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Bioseparation by Cartridge Chromatography
CHAPTER 3
BIOSEPARATION BY CARTRIDGE CHROMATOGRAPHY Haunn-Lin
Chen and Kenneth
C. Hou
AMF, Inc. Specialty Materials Group Meriden, Connecticut INTRODUCTION
The fast growing life sciences during the last decades has been drawing attention among scientists and engineers in both academe and industry, similar to the scientific advances of chemistry in the early 1900s with the consequent evolution in the chemical industry. Today, circumstances for the development of biotechnology is even more favored by its widespread and seemingly innumerable applications in medicine, food and chemical industries. Of particular interest is the increasing importance on large-scale production, separation and purification of biological products. Among all the separation methods, chromatography is one of the most effective and convenient means of bioseparation. However, the scale-up of the Chromatographie methods, especially for industrial applications, is impeded due to certain difficulties. The problem arises mainly because the scale-up process is not a simple geometrical expansion. The process cost and the time involved in operations are more frequently matters of concern. These include column packing, media wetting and equilibration, flow rate, resolution, costs of materials and equipment, etc., in association with the set-up of a Chromatographie system. Cartridge chromatography discussed in this paper is designed to fulfill those demands as a tool for preparative separation.
ANNUAL REPORTS ON FERMENTATION PROCESSES, VOL. 8
59
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
HAUNN-LINN CHEN AND KENNETH C. HOU
60 RESULT
AND
DISCUSSION
The concept of cartridge configuration prompted us to think of the physical features of the packing materials. Although the conventional gel or particulate type media have proven very successful in analytical scale, the application of cartridge design on those media may give little or even no advantage. Thus, the search for a new type of media is undoubtedly needed. This would involve the consideration of basic requirements toward a useful matrix. Generally speaking, an ideal Chromatographie packing material for the separation and purification of biological species must be: 1) 2). 3) 4) 5) 6) 7) 8) 9) 10) 11)
semi-rigid in structure to sustain moderate pressure chemically inert and physically stable high in surface area with proper pore size bio-compatible in nature with high adsorption capacity easy for equilibration and elution to reduce the operation time low in non-specific adsorption with high protein recovery autoclavable and non-toxic low in extractables high in regenerability uniform in structure to eliminate channelling effect inexpensive.
With those requirements and the specific cartridge design in mind, we decided to choose cellulose as our starting material. The idea of utilizing cellulose as ion-exchange matrix had been brought out more than a decade ago by directly linking the ion-exchange functional groups to the glucosidic chains (1,2). The media thus formed has been commercialized since then, but suffered from several inherent disadvantages. The requirement that cellulose remains in a water-insoluble form restricts the substitution reaction to a lower degree, thereby, limiting the possibility of extending the nominal capacity. The heterogeneity of the reaction is another major cause for the variability of the performance of this ion-exchange cellulose. In AMF Zetaprep media, those problems are eliminated by incorporating polymers carrying specific functional groups to the cellulose. The porosity and the rigidity of the matrix are usually counterbalanced by the amount and type of crosslinkers added. Functional groups which are responsible for protein adsorption in ion-exchangers can be either positively
CARTRIDGE CHROMATOGRAPHY
61
charged or potentially positively charged groups, such as, diethy1amino ethyl (DEAE), dimethylamino ethyl (DMAE) and their quarternized derivatives (QAE) or negatively charged, such as, sulfonyl groups (SP), phosphonyl groups (PP), carboxyl groups (CM). The mechanism of protein separation by an ion-exchanger can be simplified as an adsorptiondesorption process. It is well known that each protein with its normal conformation exhibits a unique isoelectric point (PI). A dissolved protein molecule with the pH of the solution below its PI possesses a net positive charge. For instance, the serum albumin is positively charged at pH below 4.8, the PI of albumin (see Fig. 1 ) . By the fact that opposite charges attract each other, the albumin molecules tend to associate with a matrix which carries negative charges at that pH. This type of matrix, categorized as cation exchangers, includes SP, CM and PP. (DEAE, DMAE and QAE media, on the other hand, belong to the anionexchanger group.) With the same principle, we may expect albumin molecules to be expelled off the cation exchanging matrix at pH above 4.8. Hence, simply by varying the pH, one can selectively adsorb or desorb a particular protein in accord with its PI and consequently separate it from other proteins. This adsorption-desorption treatment is, of course, oversimplified and needs a lot of modification. For instance, it does not take into account the ionization of functional groups and the variation of matrix porosity in association with the pH value. In fact, the protein binding capacity at a certain pH should be determined by the zeta potentials of both protein molecule and the ionexchange matrix at that pH, which in turn, is governed by the PI of the protein and the PK of the functional group, respectively. A theoretical prediction of the protein capacity at various pH is shown in Fig. 2 by using SP matrix interacted with albumin and γ-globulin as examples. It should be pointed out that, besides temperature, the PK of a polyelectrolyte also varies with the degree of ionization, ionic strength, the macromolecular complex formation, etc. (3), thus further complicating the situation. The porosity is another striking factor leading to the unpredictability of the protein capacity, especially when applying the matrix to huge protein molecules such as, IgG. Nevertheless, by carefully controlling the pore structure, a capacity versus pH curve of γ-globulin adsorbed by SP matrix, as shown in Fig. 3, behaves quite close to what was predicted. Figure 4 depicts the pH profiles of bovine serum albumin (BSA) adsorbed on anion exchangers. It may be seen that BSA exhibits the highest adsorption on DEAE matrix at pH 6.0 while the maximal BSA capacity of DMAE lies
HAUNN-LINN CHEN AND KENNETH C. HOU
62 ZETA POTENTIAL
PI OF ALBUMIN= 4.8 ULIN=6.9
pH DSORBED BY ION CHANGER BETA GLOBULIN ALPHA GLOBULIN ALBUMIN
Figure 1.
