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American Journal of Biochemistry and Biotechnology
Year: 2009  |  Volume: 5  |  Issue: 1  |  Page No.: 21 - 29

Classification of Pressure Range Based on the Characterization of Escherichia coli Cell Disruption in High Pressure Homogenizer

Ramakrishnan Nagasundara Ramanan, Beng Ti Tey, Tau Chuan Ling and Arbakariya B. Ariff    

Abstract: Problem statement: High pressure Homogenizer was used for cell disruption in many studies. But no work was carried out to study the characteristics of cell disruption in a wide range of pressure. Approach: The characteristics of Escherichia coli cell disruption was studied in Avestin small scale homogenizer by varying the operating pressure (50-1500 bar), cell concentration in the feed (1.39-12.51 g dry cell weight L-1) and number of passes (1-5 passes). Results: It was found that cell concentration between 1.39 g dry cell weight L-1 and 12.51 g dry cell weight L-1 has no effect on cell disruption while the pressure applied and number of passes gave different effects on cell disruption characteristics. In between 100 and 250 bar, the protein release was mainly due to point break. In this case, the variation in cell size was not significant with increasing number of passes and maximum protein release was not achieved even after many numbers of pass. However, selectivity of specific protein (interferon-α2b) was high as it is located predominantly in periplasmic region. In between 1000 and 1500 bar, the maximum protein release, maximum interferon-α2b release and drastic reduction of cell size was observed after the first pass. In subsequent passes, micronization of cell debris was observed but without much variation in protein release. There was no reduction in antigenicity of interferon-α2b even at 1500 bar. At 500 bar, the protein release and reduction of cell size were significantly increased with increasing number of passes. Conclusion: The pressure range for E. coli cell disruption was classified as low pressure range (100-250 bar), transition pressure (500 bar) and high pressure range (1000-1500 bar). The working pressure for the homogenizer could be selected by considering the operating cost and further downstream processing.

(1)

The calculation of compressor power requirement was taken from the manual supplied by the manufacturer (Avestin). Air required in Standard Cubic Feet (SCF) for different operating pressures was obtained from the manufacturer’s chart. The equation was modified to process 100 L of sample and it is given as below:

(2)

RESULTS

Effect of pressure and number of passes: Feedstock with cell concentration of 5.56 g DCW L-1 was passed up to 5 passes in six different pressures (50, 100, 250, 500, 1000 and 1500 bar). The samples were taken from every passage to analyze the amount of total protein release, the amount of IFN-α2b release and PSD. At 50 bar, there was no significant release of protein and reduction in cell viability (result not shown here). This is not surprised since the value of this pressure is below the threshold pressure for disruption to occur. Indeed, Siddiqi et al. [12] have reported that only little breakage was observed for baker’s yeast in APV homogenizer operated at pressure below 115 bar.

The maximum protein release (Table 1) of homogenization operated at 100 and 250 bar was low compare to the higher pressures (> 500 bar) even after 5 passes. The PSD for 100 bar shows negligible difference with increasing number of passes and for 250 bar, only very little variation could be seen (Fig. 1a and 2a). At 500 bar, both protein release and PSD (Fig. 1b and 2b) was varied significantly with the increase in number of passes. At 1000 and 1500 bar, sharp increase of protein release and shift of PSD (Fig. 1b and 2b) was observed after 1 pass. With increase in number of passes there was no much difference either in protein release or PSD.

Effect of cell concentration: Culture with three different cell concentrations (1.39, 5.56 and 12.51 g L-1) was passed up to 5 passes in homogenizer to investigate the effect of concentrations on low, transition and high pressure ranges. The extent of cell disruption was found by the percent of protein release, percent of reduction of cell viability and finally by PSD analysis. At high and transition pressure range, the difference in cell concentrations did not have any effect in either protein release or in reduction of cell viability (Fig. 3a and 3b). This is similar to the results published previously for different microorganism including E. coli[13-15].

Table 1: Characteristics of different cell disruption methods
Samples passed into high pressure homogenizer were denoted as pressure in bar followed by cell concentration in g L-1 and number of passes. Selective product release was calculated using equation 1. Specific surface area (SV) and volumetric mean diameter (VMD) were taken from particle size analysis. Power requirement (equation 2) and time requirement were calculated according to the manufacturer’s manual

Fig. 1a: Cumulative distribution of before distribution, osmotic shock and low pressure range after each number of passes for 5.56 DCW g L-1 of cell concentration. The legend indicates the pressure in bar followed by cell concentration in g L-1 and number of passes. The data are the average of replicates

Fig. 1b: Cumulative distribution of before distribution transition and high pressure ranges after each number of passes for 5.56 DCW g L-1 of cell concentration. The legend indicates the pressure in bar followed by cell concentration in g L-1 and number of passes. The data are the average of replicates

