Investigating the Use of Column Inserts to Achieve Better Chromatographic Bed Support Tian Lan, Spyridon Gerontas, Graeme R. Smith, and John Langdon The Advanced Centre for Biochemical Engineering, Dept. of Biochemical Engineering, University College London, London WC1E 7JE, U.K.
John M. Ward Research Department of Structural and Molecular Biology, University College London, London WC1E 6BT, U.K.
Nigel J. Titchener-Hooker The Advanced Centre for Biochemical Engineering, Dept. of Biochemical Engineering, University College London, London WC1E 7JE, U.K. DOI 10.1002/btpr.1597 Published online July 27, 2012 in Wiley Online Library (wileyonlinelibrary.com).
Chromatography plays an important role in the downstream processing of proteins. Over the past years, there has been a steady move toward the adoption of more rigid, porous particles to combine ease of manufacture with increased levels of productivity. The latter is still constrained by the onset of compression where the level of wall support becomes incapable of withstanding flow-induced particle drag. In this study, we investigate how, by the installation of cylindrical column inserts, it is possible to enhance the level of wall support. Experiments were conducted to examine the effect of the position of the insert in the column, and also of the insert dimensions on the critical velocity at which the onset of compression occurs. It was found that when installed at the bottom of the column, inserts can provide up to a 20% increase in critical velocity without significantly affecting column hydrodynamics, C 2012 American Institute of Chemical Engias measured by the level of axial dispersion. V neers Biotechnol. Prog., 28: 1285–1291, 2012 Keywords: column insert, critical velocity, wall support, compression, column efficiency
Introduction Chromatography is widely used in the purification of proteins, contributing significantly to the cost of the overall downstream process. Chromatographic processes are typically developed in laboratory columns with diameters less than 50 mm. Scale-up is conducted by increasing the column diameter, keeping the bed height and superficial velocity constant. However, this results in an increased hydrodynamic pressure drop due to loss of wall support as the majority of commercially available chromatography media are compressible.1–4 Over time, the chromatography bed will be overcompressed and the performance of the column will deteriorate. As a result, in large-scale manufacturing, chromatography columns have to be repacked periodically. A measure of column compressibility is the superficial critical velocity, defined as the maximum flow velocity the column can operate at before the pressure starts to rise without limit.5–7 For a given column geometry a high critical velocity means that the resin is less compressible and the bed height is not reduced significantly over a wide range of operating flow rates. Therefore, the column can operate at Correspondence concerning this article should be addressed to N. J. Titchener-Hooker at
[email protected]. C 2012 American Institute of Chemical Engineers V
high flow rates, potentially resulting in high levels of productivity. Wall support is a key parameter that affects the critical velocity. It derives from frictional forces acting at the column walls and is affected by the aspect ratio (diameter/ length) of the packed bed.8–10 The impact of this force is negligible when the aspect ratio of the packed bed is above 2.1 In this article, the ability of cylindrical inserts, of negligible wall thickness, to increase the critical velocity without affecting significantly column efficiency was studied. The idea of using inserts is primarily focused on providing a greater operating range for more traditional, compressible beads and hence to extend the hydraulic capability of existing equipment. The inserts were made from cylinders constructed of plastic or stainless steel, secured to the bottom of the column, and mounted concentrically. The materials of construction are fully compatible with sterile operation and CIP procedures. Recognizing, however, that the introduction of any elements within the packed structure may disturb the even flow distribution through the column, we also assessed the impact of the inclusion of inserts on column efficiency as measured by the theoretical plate number and the degree of peak asymmetry for a nonretained tracer molecule. 1285
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Figure 1. a. Stainless steel insert configurations. The support bars can be removed to enable single or double inserts to be used. Design for both XK 50 and BPG 100 columns. The inserts diameters were 15 and 30 mm, and their length was 45 mm. All supporting bars were 50 mm long. b. Plastic insert configuration. The support bars were fixed in position. The inserts diameters were 16 and 35 mm, and their length was 45 mm.
