AU679743B2 - Tumor marker control - Google Patents

Description

WO 9424569 PCT/US94/03884 Tumor Marker Control Field of the Invention This invention relates generally to the field of stable human serum based controls for use in i vitro diagnostic assays and more specifically to stable human serum based controls for use in monitoring the precision of in vitro diagnostic assays for tumor markers.

Background of the Invention Tumor markers are substances released by tumor cells into the blood stream. The tumor markers can be detected in serum or other body fluids and are useful for clinically monitoring various malignancies. The term tumor marker has been extended to include cell or tissue characteristics, such as oncogenes or abnormally expressed proteins such as enzymes, hormones, and receptors that are related to and assist in identifying the tumor type.

Clinical oncologists measure the presence and/or amount of these markers in bodily fluids to assist them in the diagnosis of the condition, as well as for prognosis and the monitoring of the treatment of the patient. Serum assays of tumor markers are commercially available.

These serum assays are performed using assay systems such as radioimmunoassay, enzyme immunoassay, fluorescence immunoassays and other clinical analysis techniques.

Controlling and monitoring the accuracy, precision, nd reliability of these assay systems is critical to ensure that the patient receives the correct treatment and that the results of the a'.;ays are medically relevant.

Currently, some human serum based controls are commercially available. Controls, generally, are in levels representing specific ranges, for example a high, low, and/or normal range. These commercially available tumor control products include Cancer Antigen Controls, from POLYMEDCO, T-MARKERS QUALITY CONTROL SERUM, from NMS Pharmaceuticals, Inc. and LYPHOCHEK® Tumor Marker Control, from BIO-RAD. Currently available commercial controls, 1 SUBSTITUTE SHEET (RULE 26) I WO 94/24569 PCT/US94/03884 however, lack many of the tumor markers that are required by the clinical oncologists. Moreover, the currently available commercial controls have limited clarity, limited lyophilized stability and limited reconstituted stability. Also, some of the commercially available controls are only two level controls High and Low). In addition, the concentrations of some of the components in the commercially available controls are either too high or too low to be completely useful.

Although methods have been published to purify some of the tumor markers many of these methods are tedious and require several steps. Each additional step results in a lower yield of the tumor marker.

Tumor markers, for use in the controls do not necessarily need the high degree of purity resulting from some of these purification schemes.

Thus, a need exists for a tumor marker control that has a wider variety of tumor markers, has discrete useful ranges of the markers and has an enhanced optical clarity and stability. A need also exists for simpler methods of purification of some of the tumor markers which result in a high yield and a purity sufficeint for the intended use.

Summary of the Invention The human based serum or plasma control according to the present invention preferably contains many of the tumor markers that are utilized by clinical oncologists to diagnose patients. The human based serum or plasma control according to the present invention has enhanced lyophilized and reconstituted stability and has enhanced optical clarity after reconstitution.

Detailed Description of the Invention The human based serum or plasma control of the present invention comprises a base of male human plasma or serum that has been lipid stripped and tumor markers. The tumor markers may include Adenocorticotropic Hormone (ACTH), Aldosterone, Alphafetoprotein (AFP), beta-2-microglobulin (B2M), CA 15-3®, CA 125®, CA 19-9®, CA 19-9® (Registered Trademarks of Centocor Diagnostics, a division of Centocor Inc.), CA 549, Carcinoembryonic Antigen (CEA), Ferritin, 2 SUBSTITUTE SHEET (RULE 26) WO 94/24569 PCT/US94/03884 Gastrin, human Chorionic Gonadotropin (hCG), beta hCG, Gamma Enolase (NSE), Prolactin, Prostatic Acid Phosphatase (PAP), Prostatic Specific Antigen (PSA), Tissue Polypeptide Antigen (TPA), Calcitonin and LD-1. A preservative system should also be include in the control.

The preservative system should include a preservative that is stable both prior to lyophilization and after lyophilization.

A preservative system is necessary in order to ensure reconstituted stability of certain markers especially enzymes that are very sensitive to proteases that are produced by microorganisms. A combination of preservatives are added. The preferred preservative system is a combination of gentamicin sulfate, cycloheximide and Proclin 300 (Rohm and Haas). The Proclin 300 is not effective after lyophilization, however, it is useful in controlling microbial growth during the manufacturing process. The gentamicin sulfate and cycloheximide are used to control growth of microorganisms after reconstitution. Sodium azide is not used mainly due to the hazard of the explosive properties of the azide.

The stability of the lyophilized control should be at least about a year and preferably at least about three years. The reconstituted stability of the majority of the components should be at least about seven days and preferably at least about fourteen days.

The base of human serum or plasma should be substantially from all male donors in order to preserve the stability of the PAP marker. In the presence of substantially all male serum or plasma, the PAP is very stable. Female serum and plasma may contain antibodies to this enzyme marker. The antibodies would effectively eliminate the PAP from the control solution. If the antibodies are successfully removed or their effects eliminated from the female serum or plasma, the resulting serum or plasma could be utilized as the base material.