ZETA POTENTIAL
+
Separations:pH
effectrprotein
ADSORPTION CAPACITY OF PROTEIN DERIVED FROM THE INTERACTION CONDITION TOWARD THE MATRIX,
ALBUMIN
Figure 2. Force of interaction between protein and matrix
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-
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(uuejß/ßai) 961 JO AllDVdVD NOIldäOSQV 1
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64
HAUNN-LINN CHEN AND KENNETH C. HOU
around 6.8. The wider useful pH range observed in DMAE can be explained by the architectural change of the polymeric matrix. It is commonly known that macromolecules with ionizable groups change their conformations in accord with the degree of ionization. This reversible transition becomes more abrupt if the polyelectrolytes carry hydrophobic moieties (4). Examples can be found in polypeptides carrying ionizable side chains. An increasing density of ionic charges destabilizes the helical form of the polypeptide and leads to a steep transition to the random coil. Neutralization will, in turn, bring the conformation back to the helix due to the hydrophobic folding. Obviously, the degree of ionization at which the abrupt conformational transition occurs is determined by the degree of hydrophobic interactions. With this concept in mind, the abrupt drop of the BSA capacity on DEAE media at pH above 7.0 can be interpreted as the result of two effects: 1) the decreasing number of protonated DEAE functional groups which are responsible for protein binding; 2) the substantial shrinkage of porosity and the increasing unavailability of the charged groups caused by the hydrophobic folding of the matrix. As would be expected, the conformational change of DMAE matrix will happen at a higher pH than that of DEAE, due to the less hydrophobic character of the dimethyl moiety. And the operable pH for OAE media should be even higher because it contains permanent positive charges (Fig. 5 ) . The cartridge has been constructed by rolling up the thin sheet matrix along a center pole, with webs between layers of paper to allow the swelling of the media. Figure 6 delineates the final feature of the cartridge. In a preparative scale, several cartridges are connected to one another in series before installing into a bigger housing. A multi-directional radial flow pattern was designed to provide a higher flow rate, as illustrated in Fig. 7. Normally, the maximal flow rate observed in a conventional packed column (D 2.5 cm, H 45 cm) is about 5 ml/min and the highest pressure the conventional matrix can sustain is about 5 psi. In case of AMF Zetaprep, the flow rate of a lOOOcc cartridge can be as high as 500 ml/min, depending on the pressure drop (Fig. 7 ) . A question may be brought up at this point that, under such high flow rate, does the protein molecule have sufficient contact time with the matrix to give an effective binding? To answer this question, several experiments were conducted by monitoring the adsorption of BSA on DEAE matrix at various flow
65
CARTRIDGE CHROMATOGRAPHY
1000
< o>
800
-J 5.0
L 6.0
7.0
8.0
9.0
PH
Figure 5.
Effect of pH on BSA adsorption by QAE and DMAE CENTER CORE WITH FLOW PATHS
INERT SUPPORT
ION EXCHANGE MEDIA
FLOW PORTS
ION EXCHANGE MEDIA
FLOW PATHS
INERT SUPPORT-
Figure 6.
Cartridge design.
HAUNN-LINN CHEN AND KENNETH C. HOU
66
rates. The result, as listed in Table 1 and Figure 8, indicates that more than 70% of the protein molecules corresponding to a maximal capacity of 12.0 g protein/cartridge were adsorbed under a flow of 100 ml/min.
