ANALYTICAL
BIOCHEMISTRY
184,165-171(1990)
A Dual Streaming Potential Device Used as an Affinity Sensor for Monitoring Hybridoma Cell Cultivations Akiyoshi
Miyabayashi
Department
Received
of
August
Biotechnology,
and Bo Mattiasson Chemical
Center, University
of
Lund, P.O. Box 124, S-221 00 Lund, Sweden
9,1989
A dual streaming potential device was used for determining the content of monoclonal antibodies in cultivation medium for hybridoma cells. Samples of culture medium were analyzed as discrete pulses, as a continuous flow of constant concentration as well as with fluctuating concentrations. Tests were done with two subclasses of IgG as well as with IgM. Finally, the analytical device was applied to the registration of production of monoclonal antibodies in a cultivation. Q 1990
Academic
Press,
Inc.
The desire to monitor relevant parameters in biotechnological processes has led to the development of a variety of sensors for substrates and products (l-4) and cell density (56). Today, such sensors are used mainly in research laboratories. Efforts to implement this technology in production conditions have also been reported. When it comes to macromolecular structures, very little progress has been reported. In the research laboratory, immunosensors designed for use in clinical chemical analysis have been described (7-9). We recently described the construction of a new analytical device, a streaming potential measuring unit to be used to specifically monitor affinity interactions (10,ll). This sensor has now been further improved and been used in conjunction with cultivation of hybridoma cells in order to monitor the production of monoclonal antibodies.
THEORY
When a porous material is placed in a streaming liquid it is possible to monitor a streaming potential over the two electrodes on each side of the porous material. With the assumption that the potential difference created between two electrodes and the ends of a capillary tube of the radius a and the length 1 for an applied pressure difference of P, the typical streaming potential equations were defined. Considering the initial conditions as a laminar flow with flow velocity of Vx at a distance x, measured from the surface, the shear along the radius of the capillary is given by Poiseville’s equation, Vx=P.(2.aex-x2) 417.1
’
where 7 is the mean viscosity of the medium. Therefore, the rate of flow in the cylindical layer (flow velocity of Vx represented by a hollow cylinder radius a - x: with thickness dr) is .a.(a
- x)Vx.dx
=2.?r.P.(2.a.x-3~‘).(a-r).d~
MATERIALS
Sepharose CL-4B (Pharmacia Fine Chemicals AB., Uppsala, Sweden) was used as the gel material packed in the electrode columns. The rabbit anti-mouse IgG (product code: Z 259) was bought from Dakopatts A. S., Glostrup, Denmark. As a reference material in some experiments, Sepharose CL-4B with immobilized mouse anti-human antibodies (Z 450 from Dakopatts) was used. Supernatants of culture media containing different 0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
Ig molecules were obtained from BioInvent Int. AB. (Lund, Sweden). In this study, IgG, IgG, , and IgM were measured. Analytical grade chemicals were used in all experiments.
4q.l The streaming current (1,) is, therefore, Izpddll s
*
dt ’
where p is the bulk charge density. 165
Inc. reserved.
166
MIYABAYASHI
If the potential
where
AND
MATTIASSON
decay in the double layer is large, then
E is the permittivity,
and
where 1c,is the surface potential. In this case, it is an extension of the Smoluchowski equation, and x was negligible as compared with a. With the boundary condition of 4 = [ at x = 0 and y = 0, dy/ dx=Oatx=O,
I, =
C.P.A.{ q-1
’
where A is the cross section area of is the zeta potential, which depends the surface of the shear between the and the sample. The relation between current and by Ohm’s law:
where & is the conductivity Therefore,
the capillary, and [ on the potential at electrolyte solution potential
of the electrolyte
is related
solution.
where p is the liquid pressure. From the above equation, it is possible to state that the method is sensitive to any ion concentration changes in the liquid medium passing through the electrode column in a single unit system. However, in the dual column system, all liquid effects can be neglected since a reference unit is employed. The difference between the two electrode columns is that one contains immobilized ligands capable of interacting with target molecules in the passing flow, whereas the other is a blank gel. The charge density of the affinity gel in the working electrode column is affected by interaction with substances passing through the column. The differential signal measured between the two electrode systems is thus solely a reflection of this difference. Keeping all other factors constant, one can thus expect to monitor a change in the zeta potential as the affinity binding takes place in the analyzing electrode column. Therefore, the system can be achieved to measure the linear potential change vs
FIG. 1.
