JPH06500396A - Quantitative analysis and monitoring of protein structure by subtractive chromatography - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Quantitative analysis and monitoring of protein structure by subtractive chromatography Group l Tomoji! amount In a broad sense, the present invention is a method for quantifying and structurally analyzing solutions containing a large number of solutes. Regarding the method. In particular, the present invention provides a subtractive frontal gray-through analysis method. is used to detect the presence and concentration of solutes in a solution, as well as to determine their structural characteristics. Concerning how to clarify.

This method regulates the quality and quantity of desired intermediates and products in the production of therapeutic substances. Efficiency in monitoring each synthesis process step to ensure on-time performance Helps raise.

In the manufacture of therapeutic substances, many steps are taken to ensure the integrity of their manufacturing processes. There are federal regulations and appropriate safety measures that must be taken to maintain manufacturing accuracy. Measures are also subject to constraints from an economic standpoint. In fact, drugs, pesticides, food additives, Manufacture pigments or other expensive substances in a multistep process. any manufacturing amount Even in the field, it is possible to quickly and accurately determine the concentration of products produced at each manufacturing or purification step. Accurate monitoring is the key to maintaining production efficiency.

On the other hand, there is no method for precisely quantifying specific solutes such as proteins or organic compounds in a mixed solution. However, these methods are time-consuming and costly to perform the analysis. It cannot be used regularly due to the complexity of analysis and analysis. These methods are °Actual manufacturing process due to inability to provide “real-time” information It is practically impossible to use this method for continuous monitoring in

The state of each manufacturing system is determined by data obtained from samples at each point in time. This is because it has to be changed considerably depending on the data.

In addition, structural isomers of proteins, such as therapeutic proteins made by recombinant Currently, the quantification and structural analysis of Although this may appear as a difference in morphology, each variety can be re-dissolved individually from the gel. It is impossible except in certain cases. Confirming the presence of structural isomers can be useful, for example, in therapeutic Biochemical synthesis of proteins, such as recombinant DNA technology, which is applied to the production of therapeutic proteins. This is extremely important in the field of development. Proteins produced by recombinant technology The quality of For example, those with reduced activity, those with harmful side effects, or those with undesirable anti-inflammatory properties. They tend to include things that are of primary origin. Structural isomers can also be used for mutational processes after protein translation. , such as variations in hydrolysis patterns, S-3 binding or protein folding. It is also generated by

In the biotechnology industry, mixed solutions - in which cell debris, various solutes, nutritional components, DNA, lipids, class II, and various proteins with similar physicochemical properties. White, some types of recombinant proteins that contain many impurities such as structural isomers It is often necessary to monitor the concentration of target substances. The concentration of the target substance in the collection solution is , usually 100g/j! However, it can sometimes be as low as 1/l. Ru. Making identification even more difficult, many of the objects are fragile. Therefore, it is more preferable to denature these solutions under relatively low shear. must be treated with minimal and shortest contact with sensitive surfaces. Ru.

One of the known methods for identifying solutes in solutions is affinity adsorption chromatography. There is a topography. This is one type of solute or several solutes in a mixed solution. A bed filled with particles that selectively adsorbs a combination of This method involves passing a mixture sample over a matrix. Next, the adsorbed table The adsorbed substances are removed by passing a solution that changes the chemical environment of the surface. Elute. The solute moves through this system through the gaps between particles or inside the particles. It flows while spreading. Typical carriers used in liquid chromatography are , soft, highly porous particles with a high surface area/volume ratio. So As a result, liquid chromatography cannot operate at pressures higher than 50 psi; Attempts to increase fluid velocity have the opposite effect of reducing separation efficiency.

High performance liquid chromatography (HPLC) Matrix hard and porous particles such as hard polymers such as range vinylbenzene. It has been adopted in Kusu. For this reason, HPCL is somewhat faster and allows for efficient division and separation. It is Noh.

Chromatography used for purification purposes typically consists of four steps: including loading, washing, elution and re-equilibration operations. What governs each stage is the fluid and static This is the movement of molecules between the surface of the matrix and the surface of the matrix. Optimum efficiency is achieved by rapid, preferably instantaneous This is achieved by intermittent mass movement and high-velocity fluid movement. For load operation for adsorption The higher the velocity of the mobile phase in the adsorption bed, the fewer molecules are adsorbed. . Once the mobile phase reaches a certain speed, the target solute will “breakthrough”. Therefore, it flows out into the effluent. That is, it passes through without being adsorbed by the matrix. and comes out in the effluent. If we limit this “breakthrough concentration” and For example, if the concentration is set to 5% of the injectate concentration, the maximum adsorption bed that the adsorption bed can handle is If you increase the adsorption bed speed any further, it will be wastefully discharged. This only results in a reduction in the filling throughput per unit surface area.

In recent years, new matrix designs have been developed to increase the separation speed of liquid chromatography. Inn is developing. For example, non-porous microparticles can be used as a matrix. This greatly increases the speed of separation, avoiding the rate-limiting diffusion step problem. It is possible to raise it to the width.

In other words, the solution flows convectively through the gaps between the particles that make up the matrix, and the solute reacts with the particle surfaces that form the walls of the gap. In this way, separation is based on diffusion. It can be done much faster than conventional chromatography systems. however , the processing capacity (processing power) will be considerably reduced.

The throughput of chromatography consists of relatively large and small porous particles. A matrix is used to suppress the convective flow inside or around the particles. You can make it bigger by doing this. This type of chromatography It is called perfusion chromatography and is patent application number 376. ．． No. 885 (July 6, 1989 issue 11), which is also disclosed as a reference of this application. and quoted here.

Parts-use chromatography technology enables high speed, high throughput, and high resolution separation becomes possible. Barfuse 7 Trinox is PerSeptive Manually available from Biosystems Inc. (Cambridge, MA) Ru.