The content of the lipids in the human serum or plasma must be reduced. The lipid content may be reduced by treating the serum or plasma with fumed silica or dextran sulfate or other known processes.

The process used to reduce the lipids must ensure that the content of cholesterol and tnglycerides in the human serum or plasma is less than 3 SUBSTITUTE SHEET (RULE 26) WO 94/24569 PCT/US94/03884 about 20 mg/dL each after processing. There are at least three reasons to reduce the lipid content.

First, the stability of added tumor markers which are easily denatured or oxidized is increased when the lipid content is reduced.

This is because when the lipids break down, they form oxidation byproducts that can interfere with the stability of some of the markers.

Moreover, the breakdown of lipids results in turbid solutions. Second, the lipid reduction aids in the reconstitution process. The lyophilized control reconstitutes immediately upon the addition of the liquid when the lipids are eliminated. The reconstitution time of the lyophilized control is delayed by between about 15 to 30 minutes if serum or plasma containing normal amounts of lipids are utilized.

Third, high levels of lipids can cause interference in measuring some of the tumor markers. Thus, reduction of the level of lipids leads to a more accurate assay result.

The serum or plasma that is utilized as the base for the control should be assayed for the tumor markers that will be added prior to the addition of those tumor markers.

Table I is a classification of the various types of tumor markers that are added to the base material. Table II lists the tumor markers and the types of cancers that are usually associated with that marker. Table III lists sources of several of the tumor markers.

The tumor markers that are added into the base material must be relatively pure that is not cross contaminated with other markers or contaminated with interfering substances. It is best to use sources of tumor markers that are native human forms; however, it has been found that many of the human source tumors produce more than one marker.

The addition of this raw source to the base material makes it difficult to formulate a control with an accurate amount of each tumor marker. In some instances, the tumor marker could end up being added in an amount that is too high to be useful for low or normal control levels.

Thus, to avoid this problem many of the tumor markers must be purified to remove cross-contamination. In addition, the tumor markers that are 4 SUBSTITUTE SHEET (RULE 26) Now U I WO 9424569 PCT/US94/03884 to be added to the base material should be assayed to determine the presence of cross-contaminants and known interfering substances.

B2M may be purified from urine that has been collected from patients having renal failure. Particulates are removed and the urine is diafiltered into an appropriate buffer and concentrated. The B2M, a protein, has an approximate molecular weight of about 11,000 daltons; thus, it can be purified using size exclusion chromatography such as gel filtration chromatography. Preferred gel materials are Ultragel ACA 54 or its equivalents. The fractions containing the B2M are pooled and concentrated to preferably at least about 1 g/dL, then the outcome of the purification can be determined using such known methods as electrophoresis. In addition, the B2M is tested by commercially available immunoassay. The B2M is stable when stored either at about 2-8 C or frozen at less than about -20 C. The resulting B2M may contain up to as much as about 70% of impurities of immunoglobulins without effecting the usefulness of the B2M..

CA 125 is a marker that is specific to ovarian cancer. This marker may be found in ascites fluid that is collected from patients with ovarian cancer. The ascites fluid contains two marker, CA 125 and TPA. The contamination level of the TPA is very high; thus, in order to add an accurate amount of each of CA 125 and TPA, the markers must be separated. Both of these markers are shed into the serum during tumor growth and due to the similarities of these markers the separation of them is difficult. It was discovered that TPA binds to a hydrophobic interaction chromatography media, Phenyl Sepharose (Pharmacia), in the presence of phosphate buffer at about a physiological pH. A phosphate buffer of about 50 mM phosphate at a pH of about 7.2 is preferred. The ascites fluid is applied onto a column of Phenyl Sepharose. The majority of the CA 125 does not bind to the Phenyl Sepharose and flows directly though the column and is collected. The column is then washed with the phosphate buffer to which has been added about 2.5 M urea. This buffer elutes the remaining CA 125. The column is then washed with the phosphate buffer to which has been added about 6 M urea. The TPA is eluted with this buffer and collected.

SUBSTITUTE SHEET (RULE 26)

~_I

WO 94/24569 PCT/US94/03884 Normally, chromatography using Phenyl Sepharose requires a high salt concentration for binding to occur. However, surprisingly, the TPA binds without a high salt concentration. Thus, it is surprising that the separation occurs because the separation is not due to the hydrophobic interaction. The separated proteins are buffer exchanged to remove the urea and are concentrated to a protein level of preferably about greater than Ig/dL. The separation of the proteins may be confirmed by assaying the separated proteins using commercially available immunoassay techniques.

CEA, CA 19-9 and TPA are often obtained from the same source; thus, they must be separated from each other. CEA is a large glycoprotein of about 200,000 daltons and is found at elevated levels in the serum of patients with colon cancer. CEA is an oncofetal antigen that is expressed during intra-uterine life and disappears after birth.