Rate
of
BSA Adsorption
Table 1. by AMF Zetaprep
Flow Rate (ml/min)
Contact Time (min)
150 100 50
1.67 2.50 5.00
BSA Adsorption 6.9 8.7 10.5
DEAE-250
(g)
%
Cartridge
Adsorption 58 73 88
It should be emphasized that the flow rate increases proportionally in a multi-cartridge set-up (Fig. 6) yet the protein-matrix contact time remains virtually unchanged, (as does the protein adsorption). This effect reflects one of the advantages of cartridge configuration which makes the preparative separation a reality. Examples illustrating the applications of ion exchange cartridges on the separation of biological substances are discussed below, The separation of nucleotides by DEAE-250 cartridges was demonstrated in Fig.10. The cartridge with nominal volume of 250 ml was pre-equilibrated by passing 0.02 M potassium phosphate buffer at pH 4.5 at 150 ml/min until the conductivity and pH of the effluent are the same as those of the buffer. The mixture of 100 mg each of AMP, ADP and ATP in 100 ml of 0.02 M potassium monophosphate buffer at pH 4.5 was introduced into the cartridge. Elution was then performed with 0.2 M phosphate dibase at pH 8.96 and flow rate of 46 ml/min. A U.V. monitoring unit was used to register the eluents eluted from the cartridge at wave length 254 nm. The adenosine, being neutral, passsed through the DEAE cartridge under the specific buffer condition without interaction as shown in first peak. AMP, being weakest in charge, came off the cartridge as the second peak followed by ADP and ATP according to their relative charge strength toward DEAE. The poor separation between ADP and ATP can be resolved by either slowing down the flow rate or performing on a cartridge with increased depth.
CARTRIDGE CHROMATOGRAPHY
67
PARTICULANT PACKED COLUMN
UNI-DIRECTIONAL FLOW
F i g u r e 7.
AMF CARTRIDGE
MULTI-DIRECTIONAL RADIAL FLOW
Comparison of flow p a t t e r n between c o n v e n t i o n a l packed column and AMF c a r t r i d g e
DI H z 0
20 H
15 H DIFFERENTIAL PRESSURE (PSI)
10 H
PHOSPHATE BUFFER OJM
5 H
FLOW (ml/min.)
Figure 8.
DEAE flow characteristics
HAUNN-LINN CHEN AND KENNETH C. HOU
68
Figure 9.
Zetaprep multicartridge system size: 24L
approximately
-ADENOSINE
CHART SPEED: I cm/hr. ELUTION CONDITION: I8.0L OF 0.2M DIBASIC >H=8.96 YIELD· 96%REC0VERY
OD'254
10
15
TIME (HOURS)
Figure 10. Separation by elution nucleotides on DEAE
CARTRIDGE CHROMATOGRAPHY
69
Separation of transferrin from gamma globulin was also performed on DEAE 250 ml size cartridge as shown in Fig. 11. The cartridge was pre-equilibrated with 0.01 M phosphate buffer at 6.8. 200 mg of gamma globulin was mixed with 50 mg of transferrin in 100 ml of 0.01 M monophosphate buffer at pH 6.8 and applied to the column at 20 ml/min rate. Gamma globulin passed through the cartridge as shown in peak A with transferrin adsorbed in the cartridge. The physically entrapped IgG were completely brought out by washing the cartridge with equilibration buffer. Applying 4.0 liters of 0.02 M sodium phosphate buffer at pH 6.5, a portion of adsorbed transferrin was eluted (peak A ) . The remaining transferrin was eluted by increasing the buffer molarity to 0.05 M and decreasing the pH to 5.9 with 1.6 liter buffer volume (peak C ) . Protein recovery in the process was over 95%. After the elution of transferrin, the cartridge was subjected to 1 M acetic acid at pH 2.7 and then 0.05 M dibasic sodium phosphate with 1 M NaCl at pH 8.2. This cycle of buffers with 3-4 volumes of cartridge each can be repeated until the O.D. 280 drops to baseline indicating the complete removal of residual proteins in the cartridge. The salt is then rinsed out with 0.01 M phosphate buffer at pH 6.8 and the cartridge is regenerated for repeated use. The purity of gamma globulin and transferrin was checked by electrophoresis on cellulose acetate plate. The transferrin thus separated shows over 80% purity and can be further purified by subjecting to the cartridge for second time. The performance of a cation exchange cartridge was also tested on sulphopropyl of 100 ml cartridge volume (SP-100). The cartridge was equilibrated with phosphate buffer of 0.01 M at pH 6.3. A synthetic protein mixture was made by mixing 750 mg human serum albumin (HSA) with 205 mg human gamma globulin (Hyg) and 100 mg cytochrome C in 55 ml 0.01 M monobasic sodium phosphate at 6.3. The flow rate of protein adsorption and elution was 10.0 ml/min. The elution pattern in Fig. 12 shows peak A as unadsorbed human albumin, peak B was eluted with 0.025 M phosphate buffer at pH 7.55. Two minor impurities/components, peak C and peak D, of cytochrome C were respectively eluted with two different buffers: (a) 0.05 M phosphate buffer; pH 7.9; 6.8 mS and (b) 0.05 pyrophosphate; pH 9; 6.2 mS. The strong interaction between cytochrome C and sulfonic groups requires addition of 25 M salt to buffer b. to elute out completely as shown in peak E.