Scheme of the flow through electrodes. 1, electrode contact; 2, Teflon fittings; 3, Teflon tube; 4, Teflon electrode support; 5,0 ring; 6, Ag/AgCl electrode; 7, Teflon net; 8, affinity gel; 9, Plexiglas tube; 10, sample passage; 11, adjustable screw; 12, electrode holder; A and D, liquid flow outputs; B and C, liquid flow inputs.
linear substance concentration changes rather than logarithmic relationships established by the Nernst equation. ELECTRODE
SYSTEM
The dual flow-through electrode system has been described previously (10,ll). To facilitate column packing and the maintainance of equal pressure in both units, the LC column type flow-through electrode was designed and constructed (Fig. 1). It consists of Ag/AgCl disk electrodes with four holes (0 1 mm) in each to allow liquid passage. All materials were selected to be autoclavable. Both electrode units are placed in an electrode column holder constructed so that the packing pressure can be adjusted by twisting a screw. The sample was injected via synchronized dual injection valves in order to let the sample pulse enter the two flow columns simultaneously,
AFFINITY
SENSOR
FOR
MONOCLONAL
Flow Injection Analysis of Immunoglobulin by Its Interaction with Anti-&G Plain Sepharose CL-4B was packed in the reference electrode column. Sepharose CL-4B to which rabbit anti-mouse IgG was immobilized was packed in the counting electrode column. Tris-HCl buffer, 0.02 M, pH 7.2, was circulated for a few hours during which the steady-state differential signal was observed. Then, pulses of 0.5 ml culture medium containing IgG, were injected, and potentials were recorded. Culture broth was applied in varying degrees of dilution (diluted with plain medium). In this experiment, no cleaning by dissociating buffer of columns between the pulses was applied. The same procedure was repeated with IgG and IgM solutions.
CO2 gas
/
I
J
8 1 FIG. 2. System set-up for the continuous analysis of IgM concentration at hybridoma cell cultivation. Details are discussed in the text. 1, magnet; 2, culture media; 3, double wall glass flask; 4, rubber sealing; 5, air filter; 6, polycarbonate hollow fiber filter; 7, pump; 8, magnetic stirrer; A and B, warm water circulation; C, cell-free sample to the electrode system.
thereby signal.
minimizing
ELECTRIC
CIRCUIT
disturbances
167
ANTIBODIES
Continuous Sampling Analysis With a flow of diluted IgG solution (dilution of 400 times), the differential signal was continuously recorded
of the differential
DESIGN
The dual isolated electrometer was built as follows: the potential changes at each unit were detected by precision, low-power FET-input electrometer operational amplifiers (AD 515L. analog Devices, Norwood, MA) with RC filters (cutoff frequency 2 Hz). Both signals were isolated by the precision hybrid isolation amplifiers (AD 295C. Analog Devices) from leakage currents between flow cells. In this stage, there were individual baseline offset adjustments. Passing through the active lowpass filter (cutoff frequency 1 Hz), the signal was individually amplified by an operational amplifier (LT 101. Linear Technology, Milpitas, CA). The differential signal, which is the difference between the reference potential and the potential from the analyzing channel, was detected by the differential stage made of the operational amplifier (LT 101). PROCEDURES
Preparation of the Sorbent Material Antibodies against IgG were immobilized to Sepharose CL-4B which first was activated by tresyl chloride (12). Both the specific sorbent and a reference sorbent containing mouse anti-human IgG were prepared by the same method.
0
1.0
2.0 Total lg sample
3.0 (mL)
4.0
FIG. 3. The calibration curves of IgG,, IgG, and IgM. (a) IgG,: 1, in culture medium; 2, diluted 2 times; 3, diluted 5 times; 4, diluted 10 times. (0) IgG: 5, in full culture medium; 6, diluted 2 times; 7, diluted 5 times; 8, diluted 10 times. (0) IgM: 9, culture broth diluted 10 times; 10, diluted 20 times; 11, diluted 50 times; 12, diluted 100 times. (m) IgGi; (13) in culture medium: 14, diluted 2 times; 15, diluted 5 times; 16, diluted 10 times. Measurements 13-16 were monitored with mouse anti-human antibodies as a reference whereas all others were measured toward plain Sepharose. IgG,, IgG, and IgM concentrations of the concentrated solutions are determined to be 63,30, and 312 pg/ml, respectively, by radio immunoassay.