Oncofetal antigens reappear in situations of repair or neoplastic growth in the organs where they appeared during gestation. Elevated levels have also been found in patients with lung, gastric, breast and pancreatic cancers. CA 19-9 is a tumor mucin antigen. Tumor mucins are high molecular weight glycoprotein from about 200,000 daltons to 1000 kDA and contain from about 25% to 80% carbohydrate. As a tumor marker CA 19.9 is elevated in patients with pancreatic cancer and gastrointestinal cancer.

One source of CEA, CA 19-9 and TPA is a cell line identified as SW 1116. SW 1116 is a human cell line developed from a colorectal carcinoma. The cancer cells excrete the antigens into a cell growth media. The cell growth medium is collected and frozen as it is produced. The cell supernatant is thawed and concentrated about times. The concentrated supernatant is buffer exchanged into buffers such as phosphate buffers at physiological pHs. The preferred buffer is 50 mM phosphate at about pH 7.2.

Although CEA, CA 19-9 and TPA are somewhat different, they are all glycoproteins and are very difficult to separate by typical chromatography methods. Precipatation methods using perchloric acid 6 SUBSTITUTE SHEET (RULE 26)

~G

WO 94/24569 PCT/US94/03884 treatment to precipatate the CEA have been suggested, however the process results in a low yield of the purified markers.

Thus, a method was developed to purify the three markers. The concentrated, buffer exchanged supernatant is applied onto a Phenyl harose column. As described for the s puigication, the TPA omds to the chromatography media without the presence of high salt.

The column is then washed with phosphate buffer and the eluant is collected in fractions. As determined by immunoassay, these fractions contain mostly CA 19-9, but selected fractions contain CEA.

The CEA/CA 19-9 fractions could be separated and further purified by affinity chromatography using processes known in the art.

In one process disclosed in Ford, et al. Immunoadsorbent Purification of Carcinoembrvonic Antigen using A Monoclonal Antibody: A Direct Comparison with a Conventional Method, Tumor Biol. Vol. 8 pages 241-250 (1987), a column is prepared which contains a media that has an antibody specific to CEA attached to the chromatography media. This column can strip out the CEA and the CA 19-9 will pass through the column. The CEA can be stripped from the column. However there are other sources of commercially available CEA; thus, it is not necessary to utilize thii method.

Since the CEA is not required to be obtained from this method, it is preferred to combine the fractions from the Phenyl Sepharose column and then wash the Phenyl Sepharose column with a phosphate buffer, preferably 50 mM phosphate at pH 7.2, containing from about 2 to 3 M urea to remove any additional CA 19-9. The eluant is collected. All of the fractions containing CA 19-9 and CA 19-9 with CEA are combined.

The colunm is next eluted with the same buffer but also containing about 6 M urea. The TPA is eluted and collected.

The CA 19-9/CEA containing pool is buffer exchanged to remove the urea and concentrated to at least about 1 g/dL. The CA 19-9 in the concentrate by freezing the concentrate. Long term freezing of the CA 19-9 results in a loss of activity of the CEA, however the activity of the CA 19-9 is preserved. Thus, the entire purification process can be simplified. The length of freezing time can be determined by testing 7 .1 SUBSTITUTE SHEET (RULE 26) WO 94/24569 PCT/US94/03884 aliquots of the concentrate for the presence of CEA by immu asay techniques. The approximate recovery can be up to 100%.

Alternatively, the fractions containing the CEA/CA 19-9 can be discarded. Then, only the fractions containing CA 19-9 are pooled and concentrated.

The TPA is also buffer exchanged and concentrated as described above. The recovery of the TPA can also be up to about 100%. Crosscontamination is determined using immunoassay techniques.

The CEA can be obtained as described above using the monoclonal antibody method or it may be obtained from other commercially available sources. The CEA should be tested for crosscontamination with immunoassay methods prior to use in a control. If contamination is detected, the CEA must be purified using one of the methods known in the art, preferably the affinity method described above.

NSE is obtained from fresh or freshly frozen human brain.

Purified NSE may be obtained commercially. The preferred method for purification is accomplished by preparing a homogenate of the brain, centrifuging the homogenate and collecting the supernatant. Next the supernatant is pelleted using 40% ammonium sulfate. The pellet is resuspended in a 10 mM Tris-phosphate buffer and dialyzed against the buffer then concentrated. The concentrate is chromatographed on DE- 52 and eluted with a 0.15 M -0.35 M NaCl gradient. The peak containing the NSE is dialyzed, lyophilized and fractionated on Sephadex G 150 or the like. Polybufferexchanger chromatofocussing is used to focus the NSE. The NSE is then eluted and finally fractionated on G-150 (Superfine).