Materials and Methods Equipment XKTM 16 (i.d.: 16 mm diameter) and XKTM 50 (i.d.: 50 mm diameter) columns were used for laboratory-scale experiments, whereas a BPGTM 100 (i.d.: 100 mm diameter) was used for pilot-scale experiments. All columns were purchased from GE Healthcare (GE Healthcare, Uppsala, Sweden). Chromatography resins Sepharose 4B, Sepharose CL-6B (GE Healthcare, Uppsala, Sweden), and PurabeadV 6XL (ProMetic Biosciences, Cambridge, UK) resins were used in this study. All are agarosebased resins with no ligands attached. The average particle size was 90 lm in all cases. Each resin had a different level of agarose crosslinked structure and hence rigidity. Experiments performed with non-crosslinked and crosslinked resins evaluated the effect of resin rigidity on bed compression. Specifically, Sepharose CL-6B and P6XL resin each contain 6% v/v crosslinked agarose to achieve a high level of rigidity. Sepharose 4B contains 4% v/v agarose, which is not crosslinked, thus it is of lower rigidity than the other two resins. In all experiments, columns were repacked with the same resin before each critical velocity determination. R
¨ KTAexplorerTM system was operating specifications. The A applied for the critical velocity determination in XK 16 columns packed with P6XL, SepharoseTM CL-6B or Sepharose ¨ KTAcross4B (maximum flow rate: 100 mL min1). The A flowTM system was applied for the critical velocity determination in XK 50 and BPG 100 columns packed with P6XL ¨ KTApilotTM (maximum flow rate: 600 mL min1) and the A system was applied for critical velocity determination in XK 50 and BPG 100 columns packed with P6XL (maximum flow rate: 800 mL min1). Inserts All inserts were purpose made and of hollow cylindrical shape constructed of either plastic or stainless steel (Figure 1). Inserts were placed concentrically within the columns and their orientation was secured by stainless steel bars (diameter: 1.5 mm) bearing on the column walls to allow precise location of the insert. It was assumed that these bars did not disturb the flow field significantly as their cross-sectional area is 3% of the cross-sectional area of the column. The wall thickness of the plastic inserts was 1 mm. The wall thickness of the stainless steel inserts was 0.1 mm. Table 1 summarizes the columns and chromatography systems used and Tables 2 and 3 gives the dimensions of all inserts used.
Pumping systems
Column packing procedure
Three different chromatography systems (GE Healthcare, Uppsala, Sweden) were used to cover the range of column
Reproducibly homogenous packing of beads was achieved by placing the inserts into the empty column before pouring
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Table 1. Summary of Basic Materials and Experimental Conditions
Column
Pump System
Diameter (mm)
ucrit
XK 16
16
¨ KTAexplorer A
XK 50
50
¨ KTAcrossflow/A ¨ KTApilot A
100
¨ KTAcrossflow/A ¨ KTApilot A
BPG 100
As & HETP ¨ KTAexplorer A ¨ KTAexplorer A
Resin
Insert Material
Sepharose 4B Sepharose CL 6B P6XL P6XL
Plastic Plastic & Stainless steel Stainless steel
P6XL
Table 2. Plastic Insert Dimensions (mm)
Table 3. Stainless Steel Insert Dimensions (mm)
Column
Diameter
Length
XK 16 XK 50
8 16 & 35
45 45
Insert Diameter Insert Length
reverse osmosis (RO) water and slurry of resin (70% v/v gravity settled bed slurry in 20% v/v ethanol) into the column. The resin was gravity settled overnight and then the adaptor was lowered 1 cm into the settled bed. Following this, the column was equilibrated in RO water by applying a 10 cm h1 flow rate for 40 min.
15 30 45 60 75 90
30
45
60
H H
H
H
H
H
H
H
H H
90
H
H
H
75
H H
H
H
H
Ticked blocks indicate those inserts fabricated for use either singly or combined.
Results and Discussion Critical velocity determination The automatic pressure step method developed by Chang et al.11 was applied for critical velocity determination. Briefly, a standard proportional-integral control algorithm was linked to the pressure and flow controller of the pump system. The pressure was increased incrementally from 0.05 ¨ KTAexplorer MPa in fixed pressure steps (0.1 MPa for the A system and 0.01 MPa for the rest of systems used). Each step lasted 5 min during which period the system sought to adjust the flow velocity to achieve the desired pressure set point. In the pressure step method,11 the critical velocity was deemed to have been reached, when a relatively small increase in velocity (15 cm h1) caused a rapid increase (three fold) in pressure drop. The maximum allowable operational pressure was set up at 70% of the column pressure safety limit. Column efficiency The number of theoretical plates and asymmetry factor for all columns were calculated by running a 1% CV pulse of 1% v/v acetone. The flow rate used for the XK 16 and XK 50 columns was 2 mL min1 and 20 mL min1, respectively. The absorbance of the tracer pulse leaving the column was detected at 280 nm. The equations to calculate plate number and asymmetry factor are given below12,13:
N ¼ 5:54
t2R 2 W1=2
(1)
where N is number of theoretical plates, tR is the retention time of the probe molecule, and W1/2 is the width of the peak at one half the maximum height.