HAUNN-LINN CHEN AND KENNETH C. HOU
70
ü
280
T pH PHOSPHATE BUFFER
Figure 1 1 .
5.9 0.05M
i
Γ
6.5 6.8 0.02M 0.01M
S e p a r a t i o n of t r a n s f e r r i n from IgG on DEAE 250 cartridge
■ TIME Cartridge Type: ZetaPrep SP-100 Sample: Mixture nf Human Serum Albumin, Human Gamma Globulin. Cytochrome C Running Buffer: 0.01 M Sodium Phosphate pH 8.5 Flow Rate: 10 ml/min Elut ion Condi t ions : Peak Λ (Albumin) unadsorbed Peak B (Gamma Globulin) 0.025 M Sodium Phosphate pH 7.55 (3.5 mS) Peaks C S D : (Cytochrome C impurities) 0.05 M Sodium Phosphate pH 7.9 then 0.05 M Sodium Pyrophosphate pH 9.0 Ppak E (Cytochrome C) 0.05 M Sodium Pyrophosphate w/0.05 M NaCl. final pH Total uroteir load =
Figure 12.
S e p a r a t i o n of p r o t e i n mixtures on SP 100 c a r t r i d g e
CARTRIDGE CHROMATOGRAPHY
71
The above cited examples on protein separation by cartridge process proves that certain technical problems ordinarily occurring in large scale operation can be resolved by multiple cartridge systems either connected in parallel or in series. Physically, the cartridge design eliminates the channeling problems that exist in packed column methods, leading to better separation. The dimensional and chemical stability of the dry paper structure reduces the time required for equilibration and elution and makes the process economically attractive. The minimal non-specific adsorption of protein in cartridge enables us to achieve high protein yield for maximum recovery. The matrix being hydrophilic due to the presence of cellulose as solid support will not denature the protein molecules. The size of macropores in matrix evidently are adequate for IgG migration. The accessibility of the charge groups located in matrix made the peripheral orientation of those groups highly efficient for ion exchange exhibited in high protein adsorption capacity. The improved chemical and physical properties in matrix made it applicable to form a cartridge. The cartridge thus formed simplified the tedious column packing procedure in conventional ion exchange processes. The feasibility on large scale protein separation by cartridge are demonstrated in this report. REFERENCES 1. 2. 3. 4. 5.
6.
7. 8.
Peterson, E.A. and H.A. Sober, J. Am. Chem. Soc., 78, 75 (1956). Parath, J. , Arkiv. Kern., 11, 97 (.1957). Morametz, H., Macromolecules in Solution, 2nd Ed., Chap. VII, John Wiley & Sons, New York (1975). Crescenzi, V., F. Quadrifoglio, and F. Delben, J. Polym. Sei., A-2, 10, 357 (1972). Peterson, E.A., "Cellulosic Ion-Exchanger," Laboratory Technique in Biochemistry and Molecular Biology, Vol. 2, CT.S. Work and E. Work, eds.), North Holland, Amsterdam. Scopes, R.K., "Quantitative Studies of Ion Exchange and Affinity Elution Chromatography of Enzymes," Anal. Biochem., 115, 8-18 (1981). Curling, J.M., (ed.), Methods of Plasma Protein Fractionation, Academic Press, New York (1980). Poison, A., G.M. Potgieter, J.F. Largier, G.E.F. Mears, and F.J. Joubert, "The Fractionation of Protein Mixtures by Linear Polymers of High Molecular Weight," Biochem. Biophys. Acta, 82, 463 (1964).