Even with the fastest chromatography techniques, such as those mentioned above, solutes are lost during the product manufacturing process. is too slow for real-time analysis. The solution being monitored is complex. and many steps are required for analysis. These known analytical techniques quickly provide the data needed to make critical adjustments in the manufacturing process. It cannot be provided with sufficient sensitivity to detect structural isomers of protein solutes. It's not a minute.

One of the objectives of the present invention is to utilize the advantages of high speed chromatography techniques to An object of the present invention is to provide a method for monitoring the concentration of solutes in real time. too Another objective is to control the concentration of therapeutic substances or other solutes during the manufacturing process or Those who can quickly, accurately and precisely monitor any stage of the purification process. It is to provide law. Yet another objective is to determine the properties of mixtures and the structures within them. It is an object of the present invention to provide a method for obtaining a profile representing the relative concentrations of isomers. Sara Another objective is to provide these methods with self-checking capabilities. be.

Main uni next day 1 The present invention relates to subtractive chromatography in this application. analysis of therapeutic and other substances based on the methods detailed in the The present invention provides a method and apparatus for rapidly performing characterization. In the present invention, A solution containing several solutes may contain binding molecules specific for one or more solutes of interest. It passes through a matrix that has matching sites (adsorption sites). used here As stated above, the “target soluble f” is loosely defined and includes all water-soluble analytes. Typical examples include proteins, such as recombinant proteins.

By analyzing the effluent flowing out from the column, structural isomers in the target solute can be determined. The presence, concentration, and overall picture can be confirmed.

In the first object of the invention, one or more solutes of interest, e.g. A mixed solution of biochemically active molecules such as proteins, polysaccharides, etc. and other solutes. , through a matrix having binding sites specific for the solute of interest. injection solution As it passes through this matrix, the target solute is adsorbed to this binding site, and Thus, the target solute is virtually completely removed from the effluent. In this process, the purpose Very small amounts of substances other than the solute may also be adsorbed non-selectively to this matrix. be. Monitor the effluent to determine the concentration of solutes.

In a preferred embodiment of the invention, ultraviolet radiation is monitored proportional to the concentration in the effluent. However, if it has the same effect, there are other methods. A kana method is also fine.

Any method that allows data on the solute concentration in the effluent to be obtained may be used.

As the effluent leaves the column, the concentration of contaminants or non-target solutes in the effluent begins to increase, and the concentration of the non-target solute in the effluent becomes equal to the concentration of the non-target solute in the raw solution. Increase and balance until they are equal. For example, consider this as a relationship between ultraviolet absorbance and time. When graphed, the analysis results at this stage can be expressed as follows, depending on the characteristics of the matrix: It becomes an upward sloping line or a vertical line, and when the solute concentration in the effluent is at its maximum, it becomes a horizontal line. Shift to line.

The solute concentration in the effluent will vary as long as binding sites in the matrix remain. It exhibits a substantially constant value throughout the flow through the matrix. However, the final , the binding sites in the matrix are saturated and the target solute is in the matrix. will be able to flow directly without being adsorbed. This condition is called a breakthrough. It will be revealed. When the target solute begins to appear in the effluent without binding to the matrix, the effluent detected as an increase in UV absorption. In this way, when the melt concentration is flat, That is, the solute in the injection solution simply enters the column without any effect on the matrix surface. , the solute concentration in the effluent is equal to the solute concentration in the injected solution. Ru.

When using a diffusion-free chromatography matrix or diffusion time When the flow velocity is small compared to It appears. In other words, when the concentration of non-target solutes in the effluent reaches an equilibrium state, one It becomes a clear flat line. then indicated by a vertical line or a sloping line approaching vertical The transition phase results in a second flat line representing the total concentration of the desired and non-target solutes. .

This difference in equilibrium concentration is directly proportional to the concentration of the solute of interest in the sample. Therefore, these equilibrium concentrations can be used to calculate the concentration of the target solute in the sample. use Additionally, the second plateau represents the sum of solutes in the injection solution. , by monitoring the sample before it enters the matrix. You can know its value.

In this way, all the information needed to calculate the target solute concentration is Or, once we reach a plateau due to impurity breakthrough, all we can gain is I can do it. The instrument scale is detected by flowing a known concentration of pure target solute to the detector. The concentration value is then carved in such a way that it directly correlates with the absorbance, for example. Flat area detected The objective base is the difference multiplied by the correlation coefficient.

It is the concentration of quality.

In another embodiment, the present invention can be used to analyze the structural profile of proteins in separate samples. It is used to detect the difference between

As used herein, the term "structural profile" refers to This refers to a specific mixing state of different molecules in a batch, and this mixing state changes from batch to batch. In addition, expression errors over time, differences in DNA sequence between cultured clones, and Mutations due to degradation by rotease or post-translational conformation or addition It changes depending on etc.

In this example, the structural isomers in the sample have different binding capacities. A matrix of immobilized binding sites is used, and the sample is applied to this matrix. A method is used to pass the . For example, use polyclonal antibodies. that claw A protein isomer acts specifically on a particular antigenic determinant based on the particular isomer of the protein.

Alternatively, affinity or substrate specificity that binds only the protein isomer of interest may be used. It is also possible to use one type of unipotent binding site with isomerism.

According to this method, after at least a portion of the matrix has been saturated with protein, Proteins in a sample can be determined by measuring the protein concentration as it passes through the matrix. Breakthrough functional characteristic values can be obtained to know the structural profile of white. Ru. By comparing this characteristic value for different samples, their Structural components can be compared indirectly.

The molecular subspecies in a mixed protein each have distinct structural characteristics, so Each subspecies has at least a number of unique antigenic determinations (epitopes). death Therefore, each fraction of protein captured in the matrix can be separated separately. ability to capture specific molecular subspecies as a population or to predominate specific molecular subspecies. It can be captured in advance. In this way, the protein sample passes through the matrix. As it passes, the various subspecies in the protein reach a state of saturated equilibrium and then pass into the effluent. Become a breakthrough. Monitoring the protein concentration of the effluent over time can The protein concentration in the effluent ranges from the baseline value, usually zero, to the protein concentration in the feed solution and the actual It can be seen that there is a certain delay before the values become the same. during this period of delay. The increase in white density reflects the unique structural profile of each protein sample. If we compare this feature in separate samples, we can see that they have the same structure. It can be determined whether This method is used, for example, to maintain product properties as specified. As a means, it can be used for periodic monitoring of product manufacturing processes.

