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Leakage stability of ligand-support conjugates under operational conditions
Leakage stability of ligand-support conjugates under operational conditions J. Lasch Institute o f Biochemistry, Faculty o f Medicine, Martin-Luther-University, Halle (Saale), G D R
and F. Janowski D e p a r t m e n t o f Chemistry, Division o f Industrial Chemistry, Martin-Luther-University, Halle (Saale), G D R
(Received 14 March 1987; revised 15 October 1987)
Leakage of ligands bound either monovalently or polyvalently to Eupergit C and macroporous glass was followed spectrophotometrically in a recirculation reactor up to 2 months under continuous operation. The data revealed a very stable fraction of bound protein desorbing only slowly from saturable noncovalent binding sites. Once this fraction was removed (which takes at least 3 days of continuous washing) conjugates with practically unlimited leakage stability were obtained.
Keywords: Ligandleakage; ligand-support conjugates; long-timeligand stability
Introduction Tesser et al.~ and Ludens et al. 2 were the first to demonstrate that ligands monovalently bound to insoluble matrices leak out during continuous operation of affinity columns. Later it was shown 3'4'5 that especially CNBr-coupled ligands are prone to be split off from the matrix. In all leakage studies it was found consistently that the presence of nucleophiles (buffers, proteins) increased the leakage rate by about one order of magnitude.Z, 3,5 It is well known that the immobilization of ligands, particularly proteinaceous ligands, on solid supports is a complex combination of various interacting forces. 6 Differentiation between these forces is difficult. Besides the covalent linkage, a combination of noncovalent forces may generate stable binding. 6 However, for molecules that are bound only by noncovalent forces, there is always the risk of interchange of already bound molecules with others in solution and/or elution in extreme milieu. This is why in properly devised leakage studies different populations of 'immobilized' protein molecules can be washed out. 7 Our efforts to develop a proteolytic enzyme reactor 8 led us to reinvestigate the leakage problem more thor312
Enzyme Microb. Technol., 1988, vol. 10, May
oughly with carriers which, from an economical point of view, would be suitable for up-scaling.
Materials and methods Materials
Azocasein was prepared from Hammarsten casein according to the method of Langner et al. 9 It had an absorption coefficient of A°i~'nm = 3.77. Eupergit C 8 , a macroporous epoxide-activated polyacrylic carrier was a gift from R6hm-Pharma (Weiterstadt, FRG). Amino Eupergit was prepared from the epoxideactivated support by reaction with 1,6-hexane diamine as described in the instructions provided by the company. Macroporous glass (PG CB), pore diameter 500 A, surface area 35 m z g-l, produced and aminoalkylated according to the method of Janowski et al. I° had a mean content of NH2-groups of 61 /xmol g 1. Coupling procedures
Azocasein was coupled to Eupergit C according to the recommendations given by the company and washed thereafter with large volumes of PBS, pH 7.4, until no azocasein was detectable photometrically in the wash© 1988 Butterworth Publishers
Leakage of ligand-support conjugates: J. Lasch and F. Janowski
Leakage measurements
n filter
cuvett~tl ~ ~
Figure 1 Schematic representation of the experimental set-up for long-term measurements of ligand leakage. The minicolumn (0.8 x 4 cm), packed with 2 ml of conjugate, was pumped at 20°C with 0.2 M phosphate buffer, pH 7.5, at a constant velocity of 5 ml h 1and turned daily about its horizontal axis so as to prevent channel formation. Total volume of the system: 5.5 ml. Increasing the pumping speed to 50 ml h -~ did not change the results
ings. Usually, I g Eupergit was reacted with 10 ml of azocasein solution in PBS (0.2-10 mg m1-1) for 72 h at 23°C. The amount of bound protein was calculated from the absorbance decrease in the coupling solution corrected for the amount found in the washings. Trinitrophenylation of amino Eupergit was achieved by reacting 1 g of the carrier derivative with I0 ml of trinitrobenzene sulfonic acid (10 mg m1-1) dissolved in saturated borate for 30 rain at room temperature. The deep red trinitrophenyl derivative was then washed thoroughly with 0.2 M NaC1. Amino glass (0.5 g) was activated with glutaraldehyde (2.5%) for 1 h, washed and incubated with 5 ml azocasein solution in phosphate buffer, pH 8.0, for 72 h.