BIOSEPARATION BY CARTRIDGE CHROMATOGRAPHY Haunn-Lin
Chen and Kenneth
C. Hou
AMF, Inc. Specialty Materials Group Meriden, Connecticut INTRODUCTION
The fast growing life sciences during the last decades has been drawing attention among scientists and engineers in both academe and industry, similar to the scientific advances of chemistry in the early 1900s with the consequent evolution in the chemical industry. Today, circumstances for the development of biotechnology is even more favored by its widespread and seemingly innumerable applications in medicine, food and chemical industries. Of particular interest is the increasing importance on large-scale production, separation and purification of biological products. Among all the separation methods, chromatography is one of the most effective and convenient means of bioseparation. However, the scale-up of the Chromatographie methods, especially for industrial applications, is impeded due to certain difficulties. The problem arises mainly because the scale-up process is not a simple geometrical expansion. The process cost and the time involved in operations are more frequently matters of concern. These include column packing, media wetting and equilibration, flow rate, resolution, costs of materials and equipment, etc., in association with the set-up of a Chromatographie system. Cartridge chromatography discussed in this paper is designed to fulfill those demands as a tool for preparative separation.
ANNUAL REPORTS ON FERMENTATION PROCESSES, VOL. 8
59
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
HAUNN-LINN CHEN AND KENNETH C. HOU
60 RESULT
AND
DISCUSSION
The concept of cartridge configuration prompted us to think of the physical features of the packing materials. Although the conventional gel or particulate type media have proven very successful in analytical scale, the application of cartridge design on those media may give little or even no advantage. Thus, the search for a new type of media is undoubtedly needed. This would involve the consideration of basic requirements toward a useful matrix. Generally speaking, an ideal Chromatographie packing material for the separation and purification of biological species must be: 1) 2). 3) 4) 5) 6) 7) 8) 9) 10) 11)
semi-rigid in structure to sustain moderate pressure chemically inert and physically stable high in surface area with proper pore size bio-compatible in nature with high adsorption capacity easy for equilibration and elution to reduce the operation time low in non-specific adsorption with high protein recovery autoclavable and non-toxic low in extractables high in regenerability uniform in structure to eliminate channelling effect inexpensive.
With those requirements and the specific cartridge design in mind, we decided to choose cellulose as our starting material. The idea of utilizing cellulose as ion-exchange matrix had been brought out more than a decade ago by directly linking the ion-exchange functional groups to the glucosidic chains (1,2). The media thus formed has been commercialized since then, but suffered from several inherent disadvantages. The requirement that cellulose remains in a water-insoluble form restricts the substitution reaction to a lower degree, thereby, limiting the possibility of extending the nominal capacity. The heterogeneity of the reaction is another major cause for the variability of the performance of this ion-exchange cellulose. In AMF Zetaprep media, those problems are eliminated by incorporating polymers carrying specific functional groups to the cellulose. The porosity and the rigidity of the matrix are usually counterbalanced by the amount and type of crosslinkers added. Functional groups which are responsible for protein adsorption in ion-exchangers can be either positively
CARTRIDGE CHROMATOGRAPHY
61
charged or potentially positively charged groups, such as, diethy1amino ethyl (DEAE), dimethylamino ethyl (DMAE) and their quarternized derivatives (QAE) or negatively charged, such as, sulfonyl groups (SP), phosphonyl groups (PP), carboxyl groups (CM). The mechanism of protein separation by an ion-exchanger can be simplified as an adsorptiondesorption process. It is well known that each protein with its normal conformation exhibits a unique isoelectric point (PI). A dissolved protein molecule with the pH of the solution below its PI possesses a net positive charge. For instance, the serum albumin is positively charged at pH below 4.8, the PI of albumin (see Fig. 1 ) . By the fact that opposite charges attract each other, the albumin molecules tend to associate with a matrix which carries negative charges at that pH. This type of matrix, categorized as cation exchangers, includes SP, CM and PP. (DEAE, DMAE and QAE media, on the other hand, belong to the anionexchanger group.) With the same principle, we may expect albumin molecules to be expelled off the cation exchanging matrix at pH above 4.8. Hence, simply by varying the pH, one can selectively adsorb or desorb a particular protein in accord with its PI and consequently separate it from other proteins. This adsorption-desorption treatment is, of course, oversimplified and needs a lot of modification. For instance, it does not take into account the ionization of functional groups and the variation of matrix porosity in association with the pH value. In fact, the protein binding capacity at a certain pH should be determined by the zeta potentials of both protein molecule and the ionexchange matrix at that pH, which in turn, is governed by the PI of the protein and the PK of the functional group, respectively. A theoretical prediction of the protein capacity at various pH is shown in Fig. 2 by using SP matrix interacted with albumin and γ-globulin as examples. It should be pointed out that, besides temperature, the PK of a polyelectrolyte also varies with the degree of ionization, ionic strength, the macromolecular complex formation, etc. (3), thus further complicating the situation. The porosity is another striking factor leading to the unpredictability of the protein capacity, especially when applying the matrix to huge protein molecules such as, IgG. Nevertheless, by carefully controlling the pore structure, a capacity versus pH curve of γ-globulin adsorbed by SP matrix, as shown in Fig. 3, behaves quite close to what was predicted. Figure 4 depicts the pH profiles of bovine serum albumin (BSA) adsorbed on anion exchangers. It may be seen that BSA exhibits the highest adsorption on DEAE matrix at pH 6.0 while the maximal BSA capacity of DMAE lies
HAUNN-LINN CHEN AND KENNETH C. HOU
62 ZETA POTENTIAL
PI OF ALBUMIN= 4.8 ULIN=6.9
pH DSORBED BY ION CHANGER BETA GLOBULIN ALPHA GLOBULIN ALBUMIN
Figure 1.