168
MIYABAYASHI
AND
MATTIASSON
0. Time (hrs) FIG. 4.
The graph
of continuous
flow analysis
of diluted
IgG solution
for 10 h. Then, buffer was circulated in order to test the stability of the signal. The effects of stepwise increases in IgG concentration in a continuous flow were also studied. In these experiments the contact time between the measuring device and the IgG containing solution was 1 h for each concentration.
Flow Injection Cultivation
Analysis of IgM through the Cell
For continuously monitoring IgM production under cell cultivation conditions, the system was set up as shown in Fig. 2. In this experiment, the total media volume of 150 ml was inoculated with 50 ml cell suspension. The inoculum was taken from an earlier cultivation and was diluted with fresh culture broth prior to inoculation. This procedure led to an initial content of Ig molecules, giving a background value when monitoring the net production in the cultivation. The cell-free sample was obtained by filtering through a polycarbonate hollow fiber filter (Gambro Fiber plasmafilter, Gambro AB, Lund, Sweden). Then, the cell-free permeate was recirculated into the batch. Samples of 50 ~1 vol were injected in the flow system. After each analysis, affinity-bound antibody was dissociated from the covalently bound antiIgG by exposure to 0.1 M glycine-HCl, pH 2.2, for 15
(dilution
of 400 times)
concentration
being
0.16 pg/ml.
min, followed by flushing with buffer. As a reference method of analysis, cell-free samples were collected, and the concentration of IgM was analyzed by radio immunoassay. RESULTS
AND
DISCUSSION
The use of streaming potential measurements for monitoring affinity interactions has been shown to be successful (10,ll). In the previous studies, various model systems were studied. Although the sensitivity of the system to changes in ionic strength in the perfusing medium was shown to be low, it is necessary to apply the analytical technique to a realistic system. For that end we chose to monitor production of monoclonal antibodies by continuous or intermittent sampling of cell-free culture medium. In the studies, a rabbit anti-mouse IgG antibody was used that cross-reacts with mouse IgM. We did not characterize this preparation further since the only important thing is that the antibody binds the molecule we wanted to quantify. The initial studies with discrete samples showed some interesting characteristics. The response was tested for varying degree of dilution of the same culture broth with unreacted broth. It was found that there is a good correlation between the signal registered and the amount of
AFFINITY
SENSOR
FOR
MONOCLONAL
169
ANTIBODIES
Time (hrs)
0-
a
4
6
6 Time (hrs)
FIG. 5. Effects of sample concentration (IgG) changes to the electrochemical signals. X2000, X800, X400, X200, Xl00 diluted IgG solutions were used respectively. (B) Random X8000, X200, X800, X8000 diluted IgG solutions were used respectively.