1,i/3
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._¢
~,, I ......f f
2
f
1
f !
2
,
~
K
z.
6
8
[j
I 60
Table 1 Initial leakage of azocasein coupled to Eupergit C
Protein bound (rag g-~)
Total release rate (/~g day -1)
2.6 6.1 14.6 26.7 37.8
354 350 524 776 794
(% day .~ ) 24.0 9.5 6.0 5.2 3.5
Tv2
Total release (3 days)
(h)
(/~g)
(%)
24 22 24 23 24
293 386 662 806 839
19.0 10.0 7.0 5.4 3.7
Table 2 Additional leakage of azocasein from the Eupergit support after exposition to 0.5% human serum albumin (HSA)
Protein bound (mg g-l)
Total release rate (/~g day -1)
2.6 6.1 14.6 26.7 37.8
154 191 175 284 294
(% day -~ ) 10.0 5.2 2.0 1.9 1.3
Tv2 (h) 20 18 19 18 20
Total release (3 days) (#g)
(%)
82 147 280 299 318
5.3 4.0 3.2 2.0 1.4
Table 3 Leakage of azocasein coupled to macroporous glass. Data in parentheses refer to controls done with non-activated amino glass
6
=
Long-term continuous leakage measurements were performed under sterile conditions in a recirculation reactor attached to an Eppendorf photometer (Figure 1). When the leakage rate had decreased to zero (after about 3 days) the system was flushed with 0.5% serum albumin and recirculation continued. The time course of the absorbance of circulating buffer was recorded and the absorbance readings converted to micromoles detached trinitrophenyl derivative, trinitrophenyl glycine serving as a reference compound, or to microgram released azocasein, using the specific absorption given above. Flushing the system at 12 h intervals with fresh buffer and adding up the amounts of free ligand in collected samples showed no difference to the closed system. As, in accordance with earlier findings in open systems 5'7, all of the ligand never leaked out, definition of half lives for 50% loss of total protein available is not feasible. Therefore, half lives of the bimodal leakage
days
Figure 2 Time course of ligand release from various ligandsupport conjugates. 1 : azocasein bound to Eupergit C, initial loading: 27 mg protein per g support. 2: azocasein bound to aminoallo/lated macroporous glass by the glutaraldehyde method, initial loading: 17 mg protein per g support. 3: azocasein adsorbed to non-activated amino glass (control), initial loading: 4 mg protein per g support
Initial release rate (%/day)
Protein bound (mg g-l)
_
0.5% HSA
4.3 (3.5) 16.7 (15,3) 36.5 (35.0)
6.5 (29.0) 4.6 (35.0) 1.8 (20.1)
30 (50) 6.4 (48.2) 4.0 (51.2)
Total release after 60 days in the presence of 0.5% HSA 20 (99) (after 3 days) 12 (100) 4 (98.5)
E n z y m e M i c r o b . T e c h n o l . , 1988, v o l . 10, M a y
313
Papers
(see F i g u r e 2) were given for each of the consecutive stages, i.e., without (Table 1) and with HSA (Table 2). They refer to loss of 50% of total protein lost, which is the characteristic time of practical interest. The initial release rates were calculated from the tangents to the release curves (Figure 2) either at time zero ( T a b l e s 1 and 3) or at the point of HSA addition ( T a b l e s 2 and 3).