Finally, AFP must be purified. AFP is an oncofetal antigen like CEA. AFP is a glycoprotein expressed in fetal liver and digestive tract.

In adults elevated levels of this antigen in serum is associated with malignant hepatoma and in some cases of ovarian and testicular cancers, The best source of this antigen is human cord serum collected at the time of birth. This serum contains high levels of AFP (about 60,000 ng/mL) and contains only one contaminating tumor marker, Prolactin.

8 SUBSTITUTE SHEET (RULE 26) WO 94/24569 PCT/US94/03884 There are methods for purifying AFP described in the art. For instance, Chudy D. and Zizkovsky A simple and rapid method for the isolation of human alpha-fetoprotein from human cord serum, Neoplasma 34 pp. 491 to 496 (1987) describes one such procedure.

For purposes of this invention, the preferred method of isolation of the AFP from the Prolactin is accomplished using ion exchange chromatography.

Using a 20 mM Tris buffer at pH 8.5 the cord serum is applied onto a cation exchange resin. At this pH and buffer strength the AFP binds to the column but the majority of the Prolactin does not bind to the column. Thus, the serum is added to the column, and the Prolactin is washed through the column. The Prolactin can be collected. The AFP can then be eluted off of the column using about 0.2 to 0.3 M sodium chloride with the buffer.

The isolated AFP is then concentrated to about Img/mL.

Recovery of the AFP in this manner can be about 100%. The AFP purified in this manner may contain large quantities of albumin.

However, this contaminant is not a problem since the serum or plasma based material contains albumin. The purified AFP can be tested using immunoassay procedures.

The other tumor markers such as ACTH, aldosterone, hCG, betahCG, CA 15-3, CA 549, Calcitonin, Ferritin, Gastrin, PAP, PSA, Prolactin and LD-1 are available commercially from several sources.

These other markers can be obtained purified or can be purified by procedures well known in the art. For each tumor marker, crosscontamination can be assessed by immunoassay techniques. The LD-1 is added as a component of LDH by detennining the amount of LD-1 present in LDH.

The solutions for the controls are formulated by first assaying the plasma or serum and all the specific tumor markers that are used to spike the plasma or serum. Table IV shows the target values for each of the specific tumor marker at each of the three levels of controls that are prepared. Calculations are performed by subtracting the concentration of each marker in the serum or plasma from the mean targeted value in 9 SUBSTITUTE SHEET (RULE 26) WO 94/24569 PCT/US94/03884 Table IV, then adding the appropriate amount of each marker to each of the three levels of controls.

The tumor markers are added to the serum or plasma according to the stability of each marker. Markers such as B2M, AFP, Prolactin, hCG, Beta-hCG, CA-15-3, CA-19-9, CA 549, CA 125, CEA, Ferritin, TPA, and LD-1 (added as LDH) may be added and adjusted within a few days of lyophilization as long as the temperature of the serum or plasma is controlled within about 2 to 10 C. If all the materials are kept at between about 2-10 C, the ACTH, Gastrin, gamma enolase, and calcitonin (markers which have short term liquid stability) may be added up to about six hours prior to lyophilization. Preferably these markers are added immediately prior to lyophilization and the additions and adjustments are done at low temperatures, that is 2-10 C.

Each of the three levels of liquid controls are lyophilized using standard methods. The bottles containing the lyophilized controls are sealed under vacuum and then stored at about 4C. The controls are reconstituted with water or other appropriate liquids such as buffers.

For ACTH, lyophilization studies are required to determine the loss of ACTH activity during the lyophilization process. Immunoassay methods are used to determine the loss of activity due to the process.

The results can be used to determine the pre-lyophilization level of ACTH that is necessary to recover a spec:ic post lyophilization level of

ACTH.

The stability of all of the markers in the lyophilized control was determined to be four weeks at a stressed temperature of 37 C. See, Table V. This is thought to correspond to about 3 years when stored at 2-8 C. A reconstituted stability of at least two weeks was found for all flarkers excepM TH, gastrin and calcitonin. See, Table VI. The ACTH, gastrin and calcitonin must be used shortly after reconstituting with liquid. It was also found that the reconstituted stability can be prolonged for 30 days for all analytes except NSE, gastrin and calcitonin by freezing aliquots of the reconstituted material at -20C. See, Table VII. The stability of the gastrin and calcitonin can be prolonged for seven days by freezing aliquots of the reconstituted material at SUBSTITUTE SHEET (RULE I WO 94/24569 PCT/US94/03884 See, Table VIII. The stability of the NSE can be extended for twenty four hours by freezing aliquots of the reconstituted material at See, Table VIII.

The following Examples are given for the purpose of illustrating the present invention: Example 1 Purification of B2M A urine concentrate was prepared by collecting urine from patients with renal failure. The urine was pooled and sodium azide at 0.02% was added as a preservative. The urine was filtered through a membrane of less than 0,3 microns to remove all particulates and microbes. The urine was then diafiltered against seven volumes of mM Tris buffer, pH 8.0 and concentrated to a volume 100 times the original volume. For example, 100 liters was concentrated to 1 liter.

