WO1992002815A2 - Quantitative analysis and monitoring of protein structure by subtractive chromatography - Google Patents
Quantitative analysis and monitoring of protein structure by subtractive chromatography Download PDFInfo
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- WO1992002815A2 WO1992002815A2 PCT/US1991/005544 US9105544W WO9202815A2 WO 1992002815 A2 WO1992002815 A2 WO 1992002815A2 US 9105544 W US9105544 W US 9105544W WO 9202815 A2 WO9202815 A2 WO 9202815A2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N2030/626—Detectors specially adapted therefor calibration, baseline
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
Definitions
- the present invention generally relates to a method for performing quantitative and structural analyses on solutions containing multiple solutes.
- the invention relates to methods for determining the presence and concentration and for characterizing the structural profile of an analyte in a solution utilizing subractive frontal breakthrough analysis.
- structural variants of proteins e.g., recombinantly produced therapeutic proteins
- recombinantly produced therapeutic proteins are not presently possible except in those instances where the variation in the a ino acid sequence or tertiary structure results in an altered activity profile or the separate species can be resolved on a gel. Determining the presence of structural variants is particularly important in the field of biosynthetic protein production, e.g., in recombinant DNA technology as applied to the manufacture of therapeutic proteins.
- Recombinantly produced proteins have .a higher rate of expression error, and can include a number of "minor" structural variants having, for example, diminished activity, harmful side effects, or undesirable antigenicity.
- Structural variants also may be produced during post translational protein modification by, for example, variation in glycosylation pattern, disulfide bonding, or protein folding.
- the concentration of a product in a mixture which may contain hundreds of contaminating species including cell debris, various solutes, nutrient components, DNA, lipids, polysaccharides, protein species having similar physiochemical properties, and structural variants of a single recombinantly produced protein. While the concentration of the target product in the harvest liquor is usually o: the order of 100 mg/1, it is sometimes as low as 1 mg/1. To complicate identification further, due to the fragility of many target solutes, they must be treated with relatively low fluid shear, and preferably with only minimal and short duration contact with potentially denaturing surfaces.
- affinity chromatography which involves passing a feed mixture over a matrix such as a packed bed of selectively sorptive particles to bind one or a subset of the solutes in the mix. Subsequent passage of solutions that modify the chemical environment at the sorbent surface results in elution of sorbed species. Solutes flow through these systems convectively in the interstitial space among the particles and diffusively within the particles.
- the media used for liquid chromatography typically comprises soft, highly porous particles having a high surface area to volume ratio. As a result, a liquid chromatography process cannot be run at pressures exceeding about 50 psi, and attempts to increase the fluid velocity are counterproductive to separation.
- HPLC High Performance Liquid Chromatography
- inorganic material such as silica or a rigid polymer such as styrene divinylbenzene.
- HPLC allows somewhat faster and higher resolution separations.
- chromatographic procedures employed for purification involve four steps: loading; washing; eluting; and re-equilibrating.
- the rate limiting step in each stage is the transport of molecules between the mobile fluid and the static matrix surface.
- Optimum efficiency is promoted by rapid, preferably instantaneous mass transfer and high fluid turnover.
- sorbent loading fewer moleculares are sorbed as the velocity of the mobile phase in the bed increases.
- breakthrough i.e., passes through the matrix without binding and appears in the effluent.
- the breakthrough concentration is limited to, for example, 5% of the inlet concentration, that limit sets the maximum bed velocity which the bed will tolerate. Further increases in bed velocity thereafter are wasteful and can only serve to decrease loading per unit surface area.
- Perfusive Chromatography One can increase chromatographic throughput by using a matrix comprising small porous part ' les having a relatively large pore diameter, so that convective flow can be induced through, as well as around, the particles.
- This type of chromatography is referred to as Perfusive Chromatography and is described in copending application serial number 376,885, filed July 6, 1989, the disclosure of which is incorporated herein by reference.
- Perfusive chromatographic techniques permit high speed, high capacity, high resolution separation.
- Perfusive matrices may be purchased from PerSeptive Biosystems, Inc. of Cambridge, Mass.
- the present invention provides a method and apparatus for rapid assay and characterization of therapeutic and other substances based on what is described herein as subtractive chromatography.
- a solution containing multiple solutes is passed through a matrix having binding sites specific for one or more target solutes.
- target solute is broadly defined and encompasses any water soluble analyte but typically is a protein such as a recombinantly produced protein.
- a feed solution containing at least one target solute for example, a biologically active molecule such as a polypeptide, protein, polysaccharide,. or the like, in admixture with other solutes, is passed through a matrix comprising binding sites specific to the target solute.
- a target solute for example, a biologically active molecule such as a polypeptide, protein, polysaccharide,. or the like
- the target solute will adsorb at the binding sites, thereby virtually eliminating any concentration of the target solute in the effluent.
