987. Myristylation of picornavirus capsid protein VP4 and its structural significance. Nature 327(6122): 482-486. Doel, T.R., Chong, W.K. 1982. Comparative immunogenicity of 146S, 75S and 12S particles of foot-and-mouth disease virus. Arch Virol 73(2): 185-191. Fry, E.E., Stuart, D.I., Rowlands, D.J. 2005. The structure of foot-and-mouth disease virus, In: Foot-and-mouth Disease Virus. Springer-Verlag, pp. 71-102. Harmsen, M.M., Van Solt, C.B., Fijten, H.P.D., Van Keulen, L., Rosalia, R.A., Weerdmeester, K., Cornelissen, A.H.M., De Bruin, M.G.M., Eblé, P.L., Dekker, A. 2006. Passive immunization of guinea-pigs with llama single-domain antibody fragments against foot-and-mouth disease. Vet. Microbiol.Submitted for publication. Logan, D., Abu-Ghazaleh, R., Blakemore, W., Curry, S., Jackson, T., King, A., Lea, S., Lewis, R., Newman, J., Parry, N., et al. 1993. Structure of a major immunogenic site on footand-mouth disease virus. Nature 362(6420): 566-568. Sobrino, F., Saiz, M., Jimenez-Clavero, M.A., Nunez, J.I., Rosas, M.F., Baranowski, E., Ley, V. 2001. Foot-and-mouth disease virus: a long known virus, but a current threat. Vet. Res. 32(1): 1-30. Van Maanen, C., Terpstra, C. 1990. Quantification of intact 146S foot-and-mouth disease antigen for vaccine production by a double antibody sandwich ELISA using monoclonal antibodies. Biologicals 18(4): 315-319.
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