7. METODOLOGÍA
7.2 METODOLOGÍA ADDIE
7.2.3 DESARROLLO DEL A.V.A
The experimental data was used to estimate total binding capacities of the packed and expanded bed rProtein A media. The binding capacities for 4D5 Fab’ are compared to the total capacities for human IgG quoted by manufacturers in Table 6.2.1. Lower capacities for Fab’ are expected as the capacities are based on the mass of antibody bound per mL of media. The mass of Fab’ is approximately one third that of whole IgG. Therefore, assuming IgG and Fab’ bind to protein A in the same ratio, approximately one third the mass of Fab’ compared to IgG will bind to the same volume of media. Furthermore, protein A binds to different sites on Fab’ and IgG, and the affinity for the Fab’ site is lower (Starovasnik et al., 1999), which will reduce the binding capacity further. Measured binding capacities of packed and expanded bed media for Fab’ were 12.5 and 13.5 mg mL'1 respectively, which is approximately one quarter that quoted for IgG (50 mg mL'1).
Chromatography Method Chromatography media Total Binding Cl Quoted value (for IgG) pacity (mg m L'1) Experimental value (for Fab’) Packed bed chromatography rProtein A Sepharose® Fast Flow 50 12.5 Expanded bed chromatography Streamline rProtein A 50 13.5
Table 6.2.1 Comparison o f experimental and quoted values fo r the total binding capacity o f packed bed and expanded bed chromatography media. Quoted values are those given by manufacturers fo r the binding o f human IgG. Experimental values are fo r 4D5 Fab
The breakthrough data for the two chromatography runs was used to estimate the Fab’ yields and dynamic binding capacities of the chromatography media at different levels of Fab’ breakthrough. The yields and capacities obtained are shown in Figures 6.2.3. and 6.2.4 respectively. For both packed and expanded bed chromatography, yields decreased and matrix dynamic capacity increased with increasing Fab’ breakthrough, as expected. However, yields and capacities were appreciably greater for packed bed chromatography.
The observed differences can be attributed to the contrasting patterns o f Fab’ breakthrough. The packed and expanded bed breakthrough curves, adjusted for differences in column volumes and the Fab’ concentration in feed streams, are compared in Figure 6.2.5. To adjust for differences in the feed concentration o f Fab’ it was assumed that the affinity of protein A for Fab’ was independent o f Fab’ concentration for the range o f Fab’ concentrations 0.1-0.4 mg mL’1.
100 ^ CD O O <D 20 30 40 % Breakthrough
Figure 6.2.3 Comparison o f Fab ’ recovery at different levels o f Fab ’ breakthrough fo r packed bed chromatography (PBA; trend shown by solid line) and expanded bed chromatography (EBA; trend shown by dotted line). PBA was perform ed using a 1 mL HiTrap rProtein A column at a flo w rate o f 40 cm hr' . EBA was perform ed using 25 mL Streamline rProtein A media in a 25 mm diameter Streamline column. Operating flo w rates fo r EBA were 185 cm h r 1 (load and wash cycles, perform ed in expanded bed mode) and 90 cm h r ' (elution cycle, perform ed in packed bed mode).
1 0 0 o TO Gi ro o "to o h- < CQ LU ■oc ro 80 60 40 < CD CL 20 O ' 10 20 30 40 % B re a k th r o u g h 50 60
Figure 6.2.4 Comparison o f media capacity at different levels o f Fab ’ breakthrough fo r packed bed chromatography (PBA; trend shown by straight line) and expanded bed chromatography (EBA; trend shown by dotted line). Media capacity is expressed as a percentage o f the total binding capacity, which was estimated from the chromatography data as 12.5 mg m L'1 (packed bed media) and 13.5 mg m L'1 (expanded bed media). PBA was performed using a 1 mL HiTrap rProtein A column at a flo w rate o f 40 cm hr'1. EBA was performed using 25 mL Streamline rProtein A media in a 25 mm diameter Streamline column. Operating flo w rates fo r EBA were 185 cm hr'1 (load and wash cycles, performed in expanded bed mode) and 90 cm hr~l (elution cycle, perform ed in packed bed mode).
100 o
f <
g > S 60 - I s « ® I 40 ■2
Q. --- 1 0 100 200 300 Column VolumesFigure 6.2.5 Comparison o f F ab’ breakthrough curves fo r packed bed (PBA) and expanded bed (EBA) chromatography. Breakthrough curves have been adjusted to account fo r differences in the concentration o f Fab ’ in the column feeds and fo r differences in column volumes. Data fo r EBA was only obtained up to 50% breakthrough.
The pattern of breakthrough for packed bed chromatography was similar to the expected or ‘traditional’ chromatography breakthrough curve, however the breakthrough pattern for expanded bed adsorption was more unusual. Fab’ concentration in the flow-through from the expanded bed immediately increased to -30% o f the feed concentration from the onset of column loading; the level of breakthrough then continued to rise but at a much slower rate compared to both the initial increase and the rate of increase in breakthrough for packed bed chromatography. The higher levels of Fab’ loss in the expanded bed flow-through accounts for the lower Fab’ yields compared to packed bed purification. In addition, the very low matrix capacities at low levels of breakthrough can also be attributed to the immediate breakthrough to -30%.