As ¼
b a
(2)
where As is the asymmetry factor, a is the distance from the leading edge of the peak to the midpoint of the peak, and b is the distance from the midpoint of the peak to the trailing edge. As has been determined at 10% of maximum peak height.
In this study, we followed an experimental approach to investigate the effect of column insert design and position on column compression and the impact on column efficiency. The position of the insert(s) in the column, their diameter, their material of construction, the aspect ratio of the column supported by inserts, and the resin rigidity were examined. Effect of insert position on critical velocity Initial studies examined the impact of insert position on the critical velocity. The three positions examined were top, middle and bottom in an XK 16 column packed with Sepharose CL-6B. The standard operating procedure for column packing was followed using a single insert 8 mm in diameter and 45 mm in length. (The bottom of insert was positioned as follows: top, 55 mm from column base; middle, 27.5 mm from column base; and bottom, resting on column base.) Figure 2 shows that there was a considerable change in critical velocity when the insert was placed at the bottom of the column. This was expected as the axial stress in the bottom region of the column is greatest and consequently this leads to more bed compression.14 Hence, an insert placed at the base of the column exerts the greatest impact. Logically then placing an insert in either the middle or top part of the column results in supporting the less compressed regions of the bed, thus failing to reduce the compression effect so significantly. The results in Figure 2 support this. A 20% increase in critical velocity was seen for the best case and provided the impetus for further studies, all of which were based on placing inserts at the base of the column. Effects of insert length on critical velocity Having determined the best location for the positioning of an insert, Figure 3 shows the effect of insert length on the critical velocity for a fixed column insert and resin combination. For the combinations studied, the following linear relationship between the increase in critical velocity and insert length presumably as a result of providing more wall support
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Figure 2. Effect of insert position on critical velocity of XK 16 column packed with Sepharose CL-6B to a bed height of 100 6 5 mm. Critical velocity without insert; 360 cm h21.
Figure 4. Effect of insert diameter on critical velocity of BPG 100 column packed with P6XL resin to a bed height of 100 6 5 mm. Critical velocity without insert; 195 cm h21.
Insert (9 mm in diameter and 45 mm in length) constructed of plastic and placed at three different positions: top (55 mm from column base), middle (27.5 mm from column base), and bottom (resting on column base) of bed prior to determination of the critical velocity. Data points are mean SD from three independent experiments.
Inserts constructed of stainless steel. Inserts all had a length of 45 mm. Data points are mean SD from three independent experiments. Line shows fit to polynomial equation: 3 2 Ducrit Dinsert þ13:7 DDinsert þ11:4 DDinsert ucrit0 ¼ 17:5 Dbed bed bed
small space within which the top adaptor may move and consequently for the column to be compressed. This may limit process productivity as the column may have to operate at relatively low linear velocities so as to avoid extensive resin compression. In our studies, we restricted the maximum insert length to 90% of the flow packed bed height.