The concentrated urine was adjusted to a total protein concentration of about 9.0 g/dL using the above buffer.

About fifty mLs of the urine concentrate was applied to an Ultragel ACA 54 column. The sample size is dependent upon the column size and is equivalent to 2.5% of the total volume of the media.

The length of the column must be about 100 cm for effective separation of the proteins. Fractions containing the B2M were combined, pooled and concentrated to about 1 g/dL.

Purification has also been accomplished on Superdex (Pharmacia). However, for this application, the purification on Ultragel ACA 54 is superior.

Purification of CA 19-9 and TPA About a fifty mL sample of a supernatant from SW1116, a cell line (supernatant available from Whitaker), was concentrated to one half the original volume. The sample was buffer exchanged three times with about fifty mL of 50 mM potassium phosphate at about pH 7.2. The final volume of the sample was about 35 mL. About twenty mLs of the sample were applied to a Phenyl Sepharose column, The column was 11 SUBSTITUTE SHEET (RULE 26) WO 94/24569 PCT/US94/03884 washed with the buffer and fractions were collected. The fractions were evaluated for CEA and CA 19-9 activity using an immunoassay.

Fractions containing CEA were pooled and concentrated and fractions containing CA 19-9 were pooled and concentrated. A buffer containing about 50 mM phosphate at pH 7.2 with increasing .nounts of urea was applied to the column. The TPA was eluted with urea at about 6M. The TPA containing fractions were pooled and concentrated then diafiltered to remove the 6M urea.

The CA 19-9 fractions undergo long term storage to remove the activity of any CEA that contaminates the CA 19-9.

Example 2 Preparation of the Controls Delipidated serum from males was filtered through the following sequences of filters: a prefilter, a 1.2 micron filter, a 0.8 micron filter, a 0.45 micron filter and a 0.22 micron filter. The filtered serum was refrigerated. Proclin 300 from Rohm and Haas was added at a concentration of 1 mL per liter of serum. The serum was divided into two pools of about 1.92 liters per pool designated as Pool 1 and Pool 2.

The serum was assayed for amounts of ACTH, Aldosterone, B2M, hCG, beta-hCG, CA 15-3, CA 19-9, CA 125, CA 549, calcitonin, CEA, Ferritin, gastrin, NSE, PAP, PSA, Prolactin, TPA, and LD-1 using immunoassay techniques.

Each of the markers were obtained through purifications as described herein or were obtained commnercially. The amount of each marker was determined. An amount of each marker necessary to reach the values given ir Table IV, Level I ranges were added to Pool 1. An amounts of each marker to reach the values given in Table IV, Level III ranges were added to Pool 2. The amounts of marker were assayed and any adjustments were made by either by adding additional amounts of marker.

Three milliliters aliquots from Pool 1 (Level 1) were filled into vials that had been chilled in a freezer for about one hour and three 12 SUBSTITUTE SHEET (RULE 261 WO 94/24569 PCT/US94/03884 milliliter aliquots from Pool 2 (Level 3) vwere filled into vials that had been chilled in a freezer for about one hour. The vials were lyophilized, sealed under a vacuum and stored at about 4C.

Example 3 Assay of Serum Based Tumor Marker Control Vials of the controls prepared in Example 2 were reconstituted with three milliliters of distilled water and inverted gently to mix. The markers contained in the Level 1 and Level 3 controls were assayed using a variety of immunoassay methods. The results are presented in Table IX.

13 SUBSTITUTE SHEET (RULE 26) __i WO 94/24569 WO 9424569PCT/UJS94/03884 CLASSIFICATION OF TUMOR MARKERS 1 Oricofetal Antigens AFP, CEA Produced during fetal development and low levels in adults. Tumors cause reexpression of these proteins.

2. Tumor associated antigens CA 19-9, CA 549, CA 15-3 Mucins(carbohydrate rich glycoproteins) excreted by the tumor cells. High molecular weight 200kda and 25 to carbohydrate.

3. Hormones Prolactin, Aldosterone, Gastrin beta 2-microglobulin, ferritin 4. Serum Proteins Enzymes PAP, NSE, LD1 TAXBLE 1 14 Sq13STITLJTE SHEET (RULE 26) WO 94/24569 ICT/US94/03884 TUMOR MARKER CONTROL CLINICAL MARKERS TUMOR MARKER SITE(S) Adenocortitropic Hormone

(ACTH)

Alphafetoprotein (AFP) Aldosterone Beta 2 Microglobulin Beta Human Chorionic Gonadotropin CA 15-3 CA 549 CA 19-9 CA 125 Carcinoembryonic Antigen

(CEA)

