Chemistry: analytical and immunological testing – Including sample preparation – Liberation or purification of sample or separation of...
Reexamination Certificate
1997-04-08
2001-01-09
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
Liberation or purification of sample or separation of...
C436S086000, C436S174000, C436S175000, C436S177000, C422S105000, C210S638000, C210S645000, C210S650000, C210S651000, C210S660000, C210S681000
Reexamination Certificate
active
06171869
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to processes for preparing protein-containing solutions for subsequent analysis using sample component separation techniques. More particularly, the present invention involves a one-step procedure for desalting and concentrating protein-containing solutions including a method for removing low molecular weight components processed in an ultrafiltration device while concentrating higher molecular weight components.
BACKGROUND OF THE INVENTION
The analysis of protein-containing solutions, in particular biological samples, often requires desalting of the solution for the removal of components that interfere with subsequent analytical techniques. Analytical techniques which may require desalting of sample solutions include those using component separation technologies such as electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry. Analysis of biological samples containing proteins using capillary electrophoresis, for instance, can be affected by the presence of various low molecular weight salts and protein degradation products. The presence of salts and other low molecular weight ions can increase the ionic strength of a sample to such an extent that the resolution upon electrophoresis decreases dramatically and/or the interpretation of the electropherograms becomes complicated due to the appearance of false peaks.
In addition to desalting, concentration of the sample is also often required since the particular component of interest may be present in small quantities. Frequently, the presence of a particular component in the sample will go undetected unless the component is concentrated to a level that is capable of being detected by the subsequent analytical technique. In the case of capillary electrophoresis, the concentration of the component in the sample may need to be substantially increased for the peak corresponding to the component on the electropherogram to be detectable.
The presence of proteins in samples of biological fluid, such as blood, serum, plasma, cerebrospinal fluid, tear, sweat, saliva and urine is a useful indicator of the presence or absence of certain disease states. Thus, the ability to identify and quantitate a variety of proteins in biological fluid can provide diagnosticians with information which may lead to the diagnosis of a variety of diseases. For example, determination of the urinary excretion of albumin, IgG, free protein human complex-forming glycoprotein (HC), alpha-1-macroglobulin, and kappa and lambda light chains allows in most cases for the classification of proteinuria in the clinically important categories. Using information obtained from the analysis of urine samples, for example, proteinuria may be classified as selective glomerular, unselective glomerular, tubular glomerular, or nontubular glomerular. The presence or absence of Bence Jones proteinuria may also be demonstrated.
The identification of monoclonal free light chains, or Bence Jones protein (BJP), is also important in the assessment of patients with multiple myeloma. Multiple myeloma may be characterized by the presence of BJP in the urine or serum. Approximately 13% of myeloma patients, however, have no BJP in their serum. Therefore, it is essential that both serum and urine be examined for the presence of BJP. The type of light chain present in the urine or serum sample may have a considerable effect on the clinical course of myeloma. For instance, lambda-type light chains are known to accompany rather aggressive myeloma, and exert their toxic effects in a shorter time than do kappa-type light chains.
Generally, identification of free light chains in urine is accomplished by analysis of urine samples with immunoelectrophoresis (IEF) or immunofixation electrophoresis (IFE). Both of these methods are labor intensive and require long execution times. Improved methods for identifying free light chains in urine involve capillary electrophoresis of samples in which the proteins of interest are subtracted from solution by being first fixed to a solid phase through antigen-antibody binding prior to electrophoresis. The identification of the specific proteins is established by their absence from the separated sample. This method is known as immunofixation electrophoresis by immunosubtraction, or IFE/s.
It is often preferable to use an instrument to analyze protein-containing solutions. The instrument should be capable of performing both routine serum protein electrophoresis (SPE) and follow-on, IFE/s testing to characterize monoclonal components detected in the initial SPE screening. One such instrument is the Paragon CZE™ 2000, available from Beckman Instruments, Inc., Fullerton, Calif. The Paragon CZE is a clinical, multi-channel automated capillary electrophoresis instrument. The unit is a dedicated analyzer for SPE and IFE testing, so that many of the traditional capillary electrophoresis parameters such as injection time, capillary rinse sequence and wash times, applied voltage, and absorbance wavelength selection have been optimized and automated to provide walk-away capability. Additional automated and optimized parameters of this instrument include sample volume in primary tubes, sample dilution and additional dilution with solid support. Using the Paragon CZE™ 2000 analyzer, the technique of immunosubtraction is used to remove a specific immunoglobulin class or type from a serum sample.
Regardless of the method used to identify proteins in solutions, and particularly urine, pretreatment of the solution to desalt and concentrate proteins in the sample solution is required. There are several classical methods for desalting or concentrating protein-containing solutions. These methods include dialysis, molecular sieve chromatography, diafiltration, ultrafiltration, precipitation, ion exchange, freeze drying, partitioning between two aqueous polymer phases, osmotic removal of water and reverse phase HPLC. These methods typically consist of two separate steps for desalting and concentration and may require specialized equipment and procedures associated with each step of desalting and concentration.
Gel filtration is one useful and mild method for desalting a protein-containing solution. The gel used in gel filtration processes consists of an open, cross-linked, three-dimensional molecular network, which can be cast in bead form for column packing and optimum flow characteristics. Pores within the beads are of such sizes that they are not accessible by large molecules, but smaller molecules can easily penetrate all the pores. Gel filtration media beneficially exhibit little protein binding and give high recoveries for even small amounts of proteins. Some of the most commonly used gel filtration support are Sephadex G-25 and BioGel P-30. Although gel filtration has many advantages, this method has several features which make it inapplicable for use in, for example, an ultrafiltration device. The primary problem with gel filtration media, particularly with polyacrylamide beads, is their softness. Even very gentle pressure, including osmotic pressures obtained during chromatography, can cause distortion, irregular packing and poor flow characteristics. Recently, Bio-Rad introduced the Bio-Spin chromatography column which is prepacked with a polyacrylamide gel matrix (Bio-Gel P-6). This ready-to-use column can be used for rapid desalting of a protein mixture at low centrifugal forces. Desalting in this manner, however, is only applicable for very small sample volumes and column volumes. Proper matrix preparation and column packing are required. The degree of desalting is further dependent on the column dimensions, shape and total volume. The optimal removal of salt that can be expected is achieved with sample volumes not exceeding 20-25% of the column volume.
Desalting of simple protein-containing solutions is often accomplished with ion exchange using, for example, a mixed bed resin. Commercially available mixed bed resins, such as IonClear BigBead (Sterogene Bioseparations, Inc., Arcadia,
Blessum Cynthia R.
Safarian Zara
Beckman Coulter Inc.
Kivinski Margaret A.
May William H.
Wallenhorst Maureen M.
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