In some cases, the characteristics of the sample can be characterized by parameters that describe its concentration (e.g. linear absorbance”) and a parameter indicating the amount of liquid leaving the matrix (e.g., flow rate If is constant, it can be expressed by plotting "time").

Alternatively, the protein can be flushed out of the matrix, the matrix washed, and The injection and measurement steps can be repeated many times. This method is particularly useful for It is most effective when the sample is an at least partially purified aqueous protein solution. Ru. This method involves flowing proteins into a matrix using a pressure gradient or an injection gradient method. The law will be taken.

The matrix is preferably a hard, non-porous, granular material with a hydrophilic surface and is Dispersed chromatography matrices are preferred. The matrix is also capillary defined by the internal surface of Protein immobilized on the surface of the matrix A, consists of protein G, and if the binding protein is immunoglobulin, - for each same operation. Binding sites can be removed from the matrix and refilled with new binding sites . Immunoglobulin and other pallor binding sites are attached to the lipophilic polymer matrix surface. Adsorbed non-selectively to surfaces and removed with a mixed organic/ionic strip solution Can be done.

In order to implement the invention and obtain clear breakthroughs, chromatography or requires effective use of electrophoresis techniques. This flows through the matrix Basically, if the speed of can be achieved.

For low flow rates, the pores of conventional HPLC or other chromatographic supports It is difficult to achieve concentration equilibrium in the effluent due to the long time required for solutes to enter and exit the effluent. Because it doesn't interfere.

However, when using conventional carriers and high flow rates, the concentration equilibrium of the effluent ( flat areas) are usually not discernible. Basically, if you want high-speed processing, This means that a diffusion-free chromatography carrier must be used. Also, it is better to make the matrix as small as possible. The samples held in the matrix Sample volume and flow rate determine the time from sample injection to breakthrough.

High flow rates and small column volumes aid rapid analysis. Such an approach By doing this, analysis can be done virtually within a minute, and even more so in less than 10 seconds. It's also easy to put it down. In practice, such short-term measurements are called “real-time” measurements. It is considered as "mu".

Quantitative analysis techniques are independent of flow rate and require that the target solute and matrix are in equilibrium. It doesn't mean that you can't do it unless you reach a certain state. Therefore, the sample matrix The sample may be injected manually, e.g. Matrix can be injected using a syringe or an electric pump. Alternatively, gradient injection may be used as in electrophoresis.

One of the major advantages of the invention is that repeatable measurements can be made without compromising process accuracy. This means that analysis is possible. Some of the binding sites in the matrix include binding, elution, Although repeated re-equilibration causes performance deterioration, the method of the present invention It generates information based on differences in the concentration of non-target solutes.

Therefore, if there are fewer binding sites, the breakthrough of the target solute will be accelerated. However, there will be no inaccurate display of concentration.

The invention can also provide self-check functionality. If the injection solution and flow A detected difference in the final equilibrium concentration of the effluent indicates that the system is operating properly. It means that there is no one.

The self-check also cleans the i-mix after the effluent reaches its final equilibrium concentration. Alternatively, the target solute can be eluted. detected from the eluent The accumulation of pulses represents the amount of bound solute of interest, which is All you have to do is correlate it with the obtained data.

Another advantage of the invention is that the method is flexible. and For example, consider a sample of a high concentration of a solute of interest passing through a matrix. In this case, the binding sites in the matrix become saturated almost immediately and Therefore, a breakthrough will occur soon. Think about this using a graph like the one above. At first, a vertical line appears, and then it connects to a horizontal flat line, and the desired melting point is reached. No information about the quality or concentration of non-target solutes appears; how can this be corrected? , simply dilute the sample with a buffer solution. Break by diluting the sample The slug is slowed down and therefore the equilibrium concentration of non-target solutes in the effluent and also the effluent A clear distinction can be made between the equilibrium concentration of the target solute and non-target solute mixture in the liquid. become. If the amount of diluent is clear, the precision and precision of the results obtained will depend on the dilution. has no adverse effects. Highly diluted samples can also be analyzed on a regular basis. can. In this case, the only potential concern with this system is that the binding site The only difference is that it takes longer to saturate. After breakthrough of target solute This is of course true for this system, since the equilibrium state of can be detected directly from the sample. This is an inconvenience only in terms of the system's self-check function.

Another feature of the invention is that the binding sites of the matrix, e.g. or polyclonal antibodies or other binding proteins using known techniques. It is possible to quickly replace the solute depending on the solute. This allows one tomato You can handle any target solute by simply preparing the mixer and measuring device. It turns out.

A further advantage of the invention is that the system can be used on a very small scale. There are many things that can be mentioned. Even small samples of microliter size can be analyzed. Ru. Moreover, traditional chromatography columns require large amounts of space to create the matrix. The column is filled with particles with a large surface area, but in this system, the inner surface of the capillary All you have to do is coat it with a binding protein. Pass the sample solution through this capillary You can achieve the same results as the analysis method described above by simply doing this.

Analytical equipment for carrying out the invention includes injection (output) for samples, effluents and buffers. Detect solute concentration in the effluent, matrix channel equipped with binding protein, and effluent. Detector that can be used to detect and convert the value obtained by the detector into a meaningful display. It is composed of air circuit devices, and it is also possible to incorporate all of these into one module. can. Alternatively, you can create inexpensive replacement Matrinox modules and use them for other needs. It is also possible to incorporate it into a device composed of necessary equipment. module is short term After using for a while, it can be disposed of and replaced. Furthermore, each has a different purpose. By preparing a module set that can combine solutes, it is possible to analyze a given target solute. It is also possible to create a device that can respond quickly to various situations.