Lung Testicular, Liver Kidney Bone Marrow Gynecological, testicular Breast Pancreas, Colorectal, Stomach Ovarian Colorectal, Breast, Lung, Stomach, Pancreas Table II SUBSTITUTE SHEET (RULE 26) I I WO 94124569 WO 9424S69PCTIUS94/03884 TUMOR MARKER CONTROL CLINICAL MARKERS TUMOR MARKER SIE Ferritin Gamma Enolase Gastrin Human Chorionic Gonadotropin (hCG) Lactate Dehydrogenase lsoenzyme (LD-1) Prolactin Prostatis Acid Phosphatase

(PAP)

P-ostate Specific Antigen

(PSA)

Tissue Polypeptide Antigen

(TPA)

Liver Lung, Drain Pancreas Testicular Brain Pituitary Prostate Prostate Bladder, Prostate, Gynecological, Lung Table'll (cont.) 16 SUBSTITUTE SHEET (RULE 26) WO 94/24569 WO 9424569PCT1US94/03884 TUMOR MARKER CONTROL ANTIGENS SOURCE B2 Microglobulin CA 15-3 CA 549 CA 19-9

CEA

TPA

Renal failure urine Breast ascites, Pleural fluid, Hybritech mouse tumor SW1 116 Supernate SWil 16 Supernate SWil116 Supernate Ovarian cancer ascites Pleural fluids Ovarian cancer ascites Pleural fluid breast Human cord serum Cord serum CA 125 Prolazctin Alpha-f etoprotein Table Ill 17 SUBSTITUTE SHEET (RULE 26) TYPICAL VALUES TUMOR MARKER CONTROL Constituent Aden ocorticotro pic Hormone Alpha-Fetoprotein Aldosterone B-2 Microglobulin Beta Human Chorionic Gonadotropin Human Chorionic Gonadlotropin CA 15-3 CA 19-9 cc CA 125 CA 549 Thyrocalcitonin Carcinoembryonic Antigen Ferritin Units pglmL nglmL pg/dL ngIL I 1.11 lU/L U/mL U/mL U/mL U/mL pglmL nglmL ng/mL Level!1 Ranges 35 (20-50) 10 (7-13) 50 (30-70) 1.50(-2) 50(-9) 50(-9) 45 (30-60) 30 (20-40) 200(0-30) 150(0-20) 30 (20-40) 4 (3-5) 30 (25-35) Level 11 Ranges 65 (50-80) 75 (65-85) 110 (95-125;i 3 (2.5-3.5) 20 (15-25) 20 (15-25) 200 (150-250) 100 (90-110) 40 (35-45) 40 (35-45) 110 (100-1 20) 150(0-20) 100 (95-105) Level IIl Ranges 400 (350-450) 250 (230-270) 700 (650-750) 12 (10-14) 450 (440-460) 450 (440-460) 240 400 (380-420) 400 (350-450) 65 (60-70) 600 (550-650) 30 (25-35) 450 (440-460) Table IV TYPICAL. VALUES TUMOR MARKER

CONTROL

0 TYPiCAL VALUES TUMOR MAR

ER

Constituent Gastrin LDH-1 Gamma Enolase Prostatic: Acid Phosphatase Prostatic Specific Antigen Prolactin Tissue Polypeptide Antigen Units pg/mi