- a limited amount of non-target solute may also non-specifically adsorb to the matrix.
- the effluent is monitored to determine its solute concentration.
- the concentration - f contaminating or "non-target" solute(s) in the effluent will increase until the concentration of non-target solute in the effluent reaches an equilibrium level equal to the concentration of non-target solute in the feed.
- this stage of the assay procedure will result in an upturned slope or vertical line, depending on the nature of the matrix, which develops into a flat, horizontal line as solute concentration in the effluent maximizes.
- the equilibrium concentration of solutes in the effluent will remain substantially constant as the feed solution is passed through the matrix as long as binding sites remain available. Eventually, however, the binding sites of the matrix become saturated, and the target solute will flow directly through the matrix without net interaction. This is referred to as breakthrough. Thus, the emergence of the target solute from the matrix will result in a detected increase in ultra-violet absorption of the effluent. Thus, when solute concentration reaches a plateau indicating that the feed is simply flowing through the column without net solute interaction with the matrix surface, the solute concentration in the effluent equals the concentration in the feed.
- the difference between these equilibrium concentrations may be used to calculate the concentration of target solute in the sample as the difference between equilibrium concentration is directly proportional to the concentration of target solute in the sample. Furthermore, since the second plateau is indicative of the additive concentration of all solutes in the feed, that value can be obtained by monitoring the sample prior to the time it enters the matrix. Thus, all information necessary to calculate the target solute concentration is available as soon as a plateau in the breakthrough of non-target or contaminating solute is reached.
- the device is calibrated by passing through the solute detector known concentrations of pure target solute so that concentration units can be correlated directly with, e.g., absorbance units. The product of the difference between the sensed plateaus and the correlation factor equals the concentration of the target solute.
- the method of the invention is used for detecting difference" in the structural profile of a protein in separate samples.
- structural profile refers to the particular mix of molecular species in a protein solution which can vary from batch to batch or over time due to expression errors, differences in DNA sequence among the clones in a culture, truncation by proteases, or differences in post translational modification resulting in variations in conformation or derivatization.
- the method comprises the steps of passing the samples through a matrix comprising immobilized binding sites which vary with respect to their binding properties to structural variants in the sample.
- polyclonal antibodies may be used, cloned variants of which are specific for a particular epitope on a particular variant of the protein.
- a single type of binding site may be used which varies in binding affinity or specificity with variants of the protein to be analyzed. This procedure can produce a breakthrough function characteristic of the structural profile of the protein in the sample as the concentration of protein exiting the matrix is measured after at least some of the binding sites have been saturated with the protein. Comparing the characteristic functions of different samples permits indirect comparison of their structural makeup.
- each subspecies has at least some unique epitopes.
- Each fraction of the binding protein in the matrix therefore will be capable of discriminating, (i.e., selectively binding) particular molecular subspecies, or of binding a molecular subspecies preferentially.
- various of its subspecies reach equilibrium saturation, and thereafter break through into the effluent. If the protein concentration of effluent is monitored over time, there is an interval over which protein concentration in the effluent increases from a baseline value, typically zero, to a value substantially identical to protein concentration in the feed.
- the protein concentration increases progressively in a way that is indicative of the particular structural profile of the protein sample.
- This function is compared for separate protein samples, one can determine whether those samples have uniform structure.
- This method can be used, for example, to monitor a product stream periodically as a means of assuring that the product remains within a predetermined specification.
- a parameter indicative of concentration e.g., U.V. absorbance
- a parameter indicative of volume of sample exiting the matrix e.g., time if flow rate is uniform
- the method is highly effective when the protein sample is an aqueous protein solution which has been at least partially purified.
- the method may involve passing the protein through the matrix by a means of a pressure gradient or a charge gradient.
- the matrix preferably is a rigid, substantially non-microporous, particulate material having a hydrophilic surface, and preferably is a perfusive chromatography matrix.
- the matrix also may be defined by the interior surface of a capillary. Where the matrix comprises surface regions comprising immobilized protein A, protein G, and the binding protein is immunoglobulin, one can remove the binding sites from the matrix after each run, and reload the matrix with H
- Immunoglobulin and other types of protein binding sites also may be non-specifically adsorbed on a hydrophobic polymer matrix surface and removed with mixed organic/ionic stripping solutions.
- the matrix should be as small as possible.
- the volume of sample that can be present in the matrix dictate the time interval between introduction of the sample and breakthrough.
- the quantitative analysis technique is independent of flow rate, and does not require the target solute and the matrix to reach equilibrium.
- the sample may be impelled through the matrix by any convenient method.
- the sample may be impelled through the matrix manually, e.g., using a syringe, by an electrically driven pump, or by a charge gradient as in electrophoresis.