ZETA POTENTIAL
+
Separations:pH
effectrprotein
ADSORPTION CAPACITY OF PROTEIN DERIVED FROM THE INTERACTION CONDITION TOWARD THE MATRIX,
ALBUMIN
Figure 2. Force of interaction between protein and matrix
.XT'
Γ
Js
ce UJ
xi
-40
T
i° is* < < Q
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o o
o o
AilDVdVD VS8
Q
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UJ Q
^^x \
\
UJ X £ Û CL — „ < tf) e»
oc 0 E 0 x m CL —
•«5
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1
o o
-'
\
J
\
CL · O -C ( Λ Ο lO X ce K UJ
V
O
E
\
\
ί
ÜL Ul
-
5u.ni2 ^ ί O
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Q X
\
o o
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(uuejß/ßai) 961 JO AllDVdVD NOIldäOSQV 1
jcebupipxe uoxq.-eD Äq
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-J
-1
< ce 0 2:
64
HAUNN-LINN CHEN AND KENNETH C. HOU
around 6.8. The wider useful pH range observed in DMAE can be explained by the architectural change of the polymeric matrix. It is commonly known that macromolecules with ionizable groups change their conformations in accord with the degree of ionization. This reversible transition becomes more abrupt if the polyelectrolytes carry hydrophobic moieties (4). Examples can be found in polypeptides carrying ionizable side chains. An increasing density of ionic charges destabilizes the helical form of the polypeptide and leads to a steep transition to the random coil. Neutralization will, in turn, bring the conformation back to the helix due to the hydrophobic folding. Obviously, the degree of ionization at which the abrupt conformational transition occurs is determined by the degree of hydrophobic interactions. With this concept in mind, the abrupt drop of the BSA capacity on DEAE media at pH above 7.0 can be interpreted as the result of two effects: 1) the decreasing number of protonated DEAE functional groups which are responsible for protein binding; 2) the substantial shrinkage of porosity and the increasing unavailability of the charged groups caused by the hydrophobic folding of the matrix. As would be expected, the conformational change of DMAE matrix will happen at a higher pH than that of DEAE, due to the less hydrophobic character of the dimethyl moiety. And the operable pH for OAE media should be even higher because it contains permanent positive charges (Fig. 5 ) . The cartridge has been constructed by rolling up the thin sheet matrix along a center pole, with webs between layers of paper to allow the swelling of the media. Figure 6 delineates the final feature of the cartridge. In a preparative scale, several cartridges are connected to one another in series before installing into a bigger housing. A multi-directional radial flow pattern was designed to provide a higher flow rate, as illustrated in Fig. 7. Normally, the maximal flow rate observed in a conventional packed column (D 2.5 cm, H 45 cm) is about 5 ml/min and the highest pressure the conventional matrix can sustain is about 5 psi. In case of AMF Zetaprep, the flow rate of a lOOOcc cartridge can be as high as 500 ml/min, depending on the pressure drop (Fig. 7 ) . A question may be brought up at this point that, under such high flow rate, does the protein molecule have sufficient contact time with the matrix to give an effective binding? To answer this question, several experiments were conducted by monitoring the adsorption of BSA on DEAE matrix at various flow
65
CARTRIDGE CHROMATOGRAPHY
1000
< o>
800
-J 5.0
L 6.0
7.0
8.0
9.0
PH
Figure 5.
Effect of pH on BSA adsorption by QAE and DMAE CENTER CORE WITH FLOW PATHS
INERT SUPPORT
ION EXCHANGE MEDIA
FLOW PORTS
ION EXCHANGE MEDIA
FLOW PATHS
INERT SUPPORT-
Figure 6.