Fig. 2a: Density distribution of before distribution, osmotic shock and low pressure range after each number of passes for 5.56 DCW g L-1 of cell concentration. The legend indicates the pressure in bar followed by cell concentration in g L-1 and number of passes. The data are the average of replicates

Fig. 2b: Density distribution of before distribution, transition and high pressure ranges after each number of passes for 5.56 DCW g L-1 of cell concentration. The legend indicates the pressure in bar followed by cell concentration in g L-1 and number of passes. The data are the average of replicates

At low pressure range, the percent reduction of cell viability was found to be similar in all cell concentrations where as the difference was observed in protein release between 1.39 g L-1 and other concentrations.

Fig. 3a: Protein release for different cell concentrations. The legend indicates the pressure and cell concentration. The data are the average of replicates. The error bars represents the standard error

Fig. 3b: Reduction of cell viability for different cell concentrations. The legend indicates the pressure and cell concentration. The data are the average of replicates analyzed after1, 3 and 5 passes. The error bars represents the standard error

This difference was gradually increased with the increment of number of passes. This might be due to the low content of maximum protein and in turn due to the difference in the dilution factor between lower and higher content of maximum protein. On the other hand, similar profiles of PSD were seen for all the concentrations in all the pressure range (Fig. 3c).

Fig. 3c: Cumulative distribution of different pressure ranges for different cell concentrations after three passes and before disruption. The legend indicates the pressure followed by cell concentration in g L-1 and number of passes. The data are the average of replicates

Maximum protein and IFN-α2b release: Maximum protein release can be achieved in the transition and high pressure range. Both maximum protein and IFN-α2b release were similar with glass bead stirring (Table 1). At 500 bar maximum protein and IFN-α2b release was achieved with 3-4 passes while the same was achieved at 1000 bar with 1-2 passes and 1500 bar with 1 pass. Loss of antigenicity of IFN-α2b was not observed even at 1500 bar. The power requirement was calculated based on compressor power requirement and tabulated in Table 1. It should be noted that the exact power requirement would be more than the calculated value as the product has to be cooled.

Selective product release: High selectivity of product (IFN-α2b) release in homogenizer was achieved at low pressure range, which gave approximately twice the value of selectivity than high pressure range. Yet, when compared to osmotic shock which releases periplasmic protein the selectivity was five times lower.

DISCUSSION

Effect of pressure and number of passes: As mentioned in the result section, the maximum protein release was not achieved even after many no. of passes at 100 and 250 bar of homogenization operation. This is contrary to Hetherington et al. [16], who reported that maximum amount of protein release is independent of pressure. However, the result of this study is similar to that reported by Limon-Lason et al. [17], who explained that it was due to the release of insoluble protein complex and peptides through micronization of cell debris at higher pressure. Cell disruption is a two step processes which involved point break of cell envelope and followed by disintegration of cell wall along with degradation of cell debris[3]. Foster[1] reported that recombinant E. coli strains needed minimum of 4 kpsi (275.9 bar) to break the cells. Perhaps below this pressure range and above the threshold pressure, the cell disruption stopped at the first step and the pressure applied was not enough to disintegrate cell wall.

The PSD results (Fig. 1a and 2a) may indicate that total disintegration of cell wall was not occurred at these pressure ranges. In fact, the bimodal distribution was observed in all the samples (Fig. 2a).The results observed is in agreement with Keshavarz et al. [13], who found that the fermented grown cultures of Rhizopus nigricans were intact after two passes of homogenization at 100 bar. Balasundaram and Harrison[18] too have reported that disruption of baker’s yeast at 138 bar has a similar PSD to that of undisrupted yeast cells (6.2-5.9 μm). This leads to the classification of this pressure range as low pressure range where total disintegration did not occur.

At 1000 and 1500 bar, the maximum protein release (Table 1) was achieved after 1 pass with two steps of disruption occurred simultaneously. Further increase in number of passes will contribute only to micronization of cell debris. Earlier reports claimed that increase in number of passes above certain pressure would cause micronization of cell debris and also reduction in viscosity[14,19]. It was also mentioned that micronization won’t reduce the PSD much as if like total cell disintegration[12]. Even though the micronization was observed in this pressure range, there was not much variation in viscosity (result not shown here). The cell concentration range (0.14% DCW to 1.25% DCW) used in this study might not be significant to see the viscosity variation. Similar result was observed by Balasundaram and Harrison[18] where 5% wet concentration of baker’s yeast was used in their study.