Results and discussion Monopoint
attachment
The trinitrophenyl amino Eupergit (150/~mol trinitrophenyl residue per gram support) showed a linear leakage up to 60 days (not shown) corresponding to a constant release rate of 0.06% per day (equal to 90 nmol per g per day), i.e., a total release of 3.6% during 60 days. Multipoint
attachment
The results obtained with polyvalently bound azocasein are assembled in T a b l e s 1 to 3 and F i g u r e 2. Inspection of all the data reveals a number of interesting phenomena: (1) ligands monovalently bound to Eupergit leak extremely slowly; (2) protein is bound very stably to both supports by non-covalent interactions (this binding is saturable, see Table 1); (3) complete removal of this protein fraction needs treatment with serum albumin; (4) amino glass (control) adsorbs nearly the same amount of protein as is bound by the glutaraldehyde method, but it can be washed off rapidly and quantitatively (Table 3); (5) after proper treatment ( F i g u r e 2) the protein-support conjugates are completely stable under continuous operation up to 2 months and (6) the double cross-linking reaction on the
314
Enzyme Microb. Technol., 1988, vol. 10, May
glass surface with dialdehyde and protein renders this support stable against hydrolysis. There exist clearly two types of leakage: (1) splitting of the anchoring bond between ligand and matrix (to this category belong monovalently as well as multivalently bound ligands with labile linkages like Schiff's base bonding and the isourea bond formed after BrCN activation 1'5) and (2) slow desorption of ligands bound by multiple non-covalent interactions. As the strongly adsorbed protein desorbs very slowly, it will not be removed by conventional washing of the conjugate on the filter funnel. Therefore, we recommend before use continuous washing on a column for at least 3 days with a dilute protein solution. We should like to stress that physical studies of immobilized proteins washed on the filter funnel for a limited time are likely to reveal biphasic behavior of physical parameters, e.g., ESR correlation times, fluorescence lifetimes, thermostability etc. Depending on the total amount of protein bound, the strongly adsorbed fraction will fall into the range of 5 to 30% ( T a b l e s 1 to 3).
References 1 2 3 4 5 6 7 8 9 10
Tesser, G. I., Fisch, H.-U. and Schwyzer, R. FEBS Lett. 1972, 23, 56-58 Ludens, J. H., De Vries, J. R. and Franestil, D. D., J. Biol. Chem. 1972, 247, 7533-7538 Wilchek,M., Oka, T. and Topper, Y. J., Proc. Natl. Acad. Sci. USA 1975, 72, 1055-1058 Kolb, H. J. et al. Proc. Natl. Acad. Sci. USA 1975, 72, 248252 Lasch, J. and Koelsch, R. Eur. J. Biochem. 1978,82, 181-186 Hofstee, B. H. J. Proc. FEBS Meeting 1979, 52, 469-482 Lasch, J. et al. Anal. Chem. Symp. Series 9: Affinity Chromatography and Related Techniques, 1982, 245-254 Lasch, J. et al. GDR Pat. WP C12P/264 292/5 (1985) Langner, J. et al. Acta Biol. Med. Germ. 1973, 31, 1-18 Janowski, F. et al. GDR Patent WP C03C/283 065/6 (1985)
and F. Janowski D e p a r t m e n t o f Chemistry, Division o f Industrial Chemistry, Martin-Luther-University, Halle (Saale), G D R
(Received 14 March 1987; revised 15 October 1987)
Leakage of ligands bound either monovalently or polyvalently to Eupergit C and macroporous glass was followed spectrophotometrically in a recirculation reactor up to 2 months under continuous operation. The data revealed a very stable fraction of bound protein desorbing only slowly from saturable noncovalent binding sites. Once this fraction was removed (which takes at least 3 days of continuous washing) conjugates with practically unlimited leakage stability were obtained.
Keywords: Ligandleakage; ligand-support conjugates; long-timeligand stability
Introduction Tesser et al.~ and Ludens et al. 2 were the first to demonstrate that ligands monovalently bound to insoluble matrices leak out during continuous operation of affinity columns. Later it was shown 3'4'5 that especially CNBr-coupled ligands are prone to be split off from the matrix. In all leakage studies it was found consistently that the presence of nucleophiles (buffers, proteins) increased the leakage rate by about one order of magnitude.Z, 3,5 It is well known that the immobilization of ligands, particularly proteinaceous ligands, on solid supports is a complex combination of various interacting forces. 6 Differentiation between these forces is difficult. Besides the covalent linkage, a combination of noncovalent forces may generate stable binding. 6 However, for molecules that are bound only by noncovalent forces, there is always the risk of interchange of already bound molecules with others in solution and/or elution in extreme milieu. This is why in properly devised leakage studies different populations of 'immobilized' protein molecules can be washed out. 7 Our efforts to develop a proteolytic enzyme reactor 8 led us to reinvestigate the leakage problem more thor312
Enzyme Microb. Technol., 1988, vol. 10, May
oughly with carriers which, from an economical point of view, would be suitable for up-scaling.