- assays can be performed repeatedly without comprising the accuracy of the process. While an unknown subset of binding sites of the matrix may be degraded with repeated sequences of binding, elution, and reequilibration, the method of the invention generates information based on concentration differences of the target and non-target solutes. Thus, the availability of fewer binding sites will translate to earlier target solute breakthrough but will not give inaccurate indications of concentration.
- the invention also affords a self checking capability. If detected concentration differs between the feed and the final effluent plateau, the system may be operating improperly. Self checking also can be implemented by washing the matrix after the final effluent plateau has been reached and then eluting the target solute. Integration of the detected pulse in the eluate will give an indication of the amount of bound target solute, which should correlate with the previous datum.
- Another advantage of the invention is that it is very flexible. Consider, for example, a situation in which a sample having high concentration o-" target solute is passed through a matrix. This may result in almost immediate saturation of the binding sites of the matrix, and therefore, almost immediately breakthrough. On a graph like that discussed above, the output will appear as a single vertical line followed by a horizontal plateau, giving no information about the concentration of target or non-target solute. To remedy this situation, the sample need only be diluted with buffer solution or the like. By diluting the sample breakthrough is delayed, thereby affording a clear distinction between the equilibrium concentration of the non-target solute in the effluent and the equilibrium concentration of the target and non-target solute together.
- binding sites on the matrix e.g., monoclonal or polyclonal antibodies or other binding proteins
- binding sites on the matrix e.g., monoclonal or polyclonal antibodies or other binding proteins
- This feature permits construction of a single matrix and assay device which can be customized for any target solute.
- a further advantage of the invention is that it can be utilized on an extremely small scale. Even microliter sized samples can be analyzed. Moreover, rater than filling a traditional chromatography column with high surface area particles to serve as a matrix, on* 1 can coat binding protein on the inner surface of a capillary tube. Passing a solution through the capillary tube can achieve the same results as those discussed above. It is contemplated that an assay device embodying the invention, including sample, eluant, and buffer ports, matrix channel ready to be activated with binding protein, detector for solute concentration in the effluent, and circuitry to convert the output of the detector to a meaningful form, all could be housed in a single module.
- an inexpensive matrix module can be produced for placement in a device comprising the other necessary components.
- the module can be discarded after a short useful life and then replaced.
- sets of modules each of which bind a different target solute will permit rapid adaptation of an assay apparatus for given target solute.
- FIG. 1 is a representative chromatogram generated in conjunction with diffusion bound chromatography at high throughput
- FIGS. 2 through 4 are various chromatograms illustrating the principles of the present invention.
- FIG. 5 and 6 are schematic representations of two embodiments of apparatus embodying the invention, in which like reference characters indicate corresponding parts;
- FIGS. 7 and 8 are chromatograms generated using the analysis technique of the present invention for measuring the concentration of Immunoglobulin in solution using a Protein A column where BSA is a contaminant;
- FIG. 9 is a chromatogram showing the results of an experiment involving the tertiary structural profile of mouse gamma globulin and demonstrating the feasibility of an embodiment of the invention.
- FIG. 10 is a representative calibration curve of milli absorbance units (mAu) versus protein (IgG) concentration in mg/ml.
- the invention provides a method for monitpring the production of a solute based on subtractive frontal breakthrough analysis.
- the concept is to exploit an affinity chromatography matrix to remove selectively at least one solute of interest from a solution, and to measure the equilibrium concentration of contaminating solutes in the effluent exiting the matrix.
- the next step involves determining the concentration of the target solute in the analyte sample from the difference between the sensed concentration of all solutes in the sample and the sensed concentration of the contaminants.
- the solute of interest is a protein, particularly a purified recombinantly produced protein, comprising an unknown number of structural variants each of which vary at least subtly in their affinity for a particular binding protein, and have at least some unique epitopes.
- the protein sample preferably substantially free of contaminants, is passed through a matrix comprising a single binding protein or immobilized polyclonal antibodies to the protein of interest.
- Variant protein molecules in the sample saturate the various clonal species of the polyclonal antibodies or compete for sites of attachment to a single type of binding site and then break through.
- Output monitoring produces a step-like plot of the breakthrough fronts characteristic of that particular sample.
- FIGS 5 and 6 schematically illustrate apparatus designed for implementing the process of the invention.
- a valve 10 directs through its output 12 either a sample from sample input 14, a buffer solution from reservoir 16 for washing and reequilibrating a chromatography matrix, or an eluent from reservoir 18 capable of inducing release of sorbed species from binding sites in a chromatography matrix.
- the output of valve 10 ultimately directs a selected solution through a chromatography matrix 20 of a nature hereinafter described in more detail, which comprises binding sites disposed about a surface and capable of selectively adsorbing an analyte or target solute sought to be determined.