The above and other advantages, objects and characteristics of the invention are described below. It will be more clearly understood by reference to the details and accompanying drawings. Numbers in the drawings correspond to parts in the preferred embodiment.

Ukosara to Sarasato Hoshizuri FIG. 1 is a typical example of a diffusion-type, high-output type chromatogram.

2 to 4 are various chromatograms for explaining the principle of the present invention.

5 and 6 are schematic diagrams of two examples of devices according to the invention, in which the numbers indicate the embodiments. It corresponds to the part number inside.

Figures 7 and 8 are chromatograms obtained using the analytical method of the present invention, in which BSA The concentration of immunoglobulin in a solution containing This is what was measured.

Figure 9 shows the experimental results and tertiary structure profile of mouse gamma globulin. 2 is a chromatogram illustrating the possibility of implementing the present invention.

Figure 10 shows typical milli-absorbance (sAu) and protein (IgG) concentration (mg/d). This is a calibration curve.

new i day Broadly speaking, the present invention is based on subtractive frontal breakthrough analysis. The present invention provides a method for monitoring emitted solutes.

The concept of the present invention is to utilize adsorption chromatography to at least Separate one or more desired solutes and remove impurities in the effluent emerging from the matrix It measures the equilibrium concentration of solute in bone. In the first example, the next step To assay the concentration of the solute of interest in the analyte, all solutes in the solution are A method that utilizes the difference between the total detected concentration and the concentration detected as an impurity in the solution. It shows. In the second example, the solute to be analyzed is a protein, especially a recombinantly produced or A purified protein that contains many structural isomers, and these individual isomers bond together. have at least slightly different affinities for the target protein, and each has at least a unique affinity. Examples of cases with unique antigenic determinants are disclosed. protein sample, preferably Substantially free of impurities are matrices that bind one type of protein. A mask that incorporates an immobilized polyclonal antibody against the protein targeted by the drug. Pass through Trix. The various protein isomer molecules in the sample are polyclonal. saturate the various clonal species of the antibody, or if the binding site is a single one, antagonize to occupy the adsorption site of and then make a breakthrough.

The output monitor displays a breakthrough frontal that shows the characteristics of each sample. Draw a step-like plot.

Equipment-1 5 and 6 are schematic diagrams of apparatus for carrying out the process of the invention. . In FIG. 5, outlet 12 of valve 10 connects the sample from sample inlet 14. or a laser pad for cleaning and re-equilibrating the chromatography matrix. -16 or from the adsorption site of the chromatography matrix. to any of the eluents from Razor Par 18 that have the ability to elute the attached solutes. It's starting to get through. The selected fluid is finally supplied from valve 10 as will be explained in more detail later. A chromatography matrix 20 having properties as described below is passed through.

This matrix is placed on the surface of the solution to be analyzed or measured. A binding site having the ability to selectively bind the target solute is arranged. model As shown at 22 in the mold diagram, between the valve 10 and the matrix 20, an additional In general, the solute concentration in the sample is sent as a signal through line 24. Detector 22 may include a detector 22 that is used in conventional chromatography equipment. Conventional equipment, such as passing a beam through a film of sample, U, V for emitting light, U for measuring the absorbance of the solute in the light source and the sample. , V, and a detector, the liquid leaving the matrix 20 is detected. Enter vessel 26. In this case, this detector detects a parameter representing the solute concentration in the effluent. A signal of this value is sent out through line 28. Detector Line 24 from 22 and line 28 from detector 26 are connected to electronic computing device 30. where, for example, the difference between the maximum absorbances detected by detectors 22 and 26 is calculated. be done. Then, the target solute concentration is determined by multiplying the difference by a conversion coefficient. child The density value is sent via line 32 to a display device 34.

If detector 22 is omitted, the operation of the apparatus of FIG. 5 will be slightly different.

Specifically, the detector 26 first detects non-target solutes or impurities that have left the matrix 20. The first flat part, which represents the concentration of the substance, is detected, and after a certain period of time has passed, the first flat part is detected. After breakthrough of the target solute, the total solute concentration is detected. As these flat areas The detected data is sent via line 28 to a computing device 30 for calculation as described above. It is processed.

FIG. 6 shows another embodiment of the invention. The operation of this device is conceptually Same as in case 5, but the effluent from matrix 7x 20 is passed through line 36. The difference is that it is returned to the detector 22. According to this method, with one detector, first , a pilot sample is run through the matrix 20 to measure the total solute concentration, and then Measure the plateau level of the effluent before breakthrough of the target solute occurs. This fruit In the example design, a reservoir is placed in line 38 between detector 22 and matrix 20. tank (not shown) or before the solute concentration in the effluent reaches a plateau. Some measure is taken to ensure that all sample is removed from detector 22. It would be necessary to do so. The signal representing the solute concentration detected by the detector 22 is as described above. is sent to computing device 30 via line 24.

The purpose of the calculation device 30 is simply to monitor the structure of the protein. You can omit it if you have it. In this case, the display device 34 represents the protein concentration in the effluent. It is devised so that the value and the value representing the amount of effluent are displayed in comparison. Kosu If so, the display plots a curve that characterizes the structural profile of the sample protein. identification of the composition of a given sample or the structural profile of a protein. It can be used as a “fingerprint” to detect changes.

Main 11 skin In both of the examples shown in Figures 5 and 6, the matrix is applied to the entire system before starting the analysis. Fill the buffer solution 16 to equilibrate the gas 20, and fill the detector 22.26 with solute bone. Make sure there is no residue. To begin quantitative analysis, introduce sample 14 into the system. Adjust valve 10 to do so. This sample can be delivered by pump or syringe. electrophoretic movement in the matrix 20 or the pressure gradient created by the is pumped into the system by an injection gradient that causes Optional implementation of Figures 5 and 6 By way of example, a value representative of the total solute concentration in the sample may be obtained at detector 22.

The sample then enters matrix 20. The target solute is immobilized in the matrix. Impurities that do not bind forcefully flow through the matrix. It begins to come out as fluid.