Effect of single insert diameter on critical velocity Figure 4 shows the effect of insert diameter on critical velocity for a fixed column/resin combination. The following empirical equation expresses the increase of critical velocity as a function of the ratio of insert to column diameter:
Ducrit ¼ 17:5 ucrit0 Figure 3. Effect of insert length on critical velocity of BPG 100 column packed with P6XL resin to a bed height of 100 6 5 mm. Critical velocity without insert; 195 cm h21. Insert constructed of stainless steel. Insert lengths (mm): 30, 45, 60, 75, and 90 and insert diameter: 30 mm. Data points are mean SD from three independent experiments. Line shows crit best fit to the linear equation:Du ucrit0 ¼ 0:08 Linsert
to the bed structure was developed:
Ducrit ¼ 0:08 Linsert ucrit0 where Ducrit is the change in critical velocity (%), ucrit0 is the critical velocity in column without insert(s), and Linsert is the insert length. However, there is a practical limit to the length of the insert. If the insert length becomes comparable to the length of the packed length of the column, there will be only a
Dinsert 3 þ13:7 Dbed Dinsert 2 Dinsert þ11:4 Dbed Dbed
where Ducrit is change in critical velocity (%), ucrit0 is the critical velocity in column without insert(s), Dinsert is the insert diameter, and Dbed is the column diameter. Inspection of Figure 4 indicates that an insert 75% the diameter of the base column is optimal. Beyond this only the small amount of resin contained in the annulus between the column wall and the insert gains effective support. By contrast, the inner part of the resin cannot be supported well. The aggregate effect is to cause the overall critical velocity to decrease for inserts [75% of column diameter. The cross-sectional area of the inner column and annulus (when pffiffiffi using one insert) are equal when Dinsert/Dcolumn ¼ 1= 2 ¼ 0.71. This is quite close to the optimal Dinsert/Dcolumn ¼ 0.75 that was found experimentally. Subsequent work will use these data to verify a structural mechanics model
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Figure 5. Effect of insert configuration on critical velocity of BPG 100 column packed with P6XL resin to a bed height of 100 6 5 mm. Critical velocity without insert; 195 cm h1. Inserts were constructed of stainless steel, length 45 mm. Data points are mean SD from three independent experiments.
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Figure 7. Effect of resin type on critical velocity in a XK 16 column with a bed height of 100 6 5 mm. Inserts constructed of plastic. Critical velocity without insert; Sepharose CL-6B resin 360 cm/h; P6XL resin 234 cm/h; Sepharose 4B 200 cm h1. Data points are mean SD from three independent experiments.
critical velocity reducing as the number of concentric inserts used increases. This may be explained by the reducing annular volumes of resin affected as the number of inserts rises and hence an asymptotic relationship between the level of improvement seen and the number of inserts used. This trend needs to be explored further for manufacturing scales of operation but was beyond the experimental capabilities to hand. Clearly, there will also be a practicable limit to the complexity of the concentric insert arrangements deployed. Effect of column aspect ratio on the critical velocity
Figure 6. Effect of scale on critical velocity change. Constant ratio of insert to column diameter packed with P6XL resin to a bed height of 100 6 5 mm. Inserts constructed of stainless steel. All inserts were 45 mm in length. Insert diameter was 35 mm (XK 50 columns) or 70 mm (BPG 100 columns). The ratio of insert to column diameter was 10:7. Critical velocity without insert; 234 cm h1 (XK 50 column) and 195 cm h1 (BPG 100 column). Data points are mean SD from three independent experiments.
developed to optimize the insert dimensions so as to obtain the highest critical velocity.
Effect of insert number on critical velocity The change in critical velocity when the column was supported by multiple inserts is explored in Figure 5. It can be seen that additional inserts serve to increase the critical velocity as more wall support is provided. However, there is a limit to this benefit with the incremental improvement in
Figure 6 shows how the critical velocity changes with an increase in the diameter of the column, while keeping the column/insert aspect ratio constant. The changes in critical velocity for the XK 50 and BPG 100 with an insert 70% of the column diameter were 6% and 8%, respectively. The greater level of improvement achieved for the larger diameter column is consistent with the relatively low level of wall support offered in the original column before addition of an insert. Effect of resin type on critical velocity Figure 7 shows the effect of the rigidity of the resin on the critical velocity in columns containing inserts. As expected, columns packed with soft resins, for example, Sepharose 4B showed the greatest level of improvement. All resin types, irrespective of rigidity, were capable of withstanding greater process velocities when inserts were included. Experimental data show, for example, that when a column was packed with the soft Sepharose 4B resin with inserts, this combination can operate at a higher critical velocity than a normal column packed with a more rigid resin such as P6XL. Inserts can allow columns to operate with more compressible material but at flow rates normally associated with operation of columns packed with rigid material.
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Figure 9. Impact of insert materials of construction on column efficiency for an XK 50 column packed with P6XL resin to a bed height of 50 6 2 mm. All inserts were 45 mm in length. Volume of inserts; plastic insert is 4 mL; stainless steel inserts is 0.14 mL. No insert ( ), plastic insert ( ), stainless steel insert ( ). a: Tracer absorbance as a function of retention volume, b: Tracer absorbance normalized as a function of column volume.