Materials and methods Materials
Azocasein was prepared from Hammarsten casein according to the method of Langner et al. 9 It had an absorption coefficient of A°i~'nm = 3.77. Eupergit C 8 , a macroporous epoxide-activated polyacrylic carrier was a gift from R6hm-Pharma (Weiterstadt, FRG). Amino Eupergit was prepared from the epoxideactivated support by reaction with 1,6-hexane diamine as described in the instructions provided by the company. Macroporous glass (PG CB), pore diameter 500 A, surface area 35 m z g-l, produced and aminoalkylated according to the method of Janowski et al. I° had a mean content of NH2-groups of 61 /xmol g 1. Coupling procedures
Azocasein was coupled to Eupergit C according to the recommendations given by the company and washed thereafter with large volumes of PBS, pH 7.4, until no azocasein was detectable photometrically in the wash© 1988 Butterworth Publishers
Leakage of ligand-support conjugates: J. Lasch and F. Janowski
Leakage measurements
n filter
cuvett~tl ~ ~
Figure 1 Schematic representation of the experimental set-up for long-term measurements of ligand leakage. The minicolumn (0.8 x 4 cm), packed with 2 ml of conjugate, was pumped at 20°C with 0.2 M phosphate buffer, pH 7.5, at a constant velocity of 5 ml h 1and turned daily about its horizontal axis so as to prevent channel formation. Total volume of the system: 5.5 ml. Increasing the pumping speed to 50 ml h -~ did not change the results
ings. Usually, I g Eupergit was reacted with 10 ml of azocasein solution in PBS (0.2-10 mg m1-1) for 72 h at 23°C. The amount of bound protein was calculated from the absorbance decrease in the coupling solution corrected for the amount found in the washings. Trinitrophenylation of amino Eupergit was achieved by reacting 1 g of the carrier derivative with I0 ml of trinitrobenzene sulfonic acid (10 mg m1-1) dissolved in saturated borate for 30 rain at room temperature. The deep red trinitrophenyl derivative was then washed thoroughly with 0.2 M NaC1. Amino glass (0.5 g) was activated with glutaraldehyde (2.5%) for 1 h, washed and incubated with 5 ml azocasein solution in phosphate buffer, pH 8.0, for 72 h.
1,i/3
1z,
._¢
~,, I ......f f
2
f
1
f !
2
,
~
K
z.
6
8
[j
I 60
Table 1 Initial leakage of azocasein coupled to Eupergit C
Protein bound (rag g-~)
Total release rate (/~g day -1)
2.6 6.1 14.6 26.7 37.8
354 350 524 776 794
(% day .~ ) 24.0 9.5 6.0 5.2 3.5
Tv2
Total release (3 days)
(h)
(/~g)
(%)
24 22 24 23 24
293 386 662 806 839
19.0 10.0 7.0 5.4 3.7
Table 2 Additional leakage of azocasein from the Eupergit support after exposition to 0.5% human serum albumin (HSA)
Protein bound (mg g-l)
Total release rate (/~g day -1)
2.6 6.1 14.6 26.7 37.8
154 191 175 284 294
(% day -~ ) 10.0 5.2 2.0 1.9 1.3
Tv2 (h) 20 18 19 18 20
Total release (3 days) (#g)
(%)
82 147 280 299 318
5.3 4.0 3.2 2.0 1.4
Table 3 Leakage of azocasein coupled to macroporous glass. Data in parentheses refer to controls done with non-activated amino glass
6
=
Long-term continuous leakage measurements were performed under sterile conditions in a recirculation reactor attached to an Eppendorf photometer (Figure 1). When the leakage rate had decreased to zero (after about 3 days) the system was flushed with 0.5% serum albumin and recirculation continued. The time course of the absorbance of circulating buffer was recorded and the absorbance readings converted to micromoles detached trinitrophenyl derivative, trinitrophenyl glycine serving as a reference compound, or to microgram released azocasein, using the specific absorption given above. Flushing the system at 12 h intervals with fresh buffer and adding up the amounts of free ligand in collected samples showed no difference to the closed system. As, in accordance with earlier findings in open systems 5'7, all of the ligand never leaked out, definition of half lives for 50% loss of total protein available is not feasible. Therefore, half lives of the bimodal leakage
days
Figure 2 Time course of ligand release from various ligandsupport conjugates. 1 : azocasein bound to Eupergit C, initial loading: 27 mg protein per g support. 2: azocasein bound to aminoallo/lated macroporous glass by the glutaraldehyde method, initial loading: 17 mg protein per g support. 3: azocasein adsorbed to non-activated amino glass (control), initial loading: 4 mg protein per g support
Initial release rate (%/day)
Protein bound (mg g-l)
_
0.5% HSA
4.3 (3.5) 16.7 (15,3) 36.5 (35.0)
6.5 (29.0) 4.6 (35.0) 1.8 (20.1)
30 (50) 6.4 (48.2) 4.0 (51.2)
Total release after 60 days in the presence of 0.5% HSA 20 (99) (after 3 days) 12 (100) 4 (98.5)
E n z y m e M i c r o b . T e c h n o l . , 1988, v o l . 10, M a y
313
Papers
(see F i g u r e 2) were given for each of the consecutive stages, i.e., without (Table 1) and with HSA (Table 2). They refer to loss of 50% of total protein lost, which is the characteristic time of practical interest. The initial release rates were calculated from the tangents to the release curves (Figure 2) either at time zero ( T a b l e s 1 and 3) or at the point of HSA addition ( T a b l e s 2 and 3).