- inte ⁇ ⁇ osed between valve 10 and matrix 20, as indicated in phantom at 22, is a solute concentration detector capable of providing a signal through line 24 representative of ⁇ 7 the concentration of solutes in the sample.
- Detector 22 may be a conventional device of the type commonly used in chromatography equipment comprising, for example, a U.V. light source which provides a beam through a film of the sample and a U.V. detector which permits measurement of absorption by solutes in the sample.
- Liquid exiting matrix 20 enters detector 26 which also measures a parameter characteristic of solute concentration, this time in the effluent, and delivers a signal representative of that quantity through line 28.
- Lines 24 from detector 22 and line 28 from detector 26 enter electronic calculator means 30, where, for example, the difference between the sensed absorption maxima in detectors 22 and 26 is calculated, and that difference is used by multiplication with a conversion factor to determine target solute concentration.
- the concentration value may be delivered through line 32 to a display 34.
- Figure 5 operates slightly differently. Specifically, detector 26 detects a first plateau representative of the concentration of non-target solutes or contaminants exiting matrix 20, and at a later time, after breakthrough of the target solutes, detects total solute concentration. Data points representative of these sensed plateaus are delivered through line 28 to calculator means 30 and process as set forth above.
- Figure 6 depicts another embodiment of the system of the invention. Its operation is conceptually identical to that a Figure 5 excepting that effluent from matrix 20 is returned to detector 22 via line 36. This permits a single detector to measure the total soJute concentration in the sample prior to its introduction into the matrix 20, and thereafter to measure the level of the plateau achieved in the effluent prior to breakthrough of the target solute. Design of this embodiment of the system may require inclusion of a liquid accumulator (not shown) in line 38 disposed between detector 22 and matrix 20, or some other means to insure that all sample has been removed from the detector 22 prior to the time solute concentration in the effluent reaches a plateau. Signals representatives of the solute concentrations sensed by detector 22 are transmitted through line 24 to calculator means 30 as disclosed above.
- Calculator means 30 may be omitted if the purpose of the device is solely to monitor protein structure.
- the display 34 is adapted to display a plot of a function representative of protein concentration in the effluent versus a function representative of effluent volume. The display thus produces a curve characteristic of the structural profile of the protein sample which can serve as a "fingerprint" of the sample which will identify a given sample composition and change if the structural profile of the protein changes.
- the system has been filled with a buffer 16 used to equilibrate matrix 20 and to assure no solute residues remain in detectors 22 or 26.
- the valve 10 is adjusted to permit sample 14 to be introduced into the system impelled by a pressure gradient created by a pump or syringe, or by means of a charge gradient to promote electrophoretic movement through the matrix 20.
- a data point indicative of the total solute concentration in the sample is sensed by detector 22. Thereafter, the sample enters the matrix 20.
- l__rget solute begins binding to the binding sites immobilized in the matrix; contaminants which do not bind pass through the matrix and emerge in the effluent.
- the buildup of contaminants in the effluent is sensed by detector 26; in the embodiment of Figure 6, the buildup is sensed by return to detector 22.
- the concentration of non-target solute(s) or contaminant(s) in the effluent stream reaches a plateau, and a signal indicative of the level of the plateau is passed to calculated 30.
- valve 10 can be switched to direct buffer from reservoir 16 through the system, thereby washing detectors 22 and 26 and matrix 20 free of non- specifically adsorbed contaminants but leaving target solute non-covalently bonded to the binding sites in the matrix.
- valve 10 is again switched to introduce eluent from reservoir 18 through the system.
- the eluent serves to elute the target solute from the matrix 20.
- the eluted target solute is detected by detector 22 (in the embodiment of Figure 6) or 26 (in the embodiment of Figure 5) as a pulse of solute. Integration of the pulse curve or other determination of the area under the curve gives an indication of the quantity of target solute bound during the assay which, again, can be correlated to the concentration derived previously.
- the system can be designed to have replaceable matrix modules, individual ones of which comprise binding sites specific for predetermined target solutes. Since accuracy of the assay is independent of flow rate, it matters not how one chooses to promote flow through the system.
- a pump may be placed anywhere in the fluid flow line.
- the sample may be placed in a syringe and simply rammed through the system.
- the calculator means or processor 30 can take various forms, and indeed, in the broader aspects of the invention, is not required.
- a conventional plotter attached to detector 22 and/or 26 would permit an operator of a production or purification s-- _ tem to determine visually by observing plural consecutive plots whether concentration of the target solute and/or the impurities is changing with time or is constant.