10
12
14
15
(A) Stepwise increase of IgG concentrations. x4000, changes of IgG concentrations. X800, X4000, x400,
170
MIYABAYASHI TABLE
AND
1
Signal Response to Different Concentrations of IgG-Containing Samples Introduced to the System (Data Were Taken from Fig. 5) Slope
(mV/h)
Sample
dilution
A
0.095 0.200 0.380 0.960 2.060 4.180
x4000 x2000 X800 x400 x200 Xl00
B
0.401 0.090 1.030 0.0475 2.150 0.404 0.050
X800 x4000 x400 X8000 x200 X800 X8000
IgG total
amount
(rg)
0.45 0.90 2.25 4.50 9.00 18.0 2.25 0.45 4.50 0.225 9.00 2.25 0.225
antibodies in the sample analyzed (Fig. 3). On plotting the values obtained at different degrees of dilutions versus the streaming potential monitored, linear relations were obtained. The lines were characterized by the following equations: y = 0.1296 + 0.1561x, R = 1.00; y = 0.0599 + 0.0687x, R = 1.00; y = -0.1143 + 0,6643x, R = 0.99 for IgG,, IgG, and IgM, respectively. These results were obtained from measurements using blank Sepharose in the reference column. The curves 13, 14, 15, and 16 were obtained for IgG, using a reference column of mouse anti-human IgG bound to Sepharose. It is thus clearly seen from these results that there was no unspecific adsorption to the unsubstituted Sepharose and that the signals obtained are due to specific interactions with the sorbent in the measuring column. Furthermore, different signals were obtained when analyzing media from cultivation of different clones. In subsequent RIAs, the concentration of IgG and IgM was determined and corrections were made for the variations in concentration. After this normalization of the values, very small differences were observed in potential registered per microgram of antibody bound. This may be interpreted as a result that the different clones have charge densities close to each other. The influences of varying charges and isoelectric points are now under investigation. Upon dilution of the culture broth with fresh medium by 400~ it was possible to monitor continuously for at least 10 h. Upon washing with buffer, the signal remained constant, indicating that the potential change registered was due to some interactions not dissociable with perfusion buffer (Fig. 4). However, upon treatment with low pH (glycine), the signal returned to the baseline level. In a series of continuous measurements, the concentration of IgG was changed stepwise and the reading was
MATTIASSON
maintained at each concentration until a stable slope in the potential vs time recording was obtained (Fig. 5). The test was performed both with a steadily increasing concentration of antibodies (Fig. 5A) and with samples where the contents varied in an unpredictable way (Fig. 5B). In Table 1, values are given on the slopes measured for the varying concentrations. The lowest concentration recorded was 10-l’ mol/liter. It is also seen that there is no carryover between the assays since the slope for the concentration of IgG is the same regardless if it was measured after a lower or a higher concentration. From this figure it is obvious that the slope of the curve reflects the concentration of antibodies in the sample analyzed. The analytical system was applied to a cultivation of hybridoma cells for production of monoclonal antibodies. Since a process of this type is fairly slow, we decided to use intermittent assays with glycine dissociation in between each assay. As shown earlier, it is also possible to monitor in a continuous mode, but under such circumstances there might be a risk of saturating the column. In the graph (Fig. 6), change in potential registered vs time is plotted, as well as change in content of monoclonal antibodies, determined by a RIA method vs time.
Time (hrs) FIG. 6. Correlations between the electrochemical signals and radio immunoassay of IgM analysis during the hybridoma cell cultivation. 0, electrochemical signal; n , radio immunoassay. Experimental details are given in the text.
AFFINITY
SENSOR
FOR
The RIA technique gives at best +5% and the streaming potential has earlier been shown to operate within ?3%. If these experimental errors were included in the figure it would be clear that the curves are more or less superimposed. This was interpreted that the streaming potential gives a measurement of the same accuracy when assaying monoclonal antibodies in culture broth. The results obtained so far in this study clearly point toward a potential use of streaming potential measurements for the monitoring of production of monoclonal antibodies. This would then open the way to better control of the process, both concerning cultivation and harvesting of the antibodies.
MONOCLONAL
171
ANTIBODIES
REFERENCES 1. Clark,
J. L., and Lyons,
C. (1962)
2. Guilbault,
G. G. (1976) Handbook ysis, Dekker, New York.
3. Guilbault, Ed.),
Ann.
G. G. (1976) in Methods 44, pp. 579-633, Academic
Vol.
N.Y.
Acad.
of Enzymatic
102,29.
Sci.
Methods
in Anal-
in Enzymology (Mosbach, Press, New York.
K.,
4. Danielsson,
B., Mattiasson, B., and Mosbach, K. (1981) in Applied Biochemistry and Bioengineering (Wingard, L. B., KatchalskiKatzir, E., and Goldstein, L., Eds.), Vol. 3, pp. 97-143, Academic Press, New York.
5. Matsunaga, crobiol.
T., Karube, I., and Suzuki, Biotechnol. 12,97-101.
6. Miyabayashi, technol.
A., Danielsson,
Tech.
S. (1980)
Eur.
B., and Mattiasson,
J. Appl.
B. (1987)
MiBio-
1,219-224.