Figure 8. Impact of stainless steel insert on column efficiency of XK 50 column packed with P6XL resin to a bed height of 120 6 5 mm. All inserts were 45 mm in length. a: HETP for no insert ( ), 16 mm diameter insert ( ), 35 mm diameter insert ( ), and 16 mm and 35 mm diameter double inserts ( ). b: Variation in plate numbers for column tested in a, the initial plate number is 3483 plates m1. c: Variation in asymmetry factor for column tested in a, the initial asymmetry is 1.25. Data points are mean SD from three independent experiments.
Impact of insert on column efficiency Figure 8 shows the column exit absorbance-time distribution curves for a single column geometry containing different types of insert. The consequence of adding an insert is to broaden the distribution, most especially by a trailing of the peak. This will result in slightly larger elution volumes, thus increasing the loading volume of the next process step.
The rate of peak broadening is, however, smaller than that of the increase in critical velocity so that the volumetric productivity continues to increase. These data are further analyzed in Figures 8b,c showing a reduction in the theoretical plate number and in the level of asymmetry as inserts are introduced. The behavior can be explained by the fact that the bed void fraction will be high close to the walls of the column and of the inserts where the tracer will flow faster. This additional level of dispersion of the tracer will tend to reduce the theoretical plate number and decrease the asymmetry factor of the column. Figure 9a shows the absorbance-time distribution curves of a tracer for a given column equipped with either plastic or stainless steel inserts. In Figure 9b, the data were normalized to account for the insert volume, since this is significantly different between the two materials (4 mL for plastic inserts and 0.14 mL for stainless steel). This normalization reduces the difference between the two sets of tracer studies as expected. The small difference remaining between the plastic insert data and that for the stainless steel probably reflects the impact of the insert thickness in causing local axial dispersion as the flow impinges the top of the insert.
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Conclusions This study presents experimental data of critical velocity and column efficiency for a range of chromatography columns (XK 16, XK 50, and BPG 100) packed with compressible material as a function of the design of concentric cylindrical inserts placed within the packed bed. The results show that inserts installed at the bottom of a column were best and could generate up to a 20% increase in critical velocity. The penalty was a small increase in dispersion. For a single insert, the plate number fell by 10% at the most, which was deemed to be insignificant compared to the improvement in throughput. We did not conduct extensive studies of CIP and sanitization but the choice of materials of construction, combined with the low additional dispersion generated by the inserts implies that operation with insert would be simple to achieve and capable of full validation. It should be noted, however, that the use of inserts in more complex, modern designs such as pack-in-place configurations may not be technically feasible and this is a potential limitation of the insert concept. The most obvious application, therefore, is the extension of the hydrodynamic range of traditional, soft matrices, placed in conventional column designs of large diameter where bed support is negligible. Future work will focus on using structural mechanics models to describe the pressure-flow rate relationship in columns supported by inserts of different shape so as to maximize the productivity and yield of the separation by optimizing the dimensions and position of the inserts without affecting the hydrodynamic efficiency of the columns.
Acknowledgments The support of the Innovative Manufacturing Research Centre (IMRC) in Bioprocessing funded by the Engineering and Physical Sciences Research Council (EPSRC) and the EPSRC Centre for Innovative Manufacture in Emergent Macromolecular Therapies are acknowledged gratefully. The IMRC and the EPSRC Centre are each part of The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, with collaboration from a range of academic partners, biopharmaceutical and biotechnology companies. Additionally, the authors thank ProMetic Biosciences Ltd, Cambridge, UK for generously providing the Purabead 6XL chromatography resin material.
Notation As ¼ asymmetry factor a ¼ distance from the leading edge of the peak to the midpoint at 10% of peak height b ¼ distance from the midpoint of the peak to the trailing edge at 10% of peak height
1291 Dinsert Dbed Linsert N tR ucrit0 Ducrit W1/2
¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼
insert diameter (mm) column diameter (mm) insert length (mm) theoretical plate number retention time critical velocity in column without insert(s) (cm h1) change in critical velocity (%) width of the peak at one half the maximum height
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