- calculator 30 may include means for storing signals representative of data points indicative of the sensed solute concentration ratios, and correlation factors, and an arithmetic calculation module which calculates target solute concentration and/or contaminant solute concentration. These data may be displayed digitally in display 34 after each assay. Alternatively, the data may be used to produce a plot of target solute concentration over time, or other desired indication of the state of the system, as a record of the dynamic behavior of the system under analysis.
- a chromatogram is generated by measuring and charting a characteristic of the effluent that varies in proportion to the concentration of detectable solute in the effluent.
- ultra ⁇ violet radiation is passed through the effluent and the degree of ultra-violet absorption is charted.
- Absorption of U.V. light in such systems is proportional to solute concentration, provided the solute is absorptive of this wavelength. It should be understood, however, that any characteristic of the effluent which is representative of the concentrations of analyte and impurities therein can be monitored for purposes of the present invention.
- the abcissa of the chromatogram of Figure 1 indicates time, and the ordinate absorption.
- the graph is divided into five periods which are labelled A, B, C, D, and E.
- the periods define stages of solute concentration in the effluent during a chromatographic loading cycle that might be encountered when passing a sample through a conventional affinity chromatography matrix housed in a column at a high rate, e.g., 1800 cm/hr. It is easy to see that due to poor resolution the boundaries between periods must be drawn rather arbitrarily.
- Initial period A represents the condition where the effluent consists entirely of buffer.
- the solute concentration begins to rise as shown in period B.
- an equilibrium concentration will be reached as depicted in period C. This will occur when non-specific binding (if any) of impurities to the matrix has stopped and target solute is being retained by binding to the matrix so that the concentration of impurities in the feed is equal to the concentrations of impurities in the effluent.
- the target solute begins to saturate the binding sites of the matrix. This results in the emergence of target solute in a gradually increasing concentration in the effluent, commonly referred to as "breakthrough", illustrated in period D.
- breakthrough illustrated in period D.
- period C is the critical information necessary to calculate the concentration of the target solute, but that the height of the plateau, and its boundaries, are far from distinct.
- the chromatogram can be far less informative, and the faster one passes the sample through the matrix, generally the more the critical plateau is marked by band spreading.
- FIG. 2 a chromatogram typical of that obtained by passing the sample very slowly through the matrix. The single most significant distinction between the graphs of FIGS. 1 and 2 is that the latter has sharply defined breakthrough points and equilibrium levels.
- Period A' of FIG. 2 corresponds to period A of FIG. 1 and is representative of the period over which buffer alone constitutes the effluent.
- the concentration of analyte in the solution also can be determined. Since, however, over repeated uses the binding capacity (the number of binding sites in the matrix) will decrease, it will more often be the case that the concentration of analyte in the solution will be determined based upon the principles discussed above. The concentration so determined, therefore, can be used in conjunction with the timing of the breakthrough step D' to determine how many binding sites remain in the matrix.
- Line F' in FIG. 2 represents the point at which solution has ceased being passed through the matrix, and the effluent once again comprises only buffer.
- a third way to determine the amount of analyte in the solution is during desorption of the analyte from the matrix by way of passing an eluent through the matrix to free the analyte from the binding sites. This process is represented in the figure by the behavior of the chromatogram during period G' . The area under the curve in this period is directly proportional to the amount of analyte bound to the matrix as of the breakthrough point D' . It is clearly possible, therefore, to check the accuracy of -the determination of target solute concentration made based on the height of step D' to that determination made based upon the area under the curve during period G' .
- Figure 3 shows a chromatogram of the type which can be produced in the apparatus of Figure 5 with optional detector 22, or in the apparatus of Figure 6.
- Period A' represents the interval when detector 22 is measuring total solute content in the sample prior to its entry into matrix 20.
- the concentration of effluent from the matrix shows a solute free state as illustrated during interval B'.
- Impurities break through at C and their concentration is represented at interval D' in Figure 3.
- target solute breaks through at E'
- total solute concentration in the eluent, indicated at F' equals the concentration indicated by the interval A' .
- target solute concentration is in hand as soon as the level of plateau D' is known with confidence, and is here indicated by way of example by a vertical dotted line.
- the time it takes for the level D' to be established is dependent on flow rate and on the volume of the column, which is proportional to length B'. Small volume columns which can be run at high flow rates are therefore preferred for rapid analysis.
- a chromatogram will show only one step. This situation is portrayed in the chromatogram depicted in FIG. 4, wherein a single breakthrough point X represents the simultaneous saturation of both analyte and impurities in the chromatography matrix. In this situation it is obviously impossible to discern equilibrium concentration levels. While the area under the ⁇ -curve in period G' will still be proportional to the amount of analyte captured by the matrix at the point of saturation, saturation has occurred so quickly that reliable determination of concentration based on saturation cannot be made. To remedy this problem, the feed solution need only be diluted with, e.g., buffer, prior to being passed through the matrix, so that breakthrough of the non-target solute can be distinguished. After dilution, a chromatogram such as that depicted in either FIG. 2 or 3 will be generated wherein an equilibrium concentration of impurities is established in the effluent before any analyte appears in the effluent.