U/L

ng/mL ng/mL ng/mL ng/mL

U/L

Level 1 Ranges 60 (50-70) 150 (130-1 70) 50 (40-60) 3 (2-4) 3 (2-4) 5 (3-7) 30 (25-35) Level If Ranges 200 (190-210) 250 (240-260) 100 (90-110,; 11 (10-12) 150(3-17) 200(8-22) 100 (90-100) Level Ill Ran-ges 400 (390-4i0) 350 (340-360) 150 (140-1 25 (23-27) 35 (33-37) 150 (140-1 500 (490-510) Table IV (cont.) WO 94/24569 WO 9424569PCTIUS94/03884 ACCELERATED STABILITY STUDIES Four weeks @37C Analyte Level I Level I Level 11 Level 11 fresh 37C fresh 37C AFP 10,2 10.3 293 293 Aldlos 79 74 800 780 132M 1.0 0.97 4.2 4.2 Gastrin 77 72 289 275 Calcitonin 38 35 270 275 ACTH 33 34 461 486 Ferritin 31 27 692 697 PAP 2.1 1.9 17 16 Prolactin 6.8 6.6 171 170 PSA 3.0 3.0 37 37 TPA 48 48 953 957 CA549 8.9 8.6 27 CA 15-3 41 41 262 245 beta hCG 2.7 2.8 475 446 hOG 2.7 2.7 460 439 CA125 18 18 356 361 CEA 2.9 2.9 57 57 NSE 14 12 65 51 LDH 286 260 782 756 Table V SUBSTITUTE SHEET (RULE 26) WO 94/24569 WO 9424569PCTIUS94/03884 RECONSTITUTED STABILITY STUDIES Fourteen davs 0 2-8C Analyte Level I Level I Level 11 Level 11 fresh 14 Days fresh 14 Days AFP 10.2 10.6 293 294 ALDOS 79 81 804 850 B32M 1.1 1.0 4.2 4.4 CA 19-9 35 33 249 241 Ferritin 31 28 693 671 PAP 2.1 2.0 16.9 16.4 Prolactin 6.8 6.3 171 170 PSA 3.0 2.6 37 32 TPA 48 46 953 957 CA549 8.9 8.9 27 CA 15-3 41 43 165 158 beta hOG 2.7 2.6 475 446 hCG 2.7 2.7 422 414 CA125 18 20 356 360 CEA 2.9 2.6 57 56 IDI 47.7 49.8 51 51 Table V 21 SUBSTITUTE SHEET (RULE 26) WO 9412.4569 WO 9424569PCT1US94/03884 FROZEN STABILITY STUDIES Thirty Days -200 Analyte Level I Level I Level 11 Level 11 2-8C -200 2-8C Aldlos 76 78 840 834 AFP 11 11 300 297 132M 0.98 0.97 4.7 4.8 ACTH 15 17 408 418 Ferritin 31 28 726 686 PAP 2.2 2.1 20 PSA 2.6 2.6 33 33 Prolactin 4.2 4.4 125 133 TPA 56 54 779 854 CA549 9.1 9.9 36 CA 15-3 23 23 100 102 beta hOG 2.6 2.6 426 438 CA125 25 25 448 474 CEA 2.6 2.6 60 61 CA19-9 55 55 276 277 LDH 252 247 756 746 LD-1 48% 48% 51% 51% Table VII 22 SUBSTITUTE SHEET (RULE 26) WO 94/24569 PTU9/38 PCTIUS94/03884 FROZEN STABILITY STUDIES Sevel, Days -200 Level I Analyte Level I Level 11 Level 11 2-8C -20C Calcitonin 101 123 98 110 322 342 313 353 FROZEN STABILITY STUDIES Twenty Four Hours (a -200 Analyte Level 1 19.sQt Level I .gnr Level 11 Level 11 -200

NSE

Table VilI 23 SUBSTITUTE SHEET (RULE 26) WO 94/24569 WO 9424569PCT[US94/03884 INSTRUMENT/METHOD COMPARISON Analvte Method U~nits I Oal I I twifo 'a~

ACTH

Aldosterone Diagnostic Products Incstar RIA Nichols Alegro RIA Clinical Assays Diagnostics Products Clinical Assays Diagnostics Products Hybritech Stratus Hybritech Tandem E Amerlex-M AFP RIA

AFP

Beta-2-microglobulin CA 15.3* CA 19-9* Abbott IMX Pharmacia Byk( Sangtec RIA CIS ELSA Sorin Gammadab Abbott IMX Byk Sangtec RIA Centocor ER CIS ELSA Centocor Hytritech Tandem-R Abbott IMX Abbott RIA Hybritech Stratus H-ybritech Tandem E Roche EIA pg/mL pg/yiL p mo I/L pg/mL pg/mL ng/mL ng/mL ng/mL ng/mL ng/mL mg/i mg/i U/mi U/mL U/mL U/ml.

U/mi U/mi U/mi U/mi.

u/mi ng/mi ng/mi n g/m i ng/mi ng/mL 2.9 5.9 8.5 9.0 10.5 1.0 1.1 30 41 30 45 26 29 51 22 461 466 69 466 875 263 238 324 284 286 4.2 4.4 146 260 178 344 171 221 289 377 108 110 56 94 CA 125* CA 549

CEA

10 4.9 3.9 2.7 2.4 3.5 Table IX 24 SUBSTITUTE SHEET (RUJLE 26) WO 94/24569 WO 9424569PCT/US94/03884 INSTRUMENT/MIETHOD COMPARISON Analvte Mathnrl I Inite I gn1"M ~I I I nu,0t I Ferritin Abbott IMX Clinical Assays (GC) Clinical Assays (GD) Diagnostics Products Clinical Assays Diagnostics Products ng/mL ng/mL ng/mL ng/mL pg/mL pg/mL Gastrin hCG Abbott IMX mIU/mL Clinical Assays mlU/mL Diagnostics Products mlU/mL Diagnostics Products (DA) Stratus Immunoassay mlU/mL Serono mlU/mL 23 23 23 25 172 56 2.4 8.4 2.9 11 5.0 4.2 3.6 2.2 2.8 1.0 760 600 548 623 460 347 387 456 385 94 460 434 453 341 433 beta hCG Abbott IMX Hybritech Tandem-R Stratus Immunoassay Medgenix RIA 100 mlU/mL mIU/mL mlU/mL ng/mL Gamma Enolase By k-S an gte c ug/L Prolactin CIS HPRLK-PR CIS ELSA Clinical Assays Diagnostics Products Hybritech Tandem-E Stratus Immunoassay mlU/L ng/mL ng/mL ng/mL n g/m L ng/mL 70 3.9 below range 3.5 7.1 4.0 4546 54 141 152 152 Table IX (cont.) SUBSTITUTE SHEET (RULE 2% WO 94/24569 WO 9424569PCTIUS94/03884 INSTRUMENT/MIETHOD COMPARISON 9z Anali to MAnthndl I InitQ II Il LAe 3 "nitQ