7. Mattiasson, B., and Nilsson, H. (1977) FEBS Z&t. 78,251-254. 8. Aizawa, M., Marioka, A., Suzuki, S., and Nagamura, Y. (1979) ACKNOWLEDGMENTS The authors thank Ms. Lena Danielsson and Prof. Hikan Hikanson at the Department of Biotechnology and Mr. Michael Mecklenburg at the Department of Pure and Applied Biochemistry at the Chemical Center in Lund for valuable discussions and help. The cooperation of Dr. Christina Glad at BioInvent Int. AB in Lund is gratefully acknowledged. This project was supported by The National Swedish Board for Technical Development.
Anal.
Biochem.
94,22-28.
9. Boitieux, J.-L., Thomas, Acta 163.309-313. 10. Glad, C., and Mattiasson, 11. Mattiasson,
B., and
D., and Desmet,
B. (1986) Miyabayashi,
G. (1984)
Biosensors A. (1988)
Anal.
2,89-100. Anal. Chim.
Chim.
Acta
213,79-89. 12. Nilsson, 402.
K., and Mosbach,
K. (1980)
Eur.
J. Biochem.
112,397-
BIOCHEMISTRY
184,165-171(1990)
A Dual Streaming Potential Device Used as an Affinity Sensor for Monitoring Hybridoma Cell Cultivations Akiyoshi
Miyabayashi
Department
Received
of
August
Biotechnology,
and Bo Mattiasson Chemical
Center, University
of
Lund, P.O. Box 124, S-221 00 Lund, Sweden
9,1989
A dual streaming potential device was used for determining the content of monoclonal antibodies in cultivation medium for hybridoma cells. Samples of culture medium were analyzed as discrete pulses, as a continuous flow of constant concentration as well as with fluctuating concentrations. Tests were done with two subclasses of IgG as well as with IgM. Finally, the analytical device was applied to the registration of production of monoclonal antibodies in a cultivation. Q 1990
Academic
Press,
Inc.
The desire to monitor relevant parameters in biotechnological processes has led to the development of a variety of sensors for substrates and products (l-4) and cell density (56). Today, such sensors are used mainly in research laboratories. Efforts to implement this technology in production conditions have also been reported. When it comes to macromolecular structures, very little progress has been reported. In the research laboratory, immunosensors designed for use in clinical chemical analysis have been described (7-9). We recently described the construction of a new analytical device, a streaming potential measuring unit to be used to specifically monitor affinity interactions (10,ll). This sensor has now been further improved and been used in conjunction with cultivation of hybridoma cells in order to monitor the production of monoclonal antibodies.
THEORY
When a porous material is placed in a streaming liquid it is possible to monitor a streaming potential over the two electrodes on each side of the porous material. With the assumption that the potential difference created between two electrodes and the ends of a capillary tube of the radius a and the length 1 for an applied pressure difference of P, the typical streaming potential equations were defined. Considering the initial conditions as a laminar flow with flow velocity of Vx at a distance x, measured from the surface, the shear along the radius of the capillary is given by Poiseville’s equation, Vx=P.(2.aex-x2) 417.1
’
where 7 is the mean viscosity of the medium. Therefore, the rate of flow in the cylindical layer (flow velocity of Vx represented by a hollow cylinder radius a - x: with thickness dr) is .a.(a
- x)Vx.dx
=2.?r.P.(2.a.x-3~‘).(a-r).d~
MATERIALS
Sepharose CL-4B (Pharmacia Fine Chemicals AB., Uppsala, Sweden) was used as the gel material packed in the electrode columns. The rabbit anti-mouse IgG (product code: Z 259) was bought from Dakopatts A. S., Glostrup, Denmark. As a reference material in some experiments, Sepharose CL-4B with immobilized mouse anti-human antibodies (Z 450 from Dakopatts) was used. Supernatants of culture media containing different 0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
Ig molecules were obtained from BioInvent Int. AB. (Lund, Sweden). In this study, IgG, IgG, , and IgM were measured. Analytical grade chemicals were used in all experiments.
4q.l The streaming current (1,) is, therefore, Izpddll s
*
dt ’
where p is the bulk charge density. 165
Inc. reserved.