- a preferred aspect of the present invention involves high speed assays, e.g., less than 10 seconds.
- the above discussed analyses can be performed in periods substantially shorter than one minute, often shorter than 30 seconds, and frequently less than 10 seconds, if one employs a small volume column containing a matrix medium of the type described below.
- the present invention also helps to increase the speed with which assays are carried out by being able to provide meaningful data with only a very small sample of feed solution.
- assays routinely can be performed on microliter sized samples.
- the chromatography matrix can be supported in a column having suitable dimensions.
- Non-porous (or very low porosity) affinity-based silica particles is non-porous (or very low porosity) affinity-based silica particles.
- a particularly advantageous matrix medium is POROSTM brand column packing materials which may be obtained commercially from PerSeptive BioSystems, Inc. (Cambridge, MA). These materials are produced through suspension polymerization techniques and classified to the desired particle size range. POROS'" columns have been shown to have significantly reduced band spreading in high speed assays, thereby allowing analysis according to the practice of the present invention to be performed in extremely short periods of time.
- the subtractive frontal analysis technique is demonstrated using a Protein A column to measure the concentration of human Immunoglobulin in solution.
- Various concentrations and purity levels of IgG were analyzed using an HP 1090 liquid chromatograph (available from Hewlett Packard, Waldbronn, GmbH) and a "chem station” (available from Hewlett Packard). Pure samples of human IgG (available from Sigma Chemical Company, St. Louis, Missouri) are prepared in concentrations ranging from 0.01 - 10 mg/ml. These samples are first pumped into a detector flow cell without a column in line, and the absorbance at equilibrium is quantified to obtain a calibration curve.
- the accuracy of this system may be checked by analyzing samples of known concentration.
- a calibration curve relating the detector absorbance and concentration for a single component is needed to obtain meaningful comparisons with the known concentration in a sample mixture.
- Figure 10 shows such a calibration plot for human gamma globulin using the 1090 diode array detector at 280 nm. Two independent sets of experiments yielded the data and correlation shown. Human gamma globulin is about 92.4% pure, as determined by subtractive frontal analysis. Therefore this factor has to be included when converting a measured absorbance to equivalent IgG concentration.
- the calibration plot also reveals the linear range to be up to about 4 mg/ml for pure IgG or about 3000 mAu total for a mixture.
- the prototype device can detect varying IgG concentration in the presence of varying amounts of contaminating protein, here BSA.
- Certain experiments detailed in Table 2 revealed conditions under which the method is less accurate. Errors result from a number of factors, including the calibration plot, the value of the absorbance at the plateau, and measurements outside the linear range. In addition, in these experiments there may also be errors in the measurement of amounts of proteins used in the test mixture. Errors due to the calibration are found in the examples in Table 1. Errors in estimating the absorbance of the front emerging from the column (i.e., the non-bound contaminant) are magnified for cases where the IgG purity is 10% or less as shown in Table 2.
- computing means 30 may be programmed to require 5 or 10 consecutive readings over an appropriate time interval to be within some small margin of error before a "plateau" is recognized .and recorded.
- B Known BSA concentration (C ⁇ SA ) in mg/ml; C: Absorbance in milli absorbance units (mAU) without the column in line (corresponding to effluent after saturation of all binding sites in column, or height of second plateau);
- 413 is BSA absorbance from calibration curve (slope);
- Mouse Gamma Globulin (Sigma). Mouse gamma globulin actually contains several subclasses of IgG which vary with respect to their binding affinity for Protein A. If a sample of 2 mg/ml protein in 10 ml PBS plus 1% MeOH is run on a 2.1 x 30 mm column at a flow rate of 0.2 m./mn followed by elution with 0.15 M NaCL + 2% Acetic Acid + 1% MeOH, a frontal chromatogram as shown in Figure 9 is produced.
- the shape of the curve is indicative of the affinity of the various IgG species in the sample for protein A on the POROS A/M column, and changes in the structural profile of protein in the sample will induce variations in this curve.
- the curve constitutes a "fingerprint" uniquely identifying this particular mix and condition of IgG species, and repetition of the procedure with other samples will produce a curve permitting one to compare the structural profile of the samples.
