Monitoring of the stability of the rhMBP

SDS-PAGE followed by western blotting analysis was used successfully to evaluate the stability of the rhMBP in different chromatographic fractions over the time and the conditions encountered during the downstream purification. In this stability study, we focused on studying the stability of the rhMBP in the final preparation. Suitably diluted aliquots of the rhMBP reference standard were incubated at room temperature and at 4 - 8 oC. The samples were taken after 0, 1, 4, 8 and 24 h and immediately frozen. All fractions were analyzed using the optimized Bio-plex immunoassay employing a calibration curve generated on the same day. The concentration of the rhMBP in each sample was determined from the calibration curve and was plotted vs the incubation time

The results showed the good stability of the rhMBP reference standard at 4 - 8 oC for up 24 h and at RT for up to 4 h which was in agreement to the previously reported results using western blotting analysis. Results confirmed the ability of the immunoassay to detect small changes in the rhMBP concentration with time. Thus the Bio-plex assay was considered suitable for studying the stability of the rhMBP in future work since it can provide a good estimate of the intact rhMBP remaining. The relative stability of the rhMBP at 4 - 8 oC indicated that the use of a refrigerated centrifuge for preparation of the rhMBP stock was essential as previously discussed in Chapter 3 (3.3.3).

Figure 30: A plot representing the change in the concentration of two rhMBP standard samples incubated at 4 - 8 o_{C and room temperature over 24 h showing the ability of the Bio-Plex assay to}

4.5 Conclusion

In this study, a CZE with an on-line sample stacking protocol was developed in order to determine the total protein concentration in chromatography fractions. The CZE assay provided a good alternative for the determination of total protein concentration without the need for using protein stains. A sandwich type, semi-automated immunoassay was developed for monitoring of the expression levels of the rhMBP in transgenic milk and milk fractions using the Bio-Plex system. The assay was considered as a total activity and stability indicating method since it determined the total amount of the intact rhMBP isoforms. The method was used successfully for monitoring of the elution pattern of the recombinant protein in the fractions obtained by column chromatography. Fingerprinting of the expression levels of the rhMBP in transgenic milk samples from each animal at different stages of the lactation cycle / over different lactation cycles can be achieved in minimal time. Theoretically, a large number of applications could be achieved employing the multiplexing capabilities of the Bio-Plex assay such as monitoring of impurities of different sources (process-related, product-related and host-related) and possibly monitoring of markers for the health condition of each animal using the same assay. Both the CZE and the Bio-plex methods formed a good testing protocol which is particularly useful for analysis of structural protein biopharmaceuticals which lack traceable enzymatic activity.

5 Capillary Isoelectric Focusing of Milk Samples

5.1 Introduction

Capillary isoelectric focusing is the automated version of conventional gel isoelectric focusing (IEF) technique where separation of a mixture of peptides or proteins is carried out according to their isoelectric points. CIEF combines the high sensitivity and high resolving power of IEF with the instrumental capabilities of CE. The capillary format allows faster separation under higher voltages, since efficient dissipation of the Joule heat generated during the electrophoresis separation is feasible (Dolnik, 2006; Mazzeo & Krull, 1991; Righetti, Gelfi, & Conti, 1997; Strelec et al., 2002).

The traditional two-step CIEF in coated capillaries was first introduced by Hjerten and co-workers (Hjerten, 1985). They believed that the use of covalently coated capillaries was necessary to eliminate the EOF in order to obtain successful focusing of the proteins and to prevent adsorption of the analytes to the charged capillary surface, thus improve the reproducibility of separations. Lack of hydrolytic stability of the covalent coat and the expensive price of coated capillaries has limited the use of CIEF to research purposes only (Hjerten & Zhu, 1985; Horvath & Dolnik, 2001; Mazzeo & Krull, 1991; Yeung, Atwal, & Zhang, 2003).

The formation of an electrical double layer is a general phenomenon observed when an electrolyte comes in contact with a charged surface. In the presence of an electric field, this electrical double layer is responsible for the bulk flow of liquid in the capillary, known as EOF, which is generally beneficial in CE. It helps mobilize bands past the detector window without contributing to zone dispersion. The use of buffer additives to establish a dynamic coat on the wall of the FS capillaries has been in use in CE applications. This approach overcomes the batch-to-batch and run-to-run variability associated with the covalently coated capillaries, enables the use of the relatively cheap uncoated FS capillaries in protein analysis applications and provides a simple tool to tune the magnitude and the direction of the EOF. Several trials have been reported to adapt this approach to CIEF

magnitude of the EOF along the pH gradient formed along the capillary, were the main limitations to obtaining high resolution and reproducible results (Dolnik, 2006; Horvath & Dolnik, 2001; T.-L. Huang & Richards, 1997; Righetti, 2004; Tang & Lee, 1997).

The rhMBP has a relatively high isoelectric point (pI ≈ 10.5) and milk contains very few basic proteins as discussed in Chapter 2 and shown in Table 2. In this study, a CIEF protocol in dynamically coated capillaries was developed for the screening of the TGmilk and the chromatographic fractions for the presence of rhMBP via its pI. Under these conditions, the ability of the CIEF technique to separate proteins according to their isoelectric points was considered a novel tool to identify peaks corresponding to the rhMBP. At first, a “one-step” CIEF protocol in dynamically coated capillaries was developed and compared to the conventional “two-step” CIEF protocol in coated capillaries. The one-step protocol was then employed in order to investigate various factors affecting the reproducibility and resolution of CIEF in dynamically coated capillaries.