166
MIYABAYASHI
If the potential
where
AND
MATTIASSON
decay in the double layer is large, then
E is the permittivity,
and
where 1c,is the surface potential. In this case, it is an extension of the Smoluchowski equation, and x was negligible as compared with a. With the boundary condition of 4 = [ at x = 0 and y = 0, dy/ dx=Oatx=O,
I, =
C.P.A.{ q-1
’
where A is the cross section area of is the zeta potential, which depends the surface of the shear between the and the sample. The relation between current and by Ohm’s law:
where & is the conductivity Therefore,
the capillary, and [ on the potential at electrolyte solution potential
of the electrolyte
is related
solution.
where p is the liquid pressure. From the above equation, it is possible to state that the method is sensitive to any ion concentration changes in the liquid medium passing through the electrode column in a single unit system. However, in the dual column system, all liquid effects can be neglected since a reference unit is employed. The difference between the two electrode columns is that one contains immobilized ligands capable of interacting with target molecules in the passing flow, whereas the other is a blank gel. The charge density of the affinity gel in the working electrode column is affected by interaction with substances passing through the column. The differential signal measured between the two electrode systems is thus solely a reflection of this difference. Keeping all other factors constant, one can thus expect to monitor a change in the zeta potential as the affinity binding takes place in the analyzing electrode column. Therefore, the system can be achieved to measure the linear potential change vs
FIG. 1.
Scheme of the flow through electrodes. 1, electrode contact; 2, Teflon fittings; 3, Teflon tube; 4, Teflon electrode support; 5,0 ring; 6, Ag/AgCl electrode; 7, Teflon net; 8, affinity gel; 9, Plexiglas tube; 10, sample passage; 11, adjustable screw; 12, electrode holder; A and D, liquid flow outputs; B and C, liquid flow inputs.
linear substance concentration changes rather than logarithmic relationships established by the Nernst equation. ELECTRODE
SYSTEM
The dual flow-through electrode system has been described previously (10,ll). To facilitate column packing and the maintainance of equal pressure in both units, the LC column type flow-through electrode was designed and constructed (Fig. 1). It consists of Ag/AgCl disk electrodes with four holes (0 1 mm) in each to allow liquid passage. All materials were selected to be autoclavable. Both electrode units are placed in an electrode column holder constructed so that the packing pressure can be adjusted by twisting a screw. The sample was injected via synchronized dual injection valves in order to let the sample pulse enter the two flow columns simultaneously,
AFFINITY
SENSOR
FOR
MONOCLONAL
Flow Injection Analysis of Immunoglobulin by Its Interaction with Anti-&G Plain Sepharose CL-4B was packed in the reference electrode column. Sepharose CL-4B to which rabbit anti-mouse IgG was immobilized was packed in the counting electrode column. Tris-HCl buffer, 0.02 M, pH 7.2, was circulated for a few hours during which the steady-state differential signal was observed. Then, pulses of 0.5 ml culture medium containing IgG, were injected, and potentials were recorded. Culture broth was applied in varying degrees of dilution (diluted with plain medium). In this experiment, no cleaning by dissociating buffer of columns between the pulses was applied. The same procedure was repeated with IgG and IgM solutions.
CO2 gas
/
I
J
8 1 FIG. 2. System set-up for the continuous analysis of IgM concentration at hybridoma cell cultivation. Details are discussed in the text. 1, magnet; 2, culture media; 3, double wall glass flask; 4, rubber sealing; 5, air filter; 6, polycarbonate hollow fiber filter; 7, pump; 8, magnetic stirrer; A and B, warm water circulation; C, cell-free sample to the electrode system.
thereby signal.
minimizing
ELECTRIC
CIRCUIT
disturbances
167
ANTIBODIES
Continuous Sampling Analysis With a flow of diluted IgG solution (dilution of 400 times), the differential signal was continuously recorded
of the differential
DESIGN
The dual isolated electrometer was built as follows: the potential changes at each unit were detected by precision, low-power FET-input electrometer operational amplifiers (AD 515L. analog Devices, Norwood, MA) with RC filters (cutoff frequency 2 Hz). Both signals were isolated by the precision hybrid isolation amplifiers (AD 295C. Analog Devices) from leakage currents between flow cells. In this stage, there were individual baseline offset adjustments. Passing through the active lowpass filter (cutoff frequency 1 Hz), the signal was individually amplified by an operational amplifier (LT 101. Linear Technology, Milpitas, CA). The differential signal, which is the difference between the reference potential and the potential from the analyzing channel, was detected by the differential stage made of the operational amplifier (LT 101). PROCEDURES
Preparation of the Sorbent Material Antibodies against IgG were immobilized to Sepharose CL-4B which first was activated by tresyl chloride (12). Both the specific sorbent and a reference sorbent containing mouse anti-human IgG were prepared by the same method.