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- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU85306/91A AU654503B2 (en) | 1990-08-10 | 1991-08-01 | Quantitative analysis and monitoring of protein structure by subtractive chromatography |
CA002088360A CA2088360A1 (en) | 1990-08-10 | 1991-08-01 | Quantitative analysis and monitoring of protein structure by substractive chromatography |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US56612190A | 1990-08-10 | 1990-08-10 | |
US566,121 | 1990-08-10 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1992002815A2 true WO1992002815A2 (en) | 1992-02-20 |
WO1992002815A3 WO1992002815A3 (en) | 1992-04-30 |
Family
ID=24261583
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1991/005544 WO1992002815A2 (en) | 1990-08-10 | 1991-08-01 | Quantitative analysis and monitoring of protein structure by subtractive chromatography |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0548178A1 (en) |
JP (1) | JPH06500396A (en) |
AU (1) | AU654503B2 (en) |
CA (1) | CA2088360A1 (en) |
WO (1) | WO1992002815A2 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0533909A1 (en) * | 1991-03-28 | 1993-03-31 | Perseptive Biosystems Inc | On-line product identification in a chromatography effluent by subtraction. |
US6054047A (en) * | 1998-03-27 | 2000-04-25 | Synsorb Biotech, Inc. | Apparatus for screening compound libraries |
WO2001022078A1 (en) * | 1999-09-22 | 2001-03-29 | Lts Lohmann Therapie-Systeme Ag | Method and device for detecting and isolating pharmacological compounds being contained in substance mixtures |
US6607921B1 (en) | 1998-03-27 | 2003-08-19 | Ole Hindsgaul | Methods for screening compound libraries |
US6613575B1 (en) | 1998-03-27 | 2003-09-02 | Ole Hindsgaul | Methods for screening compound libraries |
US6627453B1 (en) | 1998-03-27 | 2003-09-30 | Ole Hindsgaul | Methods for screening compound libraries |
WO2010151214A1 (en) | 2009-06-26 | 2010-12-29 | Ge Healthcare Bio-Sciences Ab | A method in a chromatography system |
CN112969533A (en) * | 2018-10-09 | 2021-06-15 | C技术有限公司 | Chromatographic quality control system |
CN114280208A (en) * | 2021-11-03 | 2022-04-05 | 鼎康(武汉)生物医药有限公司 | Method for measuring dynamic loading capacity of affinity chromatography filler |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4204424A (en) * | 1979-01-08 | 1980-05-27 | Phillips Petroleum Company | Chromatographic analyzer detector gain adjustment |
EP0141259A1 (en) * | 1981-03-09 | 1985-05-15 | Environmental Sciences Associates, Inc. | Improvements in liquid chromatography |
WO1990006516A1 (en) * | 1988-12-05 | 1990-06-14 | Primus Corporation | Method for determination of glycated proteinaceous species |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS56114761A (en) * | 1980-02-14 | 1981-09-09 | Shinotesuto Kenkyusho:Kk | Fractional determination method of zinc in fresh blood serum by affinity chromatography |
JPS58216954A (en) * | 1982-06-11 | 1983-12-16 | Shimadzu Corp | Low temperature high speed liquid chromatography |
JPS5984828A (en) * | 1982-11-08 | 1984-05-16 | Suntory Ltd | Stabilized immune complex and its use |
IT1150598B (en) * | 1982-12-14 | 1986-12-17 | Ines Bianchi | PROCEDURE FOR THE PURIFICATION OF SUPEROXIDE DYSMUTASE BY AFFINITY CHROMATOGRAPHY |
JPS59143958A (en) * | 1983-02-07 | 1984-08-17 | Sekisui Chem Co Ltd | Quantitative analysis of fibronectin |
JPS63236964A (en) * | 1987-03-26 | 1988-10-03 | Ube Ind Ltd | Method for diagnosing diabetes |
-
1991
- 1991-08-01 WO PCT/US1991/005544 patent/WO1992002815A2/en not_active Application Discontinuation
- 1991-08-01 EP EP19910916341 patent/EP0548178A1/en not_active Withdrawn
- 1991-08-01 JP JP51561891A patent/JPH06500396A/en active Pending
- 1991-08-01 CA CA002088360A patent/CA2088360A1/en not_active Abandoned
- 1991-08-01 AU AU85306/91A patent/AU654503B2/en not_active Ceased
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4204424A (en) * | 1979-01-08 | 1980-05-27 | Phillips Petroleum Company | Chromatographic analyzer detector gain adjustment |
EP0141259A1 (en) * | 1981-03-09 | 1985-05-15 | Environmental Sciences Associates, Inc. | Improvements in liquid chromatography |