In the embodiment of FIG. 5, the impurities that accumulate in the effluent are detected by the detector 26 and If so, it is returned to the detector 22 and detected. In both cases, the solute of interest is The effluent saturates the Trix 20 binding sites and breaks through into the effluent. The concentration of non-target solutes or impurities in the liquid reaches a plateau, and the level of this plateau is A signal representing is sent to computing device 30.

At this point, all the information necessary to calculate the concentration of the solute of interest is available and the analysis can proceed. Complete. However, to check, the target solute breaks through matrix 20. until it forms a higher plateau with other impurities, i.e. the matrix sample solution until the concentration is equal to the detected concentration in the sample before it is introduced into the Keep flowing.

At this point, as an additional self-check, if necessary, Switch valve 10 to flow buffer into the system from detector 22, 26 and and the purpose of cleaning the matrix 20 and non-covalently bonding it into the matrix. Non-selectively bound impurities are washed away, leaving only the solute. After this cleaning step , the pulp 10 is again switched to introduce eluent from the system reservoir 18. available. The target solute is eluted from the matrix 20 by this eluent. melt The separated target solute is detected by the detector 22 (in the case of the embodiment shown in FIG. 6) or 26 (in the case of the embodiment shown in FIG. 5). ), it is detected as a solute pulse signal. Multiply this pulse curve Calculating the area of the curve using a A value representing the amount of the solute of interest is obtained and is again correlated with the previously calculated concentration value. be able to.

As previously mentioned, the design of this system and the configuration of all included equipment are publicly available. It is within the scope of knowledge technology. In fact, there are many other combinations of equipment suitable for practicing the invention. You can think of it and add other functions as needed. parable For example, this system has a site that selectively binds only the predetermined target solute. The design can also incorporate interchangeable matrix modules. Ru. Since the accuracy of the analysis is independent of the flow rate, the liquid flow in this system You can choose any method. Thus, for example, the liquid flows The pump can be placed in any part of the line. Alternatively, a sample It can also be simply pumped into the system in a syringe.

The calculation means or calculator 30 can take various forms, and indeed the present invention In the broader part of the detector 22 and/or 26, this may be absent. A conventional plotter mounted on a manufacturing or refining system Is the concentration of the target solute and/or impurity bone changing over time? Visually determine whether the can do. However, the calculator 30 does not display the detected solute concentration ratio. Calculate signal and correlation coefficient as well as target solute concentration and/or contaminant solute concentration These data can be processed by incorporating methods such as mathematical calculation modules. Each time each analysis is completed, it is digitally displayed on the display device 34. Or cute You can use this data to create a time course plot of the solute concentration of interest, or to Alternatively, this system state can be displayed in another form and used as a record of the dynamic behavior of the analysis. There is also.

The chromatogram is a graph of the effluent that varies proportionally to the solute concentration detected in the effluent. Obtained by measuring characteristics and plotting them on a chart. A typical method is to use a commercially available As is widely used in chromatography equipment, the effluent is irradiated with ultraviolet light, The ultraviolet absorbance is plotted on a chart. In those systems, If the substance absorbs that wavelength, the absorbance of U, V, and light is relative to the solute concentration. Give an example. However, if it meets the purpose of the present invention, the amount contained in the effluent may be It is important to note that any characteristic value representing the analyte and impurity may be monitored. I want to be understood.

The horizontal axis of the chromatogram in FIG. 1 shows time, and the vertical axis shows absorbance.

To make it easier to understand, the graph is divided into five periods, labeled A, B, C, D, and and E. This segmented period is similar to traditional affinity chromatography. Pack the Raffy matrix into a column and run at high speed, e.g. 1800cm/hr. The dissolution of the effluent during the loftography injection cycle as experienced when flowing the pull. This indicates the stage of quality concentration. Due to the low resolution, the boundaries of each period are It will be easy to see that the line can be drawn somewhat freely.

The first period A represents the situation when the effluent is entirely buffer. spill When the liquid begins to contain the impurities in the sample, the solute concentration is expressed by period B. It starts to climb as if it is going up. Finally, the concentration is at equilibrium as it occupies period C. reach a state. This is due to the non-specific adsorption of impurities (if any) to the matrix. ) ends, the target solute begins to bind to the matrix, and the impurity concentration in the injection solution flows out. This occurs when the concentration of impurities in the liquid becomes equal. The sample flows further through the matrix. Continuing, the solute of interest begins to saturate the binding sites of the matrix.

This results in a gradual increase in the concentration of the target solute in the effluent, which is usually It is said to be a "breakthrough" and is displayed for a limited time. The adsorption site is completed. Once fully saturated (period E), the sample simply passes through the matrix and the effluent The solute concentration will be equal to the injectate concentration.

The “plateau” of period C is essential information for calculating the concentration of the target solute; Please note that the height and boundary of the flat part of the Chromatograms are extremely informative for samples containing multiple solutes with different physical properties. The lower the value and the faster the sample flows through the matrix , this important plateau band generally becomes wider.

Figure 2 is obtained when the sample passes through the matrix very slowly. A typical chromatogram is shown. One decisive difference between the graphs in Figure 1゜2 is that The latter graph shows clearly demarcated breakthrough points and equilibrium levels. This is what is being displayed. Period A′ in FIG. 2 corresponds to period A in FIG. 2 shows the period of liquid only. In Figure 2, the matrix quickly becomes free of impurities. saturated by non-specific adsorption, and at a very well-defined point during period C', the impurity It has been shown that equilibrium concentrations are reached. This is the breakthrough point B' in the figure. It is represented. The equilibrium concentration during period C' is the analyte contained in the solution. It continues to be delivered to the Rix binding sites until those binding sites are saturated.

When this state is reached, the second breakthrough point D' occurs, and the target of analysis in the effluent The concentration of the substance becomes equal to that of the injectate. The total concentration of analyte and impurity is during period E' , which is directly proportional to the equilibrium concentration in the period following the second breakthrough point D' do. Therefore, the difference in height between the flat part E' and the flat part C' is the analyte of the injected liquid. Used to calculate concentration.