0
1.0
2.0 Total lg sample
3.0 (mL)
4.0
FIG. 3. The calibration curves of IgG,, IgG, and IgM. (a) IgG,: 1, in culture medium; 2, diluted 2 times; 3, diluted 5 times; 4, diluted 10 times. (0) IgG: 5, in full culture medium; 6, diluted 2 times; 7, diluted 5 times; 8, diluted 10 times. (0) IgM: 9, culture broth diluted 10 times; 10, diluted 20 times; 11, diluted 50 times; 12, diluted 100 times. (m) IgGi; (13) in culture medium: 14, diluted 2 times; 15, diluted 5 times; 16, diluted 10 times. Measurements 13-16 were monitored with mouse anti-human antibodies as a reference whereas all others were measured toward plain Sepharose. IgG,, IgG, and IgM concentrations of the concentrated solutions are determined to be 63,30, and 312 pg/ml, respectively, by radio immunoassay.
168
MIYABAYASHI
AND
MATTIASSON
0. Time (hrs) FIG. 4.
The graph
of continuous
flow analysis
of diluted
IgG solution
for 10 h. Then, buffer was circulated in order to test the stability of the signal. The effects of stepwise increases in IgG concentration in a continuous flow were also studied. In these experiments the contact time between the measuring device and the IgG containing solution was 1 h for each concentration.
Flow Injection Cultivation
Analysis of IgM through the Cell
For continuously monitoring IgM production under cell cultivation conditions, the system was set up as shown in Fig. 2. In this experiment, the total media volume of 150 ml was inoculated with 50 ml cell suspension. The inoculum was taken from an earlier cultivation and was diluted with fresh culture broth prior to inoculation. This procedure led to an initial content of Ig molecules, giving a background value when monitoring the net production in the cultivation. The cell-free sample was obtained by filtering through a polycarbonate hollow fiber filter (Gambro Fiber plasmafilter, Gambro AB, Lund, Sweden). Then, the cell-free permeate was recirculated into the batch. Samples of 50 ~1 vol were injected in the flow system. After each analysis, affinity-bound antibody was dissociated from the covalently bound antiIgG by exposure to 0.1 M glycine-HCl, pH 2.2, for 15
(dilution
of 400 times)
concentration
being
0.16 pg/ml.
min, followed by flushing with buffer. As a reference method of analysis, cell-free samples were collected, and the concentration of IgM was analyzed by radio immunoassay. RESULTS
AND
DISCUSSION
The use of streaming potential measurements for monitoring affinity interactions has been shown to be successful (10,ll). In the previous studies, various model systems were studied. Although the sensitivity of the system to changes in ionic strength in the perfusing medium was shown to be low, it is necessary to apply the analytical technique to a realistic system. For that end we chose to monitor production of monoclonal antibodies by continuous or intermittent sampling of cell-free culture medium. In the studies, a rabbit anti-mouse IgG antibody was used that cross-reacts with mouse IgM. We did not characterize this preparation further since the only important thing is that the antibody binds the molecule we wanted to quantify. The initial studies with discrete samples showed some interesting characteristics. The response was tested for varying degree of dilution of the same culture broth with unreacted broth. It was found that there is a good correlation between the signal registered and the amount of
AFFINITY
SENSOR
FOR
MONOCLONAL
169
ANTIBODIES
Time (hrs)
0-
a
4
6
6 Time (hrs)
FIG. 5. Effects of sample concentration (IgG) changes to the electrochemical signals. X2000, X800, X400, X200, Xl00 diluted IgG solutions were used respectively. (B) Random X8000, X200, X800, X8000 diluted IgG solutions were used respectively.