Results and discussion Monopoint
attachment
The trinitrophenyl amino Eupergit (150/~mol trinitrophenyl residue per gram support) showed a linear leakage up to 60 days (not shown) corresponding to a constant release rate of 0.06% per day (equal to 90 nmol per g per day), i.e., a total release of 3.6% during 60 days. Multipoint
attachment
The results obtained with polyvalently bound azocasein are assembled in T a b l e s 1 to 3 and F i g u r e 2. Inspection of all the data reveals a number of interesting phenomena: (1) ligands monovalently bound to Eupergit leak extremely slowly; (2) protein is bound very stably to both supports by non-covalent interactions (this binding is saturable, see Table 1); (3) complete removal of this protein fraction needs treatment with serum albumin; (4) amino glass (control) adsorbs nearly the same amount of protein as is bound by the glutaraldehyde method, but it can be washed off rapidly and quantitatively (Table 3); (5) after proper treatment ( F i g u r e 2) the protein-support conjugates are completely stable under continuous operation up to 2 months and (6) the double cross-linking reaction on the
314
Enzyme Microb. Technol., 1988, vol. 10, May
glass surface with dialdehyde and protein renders this support stable against hydrolysis. There exist clearly two types of leakage: (1) splitting of the anchoring bond between ligand and matrix (to this category belong monovalently as well as multivalently bound ligands with labile linkages like Schiff's base bonding and the isourea bond formed after BrCN activation 1'5) and (2) slow desorption of ligands bound by multiple non-covalent interactions. As the strongly adsorbed protein desorbs very slowly, it will not be removed by conventional washing of the conjugate on the filter funnel. Therefore, we recommend before use continuous washing on a column for at least 3 days with a dilute protein solution. We should like to stress that physical studies of immobilized proteins washed on the filter funnel for a limited time are likely to reveal biphasic behavior of physical parameters, e.g., ESR correlation times, fluorescence lifetimes, thermostability etc. Depending on the total amount of protein bound, the strongly adsorbed fraction will fall into the range of 5 to 30% ( T a b l e s 1 to 3).
References 1 2 3 4 5 6 7 8 9 10
Tesser, G. I., Fisch, H.-U. and Schwyzer, R. FEBS Lett. 1972, 23, 56-58 Ludens, J. H., De Vries, J. R. and Franestil, D. D., J. Biol. Chem. 1972, 247, 7533-7538 Wilchek,M., Oka, T. and Topper, Y. J., Proc. Natl. Acad. Sci. USA 1975, 72, 1055-1058 Kolb, H. J. et al. Proc. Natl. Acad. Sci. USA 1975, 72, 248252 Lasch, J. and Koelsch, R. Eur. J. Biochem. 1978,82, 181-186 Hofstee, B. H. J. Proc. FEBS Meeting 1979, 52, 469-482 Lasch, J. et al. Anal. Chem. Symp. Series 9: Affinity Chromatography and Related Techniques, 1982, 245-254 Lasch, J. et al. GDR Pat. WP C12P/264 292/5 (1985) Langner, J. et al. Acta Biol. Med. Germ. 1973, 31, 1-18 Janowski, F. et al. GDR Patent WP C03C/283 065/6 (1985)