Furthermore, if the capacitance of the matrix is known, the matrix can be Analytes in solution can be determined by monitoring the amount of solution flowing through the trix. The concentration can also be known. However, with repeated use, the coupling capacity ( As the number of adsorption sites in the matrix decreases, the solution is usually Determine the analyte concentration in the liquid. The concentration and breakthrough achieved in this way - Using the time point at which D′ occurs, how many binding sites remain in the matrix? You can find out if there are any.

Line F' in Figure 2 is the point at which the solution stops flowing through the matrix and the effluent is This shows the point in time when only the buffer solution was used again. Third step to measure the amount of analyte in the solution The method uses an eluent to elute the analyte from the adsorption sites of the matrix. It is to take advantage of the flowing elution period. This process is shown in the chromatogram of period G' in the figure. It is represented by the movement of grams. The area under the curve for this period is the matrix is directly proportional to the amount of analyte adsorbed to the breakthrough point D'. It is in. Therefore, the target solute concentration determined based on the height of step D' is The accuracy can be checked by the target solute concentration in terms of the area under the curve of period G'. is clearly possible.

FIG. 3 shows the case where an arbitrary detector 22 is attached to the device of FIG. 5 or the device of FIG. 6 is used. This is a chromatogram when During period A', the sample is entered into matrix 20. This is the period during which the total solute concentration in the sample is measured by the detector 22 before the sample is detected.

Exiting the detector 22, the sample enters the matrix 20 and the sample solution enters the matrix. While displacing the buffer in Trix, the concentration of the effluent coming out of the matrix The degree indicates a state in which no solute is contained, as represented by period B'. impure The substance breakthroughs at C' in FIG. 3, and its concentration is represented by period D'. Ma The flow to Trix progresses, the target solute breaks through at E', and the effluent The total solute concentration, or the concentration denoted by F', is the same as the concentration denoted by period A'. be equal. The data necessary to know the concentration of the target solute is the level of the flat part D'. As soon as the indicated by a line. The period required to determine level D′ depends on the flow rate and column capacity. and is proportional to the length of B'. Therefore, for quick analysis.

Small volume columns that allow high flow rates are preferred.

Chromatography matrices tend to lose their performance with repeated use. However, this does not affect the principles of the present invention. This does not affect the accuracy of the data obtained based on This means that the breakthrough of the target solute becomes faster, and the breakthrough of the target solute becomes faster, resulting in flat area C' in Figure 2 or flat area C' in Figure 3. Only the length of the flat portion D' becomes longer. Breakthrough showing the concentration of impurities There is no loss in plateau height, maximum solute concentration change, or accuracy. stomach.

However, if the number of binding sites is insufficient, the However, one problem may arise. That is, if the analysis in solution When the concentration of the target substance is extremely high compared to the number of adsorption sites or when the flow rate is extremely fast, The adsorption sites are saturated almost instantaneously, in which case the chromatogram shows two bright spots. Showing a single step without clearly demarcating the steps Ru. This state is shown in the chromatogram in Figure 4, and the chromatography matrix The inside of the gas is simultaneously saturated with both the analyte and the impurity. Each breakthrough is represented as a point X. In this state, the equilibrium concentration is It is also clearly impossible to distinguish between levels. Area under the curve of G′ between wJ is still proportional to the amount of analyte trapped in the matrix at saturation. However, saturation occurs so quickly that reliable concentration determinations based on saturation are not possible.

To solve this problem, dilute the injection solution with a buffer or other solution before passing it through the matrix. Just explain it. In this way, breakthroughs in solutes other than the target substance can be distinguished. become. After dilution, as shown in Figure 2 or Figure 3, the effluent is not contaminated. The equilibrium concentration of the pure substance is clearly observed before the appearance of the analyte in the effluent. You can get a Romagram.

As stated throughout this specification, the excellence of the present invention is that it allows rapid analysis, For example, within 10 seconds. The analysis described above can be performed in a time considerably shorter than 1 minute. It can be done in a few minutes, often less than 30 seconds, or in a matrix like the one below. This can sometimes be done in less than 10 seconds using a small volume column packed with a solid support.

The present invention also uses very small sample volumes of feed fluid, which speeds up and saves analysis. For example, microliter size It is also possible to perform regular analyzes using samples of for this purpose supports the chromatography matrix in a column of appropriate size. let For extremely small samples, coat internal surfaces with binding proteins It is possible to pass the sample through a capillary tube.

The matrices commonly used today are non-porous (or highly porous). low affinity type silica particles. Particularly useful matrices The medium is Perseptive Biosystems Inc. (Cambridge) FORO3T” brand column backing material available on the market from MA). Ru. These materials are produced by suspension polymerization techniques and are Classified by size range. FORO5” columns provide band broadening in high-speed analysis. It has been shown that there is very little difference in It is suitable for implementing the present invention.

The invention will be better understood by the following non-limiting examples.

Actual situation ■d To measure human immunoglobulin concentration in solution using brotiin A column FIP 1090 liquid chroma demonstrating subtractive frontal analysis method tography (Hewlett Packard.

available from Waldbronn G*bH) and “chew 5tatio” n” (available from HewIett Packard) at various concentrations and The purity of IgG was analyzed. Human IgGGC5igma Che#1cal Com Pany, St. Louis, Missouri) The pull was adjusted in the concentration range 0.01 to 10 μ/Id. These samples should be loaded first. pump into the detector flow cell without passing through the ram to obtain a calibration curve. Equilibrium absorbance was quantified. Next, various amounts of BSA (Sigma Che+5ic 250μj mixed with al　Company)! 10mM IgG sample Mix with sodium phosphate and 150mM sodium chloride solution to pH 7.4. , 2. 1X30K POROSTMA/M column, this is a commercially available Coat a thin film of protein A on 20μm polystyrene/divinylbengaf grains using a ram. I passed it through a plated one. As an example, 0.5■/Id and 0 ．． 5 u/di of BSA mixed with a flow rate of 1 m+/sin and 0.17/ Figures 7 and 8 show the absorbance flat lines obtained when flowing at la i n, respectively. vinegar. In the profile, BSA and IgG breakthroughs can be clearly distinguished. It has been shown that