However, slightly different observation on the PSD at this pressure range were reported by other researchers[15,20]. Bury et al. [15] reported that at 1350 bar, their product release was increasing up to 3 pass but then similar release was noticed at 2000 bar in 1 pass. This is due to the employment of gram positive microorganism (Lactobacillus delbrueckii ssp. bulgaricus) in their study which needs high strength to disrupt the cell wall. At 1600 bar, Van Hee et al. [20] observed an increase of IB with increasing number of passes for the disruption of Pseudomonas Putida without any note of soluble protein content. IB is not the original content of soluble component and has increased due to the micronization of cell debris which was in agreement with Limon-Lason et al. [17]. So this pressure range can be classified as high pressure range where the maximum protein release and lower particle size can be achieved after the first pass of homogenization.

At 500 bar, major portion of PSD (Fig. 1b and 2b) still could be seen near to the whole cell region leading to trimodal distribution curve after 1 pass. With the increase in number of passes the curve became bimodal which is different from the bimodal curve of low pressure range. Recently, Balasundaram and Harrison[18] mentioned that they had observed bimodal distribution for baker’s yeast after 5 pass at 414 bar. Since both the characteristics of low pressure and high pressure range was observed this could be classified as transition pressure range.

Maximum protein and IFN-α2b release: The result shows that the maximum protein and IFN-α2b release could be achieved above low pressure range but depends on both the pressure applied and number of passes in homogenization operation. Increasing the number of passes increases the running time and also makes the cell disruption process as batch wise rather than continuous mode. In contrast, increasing the pressure reduces the volumetric flow rate but also reduces the number of passes. The difference in power requirement between 500-1000 and 1000-1500 bar was similar but the difference in process time was increased drastically.

While comparing the protein release (Table1) and the PSD analysis (Fig. 1b and 2b) it is clear that micronization of cell debris was not necessary for maximum protein release and also for maximum IFN-α2b release. Yet it depends on the further downstream operation that follows the cell disruption. High particle size with low viscosity would be useful for centrifugal separation and dead-end filtration[1,14], but low particle size accompanied with low viscosity would be beneficial for anionic expanded bed adsorption[18,21-24]. On the other hand, the characteristics of homogenates is not an affecting factor in cross flow filtration[19].

Selective product release: The result shows that even at low pressure range, release of protein was not limited to periplasmic area. SDS-PAGE analysis (Fig. 4) shows that the profile of low pressure range was similar to high pressure range. In both cases, high molecular weight proteins were observed in higher concentration than osmotic shock. This is in line with other mechanical disruption[25-27].

Fig. 4: SDS-PAGE passed through different stages of homogenizer along with osmotic shock and glass bead stirring. Legend of samples passed into high pressure homogenizer were denoted as pressure in bar followed by cell concentration in g L-1 and number of passes. The approximate amount of protein loaded in all the sample wells was between 8 and 15 μg

For example, Balasundaram and Harrison[27] had observed 67% β-galactosidase (cytoplasmic high molecular weight protein) with 48% of total protein in their optimized hydrodynamic cavitation method for cell disruption. Middelberg[5] pointed out that disruption through mechanical method is non specific and hence selective product release is not limited to the release of periplasmic protein release. In case of E. coli, the strength depends mainly on outer cell wall which consists of peptidoglycan layer. Once broken, the inner cell wall does not have enough strength to resist unless it is stabilized osmatically. High selective protein release would be generally preferred for further downstream processing as it reduces the impurities in chromatography system and also easier for centrifugation and dead end filtration as the cells are still intact. However, the selective product release conducted at lower pressures was captured only low product in expanded bed mode operation due to the higher interaction between yeast cell debris and anionic beads[18].

CONCLUSION

This study demonstrated that the cell disruption characteristics varied differently with different pressure ranges. At low pressure range, the cells were experienced point-break losing the soluble content partly but without the total disintegration of cell wall. Selective product was achieved in this range but maximum protein release might not be possible even after many numbers of passes. At transition pressure range the protein release and the PSD varied significantly, with the increment in number of passes leading to maximum protein release and micronization of cell debris. At high pressure range, the maximum protein release and the total disintegration was attained after the first pass and further increase in passes will cause only micronization of cell debris. The fact that the release of protein and the reduction of particle size did not tally each other was clearly due to the difference in cell disruption characteristics at different pressure ranges. The selection of process condition shall be based on the subsequent downstream operation to be employed, optimal power and time requirement.

ACKNOWLEDGEMENT

This study was funded by the Ministry of Science, Technology and Innovation, Malaysia under the SR IRPA research grant (Project Number: 03-02-04 SR2010 SR0008/05). CFU experiment was performed by Ms. Azulia Zoolkiflie and Mr. Sam Tek Sing which was their part of undergraduate study. Ramanan is a recipient of graduate research fellowship from University Putra Malaysia.

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