Breakthrough from the expanded bed chromatography column was only monitored during one purification run, however similar patterns of breakthrough have also been observed during the expanded bed purification of alcohol dehydrogenase from yeast homogenates (personal communication, N. Willoughby). Losses o f Fab’ during column loading in the experiments described above may have been exaggerated as a result o f using a minimal volume of chromatography media. Increasing the volume of media will increase the column residence time, allowing more time for Fab’ binding and thereby reducing Fab’ losses in the column flow-through. Use of a small bed volume may also have resulted in streaming, with some product bypassing the bed completely. Such an effect would have further exaggerated Fab’ losses in the breakthrough.
When specifying operating conditions for a chromatographic purification process, there generally has to be a trade off between maximising the utilisation of expensive chromatography media (achieved by operation at high breakthrough) and minimising the loss of a high value pharmaceutical product (achieved by operation at low breakthrough). The trade off between product yield and matrix utilisation can be determined from a plot combining product recovery and matrix capacity data. Figure 6.2.6 shows such a plot for the expanded bed purification of 4D5 Fab’. The operating conditions which maximise matrix utilisation whilst minimising product losses are determined from the intersection of the two sets of data, in this case 50%
breakthrough. Operation at lower than 50% breakthrough will increase yields by reducing Fab’ losses, however the chromatography media will not be used to optimum capacity. Operation at higher than 50% breakthrough will improve media utilisation but at the expense o f product yield. Economic data relating matrix costs and product value is also required to provide a more detailed cost analysis o f the process and allow identification o f the optimal operating conditions.
100 100 ■ 0 > o o 0 40 40 -O ro L1_ 20 50 10 30 40 o 0 o 0 "O 0 o % Breakthrough
Figure 6.2.6 Effect o f the level o f breakthrough on F a b ' yield and matrix dynamic binding capacity fo r the expanded bed purification o f 4D5 F a b ' from E. coli unclarified periplasmic extracts. Such information, combined with cost data fo r product revenue and matrix price, could form the basis o f an economic analysis fo r
Finally, the degree o f process stream purification attained using packed and expanded bed chromatography was compared. Similar levels of purification were achieved, shown by the similar purification factors (Table 6.2.2). Comparable levels of Fab’ purity was also illustrated by SDS-PAGE analysis of the column eluates (Figure 6.2.7). Process Stream PE Specific Fab’ (mg mL'1) SA Purification factor (-) EE Specific Fab’ (mg m L'1) JA Purification factor (-) Column feed 0.078 - 0.101 - Column eluate 1.1 14 1.26 12
Table 6.2.2 Comparison o f process stream purification achieved using packed bed (PBA) and expanded bed (EBA) affinity purification o f 4D5 Fab
On the basis of results from this study, packed bed affinity chromatography appears to be the more efficient purification method because of the higher Fab’ yields and matrix capacities at low levels of breakthrough. However, the study does not consider additional processing factors such as operating time and process costs. A major advantage o f expanded bed adsorption is the fact that it can be performed on unclarified feedstocks. Packed bed adsorption requires clarification of the process stream prior to application to the column, which can add considerable time and cost to the process, and will result in reduced process yields. A disadvantage of expanded bed adsorption however is the high cost of the chromatography media and the requirement for large volumes of buffers during column equilibration and washing, which will further increase the operational costs. A more detailed comparison of packed and expanded bed purification processes, which includes reference to process yields and operating time, is given in Chapter 8.
66.2 kD a
m i|
97.4 kDa * 66.2 kDa 11' mtKKKKm 45 kDa --- ► 31 kDa --- ► 21.5 kDa(a) Packed bed chromatograp
m
:::
mm
mm
ly (b) Expanded bed chromatography
Figure 6.2.7 SDS-PAGE analysis o f Fab' purity following packed bed (a) and expanded bed (b) protein A affinity’ purification o f 4D5 Fab \
(a) Packed bed affinity purification o f 4D5 Fab ’ Lane 1 Low molecular weight markers
Lane 2 Purified 4D5 Fab ' standard
Lane 3 Column load (clarified E. coli periplasmic extract) Lane 4 Column eluate (purified 4D5 Fab )
(b) Expanded bed affinity> purification o f 4D5 Fab ’ Lane 1 Low molecular weight markers
Lane 2 Purified 4D5 Fab ’ standard
Lane 3 Column load (unclarified E. coli periplasmic extract) Lane 4 Column eluate (purified 4D5 Fab ’)
6.2.5 Summary
Packed bed and expanded bed affinity purification of 4D5 Fab’ have been compared on the basis of Fab’ yield, matrix capacity and the degree o f process stream purification achieved.
Fab’ yields and dynamic binding capacities were greater for packed bed chromatography compared to expanded bed chromatography. Differences were attributed to the contrasting patterns of Fab’ breakthrough, with considerably greater losses of Fab’ in the flow-through from the expanded bed column. Estimates o f the total binding capacity of the two matrices were similar; capacities of 12.5 mg mL’1 and 13.5 mg m L'1 were obtained for packed and expanded bed media respectively. The degree of purification was also similar for the two processes; a purification factor o f 14 was recorded for packed bed purification and 12 for the expanded bed process.
This study provides only an initial insight into the differences between the chromatographic processes. A detailed assessment of processing time and costs would also be required to give a more comprehensive view of the advantages and disadvantages of the process alternatives.