The accuracy of this system can be checked by analyzing samples of known concentration. Wear. In order to make meaningful comparisons with known concentrations in mixed samples, A calibration curve for the concentration of a single component and the absorbance by the detector is required. Using a 1090 diode array detector, we detected human gnoglobily at a wavelength of 2BOn-. Such a calibration curve is shown in which the shown here in two independent and separate experiments. The data and correlation values were obtained. The purity of human gamma globulin is determined by It was about 92.4% according to the bifrontal analysis. Therefore, the absorbance reading This factor must be taken into account when converting into the corresponding IgG concentration value. This calibration curve plot shows that the linear range for pure IgG is 4. It can be seen that up to /i, and in the case of mixtures, up to about 3000 mAu in total. .

Using a mixture of IgG of different concentrations and purities (using BSA as an impurity) A series of experiments were conducted. Comparison of expected value of absorbance and actual column absorbance and Table 1 From the modification of the non-retentive plateau values as shown in and the explanation below, the IgG Accurate estimation of concentration is possible.

It is clear from the data that varying amounts of impure protein, here BSA, are present. However, this prototype device can detect various types of IgG at once. This person The method was also found to be somewhat inaccurate under the experimental conditions listed in Table 2. The error is This is due to many factors, including the calibration curve plot, the plateau absorbance value, and the direct Examples include values measured at areas other than the line area. Furthermore, in these experiments There may also have been errors in measuring the amount of protein used in the test mixture. Calibration Errors due to this can be seen in the examples in Table 1. As shown in Table 2, the columns The error in estimating the absorbance at the front end appearance (i.e., unadsorbed impurities) from the IgG concentration is is increased when it is 10% or less. For example, the plateau absorbance estimate If the error is 5%, the IgG concentration estimation for a 10% pure 1gG sample will be 45% % error will occur. Even with the same plateau error of 5%, the sample is 50% pure. In this case, the error in the concentration estimation is only 5%. Error due to deviation from straight line section is clear from Table 2. In this case, a simple solution is to dilute the sample appropriately That's true. Or alternatively, a slightly different wavelength (higher than 2B0ns or is low), the response signal will be weaker, so it can be applied to highly concentrated samples.

Overall, these data demonstrate the usefulness and practicality of this analytical method. . The design of commercially based equipment includes various industrial designs to increase accuracy and ease of use. It is better to adopt the principle of regular use. For example, computer device 30 may Take 5 to 10 consecutive readings at regular intervals until a “flat area” is recognized and recorded. All you have to do is create a program that minimizes the error before the error occurs.

Standard deviation 4.73 1J doctor A: Known IgG concentration (CIuG) mg/ml; B: Known BS^ concentration (Ci ta)s+g/d　;C: Absorbance without passing through the column (milli absorbance unit, m Au) (equivalent to the effluent after all binding sites of the column have been saturated, or the second flat part height); D: Absorbance (a+Au) when passing through the column (corresponds to the absorbance of BSA alone, all i.e. the first plateau caused by BSA breakthrough); E: Absorbance “expected” value without passing through the column, calculated as follows; (C1l@) (726mAu) + (Cmsa) (413mAu), where 726+sAu is the IgG absorbance coefficient obtained from the calibration curve in Figure 1O, and 41 3 ■ Au is the BSA absorbance coefficient obtained from the slope of the calibration curve; F: Detected IgG concentration -(C-D/726 Xo, 924), where 0.924 is the amount of absorbed IgG percentage; and Although some band expansion can be seen at the second IgG-containing front end in Figure 7, Figures 7 and 8 A 10-fold speed difference as shown in Fig. 2 has the disadvantage of delaying the appearance of the second plateau. It can be said to be a sufficient complement. In fact, as mentioned above, the original absorption of the test solution If the concentration is known, a second plateau is not necessary to calculate the concentration of the sample. Therefore, it does not matter even if the second front end band expands.

IjiJL Shadow In order to carry out the assay of the structural profile of proteins according to the invention, a column of the type described above is used. A protein A column was loaded with mouse gamma globulin (SfgA). Ma Ugamma globulins actually have different adsorption affinities for protein A. Contains some IgG subclasses. 101d PBS and 1% Menu A sample containing 2 d/d of protein in 2.1 d/e+in at a flow rate of 0.2 Pour it into a 30x column and add 0.15M NaCl + 2% acetic acid + 1% MeOH. When developed, a frontal chromatogram as shown in FIG. 9 is obtained. curve The shape of the protein A of the POROS A/M column corresponds to the various types in the sample. It shows the affinity of IgG species, and it shows the affinity of IgG species, and it shows that the protein structure profile of the sample is different. If so, the shape of the curve will be different. In this way, this curve It constitutes a “fingerprint” that clearly identifies a particular mixture and its condition, making it difficult for other samples to By repeating this method for pulls, it is possible to compare the structural profiles of the samples. possible curves can be obtained.

Other embodiments are within the scope of the following claims.