Cartridge design.
HAUNN-LINN CHEN AND KENNETH C. HOU
66
rates. The result, as listed in Table 1 and Figure 8, indicates that more than 70% of the protein molecules corresponding to a maximal capacity of 12.0 g protein/cartridge were adsorbed under a flow of 100 ml/min.
Rate
of
BSA Adsorption
Table 1. by AMF Zetaprep
Flow Rate (ml/min)
Contact Time (min)
150 100 50
1.67 2.50 5.00
BSA Adsorption 6.9 8.7 10.5
DEAE-250
(g)
%
Cartridge
Adsorption 58 73 88
It should be emphasized that the flow rate increases proportionally in a multi-cartridge set-up (Fig. 6) yet the protein-matrix contact time remains virtually unchanged, (as does the protein adsorption). This effect reflects one of the advantages of cartridge configuration which makes the preparative separation a reality. Examples illustrating the applications of ion exchange cartridges on the separation of biological substances are discussed below, The separation of nucleotides by DEAE-250 cartridges was demonstrated in Fig.10. The cartridge with nominal volume of 250 ml was pre-equilibrated by passing 0.02 M potassium phosphate buffer at pH 4.5 at 150 ml/min until the conductivity and pH of the effluent are the same as those of the buffer. The mixture of 100 mg each of AMP, ADP and ATP in 100 ml of 0.02 M potassium monophosphate buffer at pH 4.5 was introduced into the cartridge. Elution was then performed with 0.2 M phosphate dibase at pH 8.96 and flow rate of 46 ml/min. A U.V. monitoring unit was used to register the eluents eluted from the cartridge at wave length 254 nm. The adenosine, being neutral, passsed through the DEAE cartridge under the specific buffer condition without interaction as shown in first peak. AMP, being weakest in charge, came off the cartridge as the second peak followed by ADP and ATP according to their relative charge strength toward DEAE. The poor separation between ADP and ATP can be resolved by either slowing down the flow rate or performing on a cartridge with increased depth.
CARTRIDGE CHROMATOGRAPHY
67
PARTICULANT PACKED COLUMN
UNI-DIRECTIONAL FLOW
F i g u r e 7.
AMF CARTRIDGE
MULTI-DIRECTIONAL RADIAL FLOW
Comparison of flow p a t t e r n between c o n v e n t i o n a l packed column and AMF c a r t r i d g e
DI H z 0
20 H
15 H DIFFERENTIAL PRESSURE (PSI)
10 H
PHOSPHATE BUFFER OJM
5 H
FLOW (ml/min.)
Figure 8.
DEAE flow characteristics
HAUNN-LINN CHEN AND KENNETH C. HOU
68
Figure 9.
Zetaprep multicartridge system size: 24L
approximately
-ADENOSINE
CHART SPEED: I cm/hr. ELUTION CONDITION: I8.0L OF 0.2M DIBASIC >H=8.96 YIELD· 96%REC0VERY
OD'254
10
15
TIME (HOURS)
Figure 10. Separation by elution nucleotides on DEAE
CARTRIDGE CHROMATOGRAPHY
69
Separation of transferrin from gamma globulin was also performed on DEAE 250 ml size cartridge as shown in Fig. 11. The cartridge was pre-equilibrated with 0.01 M phosphate buffer at 6.8. 200 mg of gamma globulin was mixed with 50 mg of transferrin in 100 ml of 0.01 M monophosphate buffer at pH 6.8 and applied to the column at 20 ml/min rate. Gamma globulin passed through the cartridge as shown in peak A with transferrin adsorbed in the cartridge. The physically entrapped IgG were completely brought out by washing the cartridge with equilibration buffer. Applying 4.0 liters of 0.02 M sodium phosphate buffer at pH 6.5, a portion of adsorbed transferrin was eluted (peak A ) . The remaining transferrin was eluted by increasing the buffer molarity to 0.05 M and decreasing the pH to 5.9 with 1.6 liter buffer volume (peak C ) . Protein recovery in the process was over 95%. After the elution of transferrin, the cartridge was subjected to 1 M acetic acid at pH 2.7 and then 0.05 M dibasic sodium phosphate with 1 M NaCl at pH 8.2. This cycle of buffers with 3-4 volumes of cartridge each can be repeated until the O.D. 280 drops to baseline indicating the complete removal of residual proteins in the cartridge. The salt is then rinsed out with 0.01 M phosphate buffer at pH 6.8 and the cartridge is regenerated for repeated use. The purity of gamma globulin and transferrin was checked by electrophoresis on cellulose acetate plate. The transferrin thus separated shows over 80% purity and can be further purified by subjecting to the cartridge for second time. The performance of a cation exchange cartridge was also tested on sulphopropyl of 100 ml cartridge volume (SP-100). The cartridge was equilibrated with phosphate buffer of 0.01 M at pH 6.3. A synthetic protein mixture was made by mixing 750 mg human serum albumin (HSA) with 205 mg human gamma globulin (Hyg) and 100 mg cytochrome C in 55 ml 0.01 M monobasic sodium phosphate at 6.3. The flow rate of protein adsorption and elution was 10.0 ml/min. The elution pattern in Fig. 12 shows peak A as unadsorbed human albumin, peak B was eluted with 0.025 M phosphate buffer at pH 7.55. Two minor impurities/components, peak C and peak D, of cytochrome C were respectively eluted with two different buffers: (a) 0.05 M phosphate buffer; pH 7.9; 6.8 mS and (b) 0.05 pyrophosphate; pH 9; 6.2 mS. The strong interaction between cytochrome C and sulfonic groups requires addition of 25 M salt to buffer b. to elute out completely as shown in peak E.