10
12
14
15
(A) Stepwise increase of IgG concentrations. x4000, changes of IgG concentrations. X800, X4000, x400,
170
MIYABAYASHI TABLE
AND
1
Signal Response to Different Concentrations of IgG-Containing Samples Introduced to the System (Data Were Taken from Fig. 5) Slope
(mV/h)
Sample
dilution
A
0.095 0.200 0.380 0.960 2.060 4.180
x4000 x2000 X800 x400 x200 Xl00
B
0.401 0.090 1.030 0.0475 2.150 0.404 0.050
X800 x4000 x400 X8000 x200 X800 X8000
IgG total
amount
(rg)
0.45 0.90 2.25 4.50 9.00 18.0 2.25 0.45 4.50 0.225 9.00 2.25 0.225
antibodies in the sample analyzed (Fig. 3). On plotting the values obtained at different degrees of dilutions versus the streaming potential monitored, linear relations were obtained. The lines were characterized by the following equations: y = 0.1296 + 0.1561x, R = 1.00; y = 0.0599 + 0.0687x, R = 1.00; y = -0.1143 + 0,6643x, R = 0.99 for IgG,, IgG, and IgM, respectively. These results were obtained from measurements using blank Sepharose in the reference column. The curves 13, 14, 15, and 16 were obtained for IgG, using a reference column of mouse anti-human IgG bound to Sepharose. It is thus clearly seen from these results that there was no unspecific adsorption to the unsubstituted Sepharose and that the signals obtained are due to specific interactions with the sorbent in the measuring column. Furthermore, different signals were obtained when analyzing media from cultivation of different clones. In subsequent RIAs, the concentration of IgG and IgM was determined and corrections were made for the variations in concentration. After this normalization of the values, very small differences were observed in potential registered per microgram of antibody bound. This may be interpreted as a result that the different clones have charge densities close to each other. The influences of varying charges and isoelectric points are now under investigation. Upon dilution of the culture broth with fresh medium by 400~ it was possible to monitor continuously for at least 10 h. Upon washing with buffer, the signal remained constant, indicating that the potential change registered was due to some interactions not dissociable with perfusion buffer (Fig. 4). However, upon treatment with low pH (glycine), the signal returned to the baseline level. In a series of continuous measurements, the concentration of IgG was changed stepwise and the reading was
MATTIASSON
maintained at each concentration until a stable slope in the potential vs time recording was obtained (Fig. 5). The test was performed both with a steadily increasing concentration of antibodies (Fig. 5A) and with samples where the contents varied in an unpredictable way (Fig. 5B). In Table 1, values are given on the slopes measured for the varying concentrations. The lowest concentration recorded was 10-l’ mol/liter. It is also seen that there is no carryover between the assays since the slope for the concentration of IgG is the same regardless if it was measured after a lower or a higher concentration. From this figure it is obvious that the slope of the curve reflects the concentration of antibodies in the sample analyzed. The analytical system was applied to a cultivation of hybridoma cells for production of monoclonal antibodies. Since a process of this type is fairly slow, we decided to use intermittent assays with glycine dissociation in between each assay. As shown earlier, it is also possible to monitor in a continuous mode, but under such circumstances there might be a risk of saturating the column. In the graph (Fig. 6), change in potential registered vs time is plotted, as well as change in content of monoclonal antibodies, determined by a RIA method vs time.
Time (hrs) FIG. 6. Correlations between the electrochemical signals and radio immunoassay of IgM analysis during the hybridoma cell cultivation. 0, electrochemical signal; n , radio immunoassay. Experimental details are given in the text.
AFFINITY
SENSOR
FOR
The RIA technique gives at best +5% and the streaming potential has earlier been shown to operate within ?3%. If these experimental errors were included in the figure it would be clear that the curves are more or less superimposed. This was interpreted that the streaming potential gives a measurement of the same accuracy when assaying monoclonal antibodies in culture broth. The results obtained so far in this study clearly point toward a potential use of streaming potential measurements for the monitoring of production of monoclonal antibodies. This would then open the way to better control of the process, both concerning cultivation and harvesting of the antibodies.
MONOCLONAL
171
ANTIBODIES
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