WO1990006516A1 (en) * | 1988-12-05 | 1990-06-14 | Primus Corporation | Method for determination of glycated proteinaceous species |
Non-Patent Citations (3)
Title |
---|
BIOTECHNOLOGY vol. 8, no. 3, March 1990, NEW YORK US pages 203 - 206; AFEYAN ET AL.: 'perfusion chromatography an approach to purifying biomolecules' * |
JOURNAL OF BIOLOGICAL CHEMISTRY. vol. 259, no. 6, 1984, BALTIMORE US pages 3796 - 3799; YIN CHANG LIU ET AL.: 'quantitative analysis of protein: immobilized dye interaction' * |
JOURNAL OF CHROMATOGRAPHY. vol. 512, 20 July 1990, AMSTERDAM NL pages 365 - 376; LLOYD ET AL.: 'preparative high-performance liquid chromatography on a unique high-speed macroporous resign' * |
Cited By (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0533909A1 (en) * | 1991-03-28 | 1993-03-31 | Perseptive Biosystems Inc | On-line product identification in a chromatography effluent by subtraction. |
EP0533909A4 (en) * | 1991-03-28 | 1993-06-30 | Perseptive Biosystems, Inc. | On-line product identification in a chromatography effluent by subtraction |
US6720190B1 (en) | 1998-03-27 | 2004-04-13 | Ole Hindsgaul | Methods for screening compound libraries |
US6627453B1 (en) | 1998-03-27 | 2003-09-30 | Ole Hindsgaul | Methods for screening compound libraries |
US6054047A (en) * | 1998-03-27 | 2000-04-25 | Synsorb Biotech, Inc. | Apparatus for screening compound libraries |
US6387257B1 (en) | 1998-03-27 | 2002-05-14 | Ole Hindsgaul | Apparatus for screening compound libraries |
US6395169B1 (en) | 1998-03-27 | 2002-05-28 | Ole Hindgual | Apparatus for screening compound libraries |
US6607921B1 (en) | 1998-03-27 | 2003-08-19 | Ole Hindsgaul | Methods for screening compound libraries |
US6613575B1 (en) | 1998-03-27 | 2003-09-02 | Ole Hindsgaul | Methods for screening compound libraries |
US6656739B2 (en) | 1998-03-27 | 2003-12-02 | Ole Hindsgaul | Methods for screening compound libraries |
US6649415B1 (en) | 1998-03-27 | 2003-11-18 | Ole Hindsgaul | Methods for screening compound libraries |
US6723235B2 (en) | 1998-03-27 | 2004-04-20 | Ole Hindsgaul | Apparatus for screening compound libraries |
US6355163B2 (en) | 1998-03-27 | 2002-03-12 | Ole Hindsgaul | Apparatus for screening compound libraries |
AU777927B2 (en) * | 1999-09-22 | 2004-11-04 | Lts Lohmann Therapie-Systeme Ag | Method and device for detecting and isolating pharmacological compounds being contained in substance mixtures |
WO2001022078A1 (en) * | 1999-09-22 | 2001-03-29 | Lts Lohmann Therapie-Systeme Ag | Method and device for detecting and isolating pharmacological compounds being contained in substance mixtures |
US7232690B1 (en) | 1999-09-22 | 2007-06-19 | Lts Lohmann Therapie-Systeme Ag | Method and device for detecting and isolating pharmacological compounds being contained in substance mixtures |
CN100420945C (en) * | 1999-09-22 | 2008-09-24 | 罗曼治疗系统股份公司 | Method and device for detecting and insolating pharmacological compounds being contained in substance mixtures |
EP2446257A1 (en) * | 2009-06-26 | 2012-05-02 | GE Healthcare Bio-Sciences AB | A method in a chromatography system |
WO2010151214A1 (en) | 2009-06-26 | 2010-12-29 | Ge Healthcare Bio-Sciences Ab | A method in a chromatography system |
EP2446257A4 (en) * | 2009-06-26 | 2014-09-10 | Ge Healthcare Bio Sciences Ab | A method in a chromatography system |
EP2446257B1 (en) | 2009-06-26 | 2020-06-03 | GE Healthcare Bio-Sciences AB | A method in a chromatography system |
EP3702775A1 (en) * | 2009-06-26 | 2020-09-02 | Cytiva Sweden AB | Method for determining binding capacities in a chromatography system |
CN112969533A (en) * | 2018-10-09 | 2021-06-15 | C技术有限公司 | Chromatographic quality control system |
CN114280208A (en) * | 2021-11-03 | 2022-04-05 | 鼎康(武汉)生物医药有限公司 | Method for measuring dynamic loading capacity of affinity chromatography filler |
CN114280208B (en) * | 2021-11-03 | 2024-05-24 | 鼎康(武汉)生物医药有限公司 | Method for measuring dynamic load of affinity chromatography packing |
Also Published As
Publication number | Publication date |
---|---|
AU8530691A (en) | 1992-03-02 |
CA2088360A1 (en) | 1992-02-11 |
AU654503B2 (en) | 1994-11-10 |
JPH06500396A (en) | 1994-01-13 |
EP0548178A1 (en) | 1993-06-30 |
WO1992002815A3 (en) | 1992-04-30 |
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