HAUNN-LINN CHEN AND KENNETH C. HOU
70
ü
280
T pH PHOSPHATE BUFFER
Figure 1 1 .
5.9 0.05M
i
Γ
6.5 6.8 0.02M 0.01M
S e p a r a t i o n of t r a n s f e r r i n from IgG on DEAE 250 cartridge
■ TIME Cartridge Type: ZetaPrep SP-100 Sample: Mixture nf Human Serum Albumin, Human Gamma Globulin. Cytochrome C Running Buffer: 0.01 M Sodium Phosphate pH 8.5 Flow Rate: 10 ml/min Elut ion Condi t ions : Peak Λ (Albumin) unadsorbed Peak B (Gamma Globulin) 0.025 M Sodium Phosphate pH 7.55 (3.5 mS) Peaks C S D : (Cytochrome C impurities) 0.05 M Sodium Phosphate pH 7.9 then 0.05 M Sodium Pyrophosphate pH 9.0 Ppak E (Cytochrome C) 0.05 M Sodium Pyrophosphate w/0.05 M NaCl. final pH Total uroteir load =
Figure 12.
S e p a r a t i o n of p r o t e i n mixtures on SP 100 c a r t r i d g e
CARTRIDGE CHROMATOGRAPHY
71
The above cited examples on protein separation by cartridge process proves that certain technical problems ordinarily occurring in large scale operation can be resolved by multiple cartridge systems either connected in parallel or in series. Physically, the cartridge design eliminates the channeling problems that exist in packed column methods, leading to better separation. The dimensional and chemical stability of the dry paper structure reduces the time required for equilibration and elution and makes the process economically attractive. The minimal non-specific adsorption of protein in cartridge enables us to achieve high protein yield for maximum recovery. The matrix being hydrophilic due to the presence of cellulose as solid support will not denature the protein molecules. The size of macropores in matrix evidently are adequate for IgG migration. The accessibility of the charge groups located in matrix made the peripheral orientation of those groups highly efficient for ion exchange exhibited in high protein adsorption capacity. The improved chemical and physical properties in matrix made it applicable to form a cartridge. The cartridge thus formed simplified the tedious column packing procedure in conventional ion exchange processes. The feasibility on large scale protein separation by cartridge are demonstrated in this report. REFERENCES 1. 2. 3. 4. 5.
6.
7. 8.
Peterson, E.A. and H.A. Sober, J. Am. Chem. Soc., 78, 75 (1956). Parath, J. , Arkiv. Kern., 11, 97 (.1957). Morametz, H., Macromolecules in Solution, 2nd Ed., Chap. VII, John Wiley & Sons, New York (1975). Crescenzi, V., F. Quadrifoglio, and F. Delben, J. Polym. Sei., A-2, 10, 357 (1972). Peterson, E.A., "Cellulosic Ion-Exchanger," Laboratory Technique in Biochemistry and Molecular Biology, Vol. 2, CT.S. Work and E. Work, eds.), North Holland, Amsterdam. Scopes, R.K., "Quantitative Studies of Ion Exchange and Affinity Elution Chromatography of Enzymes," Anal. Biochem., 115, 8-18 (1981). Curling, J.M., (ed.), Methods of Plasma Protein Fractionation, Academic Press, New York (1980). Poison, A., G.M. Potgieter, J.F. Largier, G.E.F. Mears, and F.J. Joubert, "The Fractionation of Protein Mixtures by Linear Polymers of High Molecular Weight," Biochem. Biophys. Acta, 82, 463 (1964).