Figure 1 Figure 2 Figure 3 Figure 4 waste Figure 6 m1ll ill/ mll/ ! 1.11,1125.1°s, PCT/11591105544

Claims (40)

[Claims] 1. Analysis of samples containing one or more impurities and one solute of interest A method comprising the steps of: A) a binding site having specificity for a solute of interest; Pass the sample through a matrix with During the process, the first plane where the effluent that does not contain the target solute represents the concentration of impurities. reaching an equilibrium concentration; B) first data representing said first equilibrium concentration; and obtain second data representing the total solute concentration; and C) calculate the difference between the first and second data. The difference is proportional to the concentration of the analyte in the sample. 2. In the method of claim 1, the second data is stored in the matrix as a sample. A method in which the solute concentration in the sample is monitored and obtained before passing through the sample. 3. In the method of claim 1, the above second data is method obtained from the effluent after being saturated with. 4. In the method of claim 1, the following steps are required to obtain the first and second data. Methods that include step: The ultraviolet absorbance of the effluent is monitored and the above absorbance value is It is proportional to the concentration of impurities in the sample. 5. The method of claim 1, wherein the solute is protein. 6. The method of claim 1, wherein the binding site is an antibody binding site. 7. In the method of claim 1, the matrix comprises protein A, protein G, its analogs, or fragments thereof, and the Doki binding site is an immunoglobulin. Something made up of Bling. 8. The method of claim 1, wherein the matrix is rigid and substantially non-porous. Particulate matter. 9. In the method of claim 1, the matrix is a perfusive chromatograph. What is the Raffi Matrix. 10. In the method of claim 1, the difference between the first and second data is tested. A method in which the step includes: a first electric current representing the value of the first data above; emitting a second electrical signal representing a value of said second data; electrically comparing the first and second signals; determining the concentration of the solute of interest based on the electrical comparison; Calculate the degree electrically. 11. The method of claim 10, including an additional step of displaying the concentration of the target solute. How to do it. 12. The method of claim 1 comprising the following additional steps: washing the matrix; Elution of the target solute adsorbed to the matrix; and detection of the amount of target solute in the eluent . 13. Cream 1 method, including the following additional steps: from the matrix Elution of target solute; washing the matrix; and Repeat steps A, B and C for second sample. 14. The method of claim 1, wherein the matrix has reverse adsorption sites. a method comprising the following additional steps: removal of the binding site from the matrix. removal; and reloading the matrix to new binding sites. 15. In the method of claim 1, the supernatant in the effluent is removed before the target solute appears in the effluent. Steps are taken to create an appropriate solute concentration such that the impurity reaches an equilibrium concentration. A method that includes an additional step of diluting the sample before step A. 16. Purpose in a sample consisting of multiple solutes containing one or more impurities Equipment for assaying solutes, including: a matrix having sites that specifically bind the solute of interest; If the effluent represents the concentration of impurities while the target solute is loaded into the matrix, means for flowing the sample through the matrix to reach a first equilibrium concentration; Means of obtaining primary data representing the equilibrium concentration; The difference between the solute and the second data representing the solute concentration is verified, and the difference is determined as the concentration of the target solute in the sample. A means of verification that is proportional to the degree of 17. In the apparatus of claim 16, the means for flowing the sample is a matrix. includes a method for creating a pressure gradient in the gas. 18. In the apparatus of claim 16, the means for flowing the sample is a matrix. Includes a method for creating an injection gradient in the system. 19. 17. The apparatus of claim 16, wherein said matrix is rigid and substantially non-polymerized. consisting of porous granular material. 20. The apparatus of claim 16, wherein the matrix is a perfusate chroma. What is the topography matrix? 21. In the device of claim 16, the matrix comprises protein A, protein InG, analogues or fragments thereof. 22. The apparatus of claim 16, wherein the means for obtaining the data is based on the purple color of the effluent. Includes monitoring of external radiation absorption. 23. In the device of claim 16, the matrix is contained within the column. What to do. 24. The apparatus of claim 16, wherein the matrix is a capillary tube. arranged on the inner surface of 25. In the device of claim 16, the difference between the first data and the second data is tested. The above-mentioned method comprising the following means using a computer; means for producing a first electrical signal representative of the first data value; means for producing a second electrical signal representative of the second data value; Means for testing the difference between the first and second data; and from that difference the target solute concentration. A means of calculating degrees. 26. The device according to claim 25, further comprising means for displaying the concentration. of. 27. In the apparatus of claim 16, further, an eluent is caused to flow through the matrix. including means. 28. In the apparatus of claim 16, further, after step C, the above matrix so that steps A, B, and C can be repeated for a second sample. including means for balancing the sea urchin. 29. How to determine the structural profile of protein samples that are mixtures of structural isomers and includes: The sample is transferred to a matrix containing immobilized binding sites for capturing antigenic determinants of the above proteins. Trix and after at least some of the binding sites have been saturated, the sample sufficient to obtain breakthrough properties characterizing the structural profile of the protein in the pull. Measure one parameter that represents the concentration of protein leaving the matrix at a given time point. . 30. of the structural profile of a protein in separate samples consisting of a mixture of structural isomers. A method for detecting differences, including: the matrix containing immobilized binding sites for at least one of the binding sites. a portion of which is saturated by the protein, characterizing the structural profile of the protein in the sample. from the matrix after a certain period of time necessary to obtain breakthrough properties. A sample breakthrough characteristic is determined by measuring one parameter that represents the concentration of protein released. Compare gender characteristics. 31. The method of claim 29 or 30 further includes a parameter thereof representing concentration; An additional screen that plots a parameter representing the amount of sample leaving the matrix. Methods including steps. 32. In the method of claim 29 or 30, the protein sample is purified. A water-soluble protein solution. 33. In the method of claim 29 or 30, the binding site is One that is a polyclonal antibody that is potent against the pull. 34. In the method of claim 29 or 30, the binding site is a monopotent type, One that has different adsorption properties depending on the isomer in the protein sample. 35. The method of claim 29 or 30, wherein the matrix is hard, substantially A particulate material that is essentially non-porous and has a hydrophilic surface. 36. The method of claim 29 or 30, wherein the matrix is perfumed. What is a Sibu chromatography matrix. 37. The method of claim 29 or 30, wherein the matrix is a capillary. – consists of the inner surface of the tube. 38. The method of claim 29, including the additional steps of: elute the protein from the matrix; wash the matrix; and One that repeats the steps of flowing the sample and measuring it. 39. The method of claim 38, wherein the matrix is free from adsorption sites in reversed phase. a method comprising the following additional steps: removing the binding site from the matrix; and Recombine new adsorption sites. 40. In the method of claim 29 or 30, the above step of flowing the sample , those performed by pressure gradients or injection gradients.