PAP

PSA

Clinical Assays Hybritech Tandem-E Hybritech Tandem-fl Hybritech Stratus Abbott IMVX Hybritech Stratus Hybritech Tandem-R Byk-Sangtec Diagnostic Products Incstar, 11 RIA ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/lL pg/mL pg/mL 1.1 2.1 2.6 2.6 4.6 8.1 2.4 59 106 978 173 367

TPA

Calcitonin 61 129 CA 15-3, Ca 19-9, CA 125 are trademarks of Centocor Diagnostics, a division of Centocor Table IX (cont.) 26 SUBSTITUTE SHEET (RULE 26)

Claims (5)

1. A control for the determination of tumour markers, said control having enhanced optical clarity after reconstitution, comprising a mixture of: a base material comprising human serum or plasma having reduced lipids, a-plurality of relatively pure tumour markers, and a preservative system for ensuring reconstituted stability of the markers, with the proviso that when one of the tumour markers is PAP, anti-PAP S. antibodies must not be present in amounts that substantially affect the concentration of PAP.

2. The control of claim 1 wherein the control is lyophilized. e

3. The control of claim 1 or claim 2 wherein the serum or plasma is S* essentially free of antibodies to PSA. r*

4. The control of claim 1 wherein the tumour markers comprise at least gastrin, gamma enolase, CA 16-3, and CA 549. S*

5. The control of claim 1 wherein the tumour markers comprise at least PSA wherein the PSA in the control has a reconstituted stability of at least thirty days at about -20 0 C. Dated this 11 April 1997 DADE INTERNATIONAL INC Patent Attorneys for the Applicant ,I PETiR MAXWELL ASSOCIATES 1N1'C'RNA'TION AL S~eAICIEI~ REPO)l' Itraioa ~lcai O PCT/US 94/03884 A. CL.ASSIFICATION OF SUIE'IICMA'ITA IPC 5 G0IN33/96 GOIN33/574 According to International Patent Classification (IPC) or to both national classification and IPC Minimum documentation searched (clawufieation system followed by classification symbols) IPC 5 GOIN Documentation searched other tan minimum documentation to thc extent that such documents are included in the fields searcbed IElccronic data base consulted during the international search (nasme of data base and, where practal, search termsi uised) C. l)OCUMIINTS CONSIDEIREDI TO OFi RELEIVANT Category Citation of document, with indication, where appropriat! ui the relevant passages Relevant to claim No, A EP,A,0 040 058 (CORNING GLASS WORKS) 18 1,2 November 1981 see page 6, line 22 -line 32 ,ee page 10, line 14 -page 11, line 4 see abstract A WO,A,91 16632 (BOARD OF REGENTS, THE 1,2 UNIVERSITY OF TEXAS SYSTEM) 31 October 1991 see page 20, line 10 -line 21 [IFurther documents art listed in the continuation of box C. Patent family members are listed in annex. Specal categories of cited documents, later document published after the International filing date th tof t artwhibor priority datc and not in conflict with the application but document dcflning te general state ofte arhc is not cited to understand the principle or theory underlying the considered to be of partictlar relevance invention earlier document but published on or after the international 'X document of particular relevanct; the claimed inveniton iling date cannot be conidred novel or cannsot be considered to Sdocument which may throw doubts on priority claim(s) or involve an inventive step when the document it taken alone %vhich is cited to establish the publication date of another document of particular relevance; the claimed invention citation or other special reason (as speified) cannot hse considered to involve an inventivestep when the document referring to an oral disclosure, use, exhibition or document 3s combined with one or miore other such docuw other means ment, suchwcmbination being obvious to a person tkilled document published prior to the international iling date but in the art. later "ht the priority date claimed W document membnr of the same patent family Date of the actual completion of the international search Date of mailing of Ut intemautiona'i search report 21 JO 199429, 07, 94 Name andI mailing address of the ISA Authonired officer TeL 31-70) 340-2O40, Tix. 31 6S1 cponrd,Grfih 0 r t ow PC'rl*N2ib liccoind ihttt) ()uiy 1092) pagje 1 of2 INTERNATIONAL SE ARCH- RE PORT Internaltional OPP11Cation No, PCT/US 94/03884 C.(Contintiaton) DOCUMLNTS CONS IDURIII) TO 1311 ILEIVANT Category' Citation of document, with indication, where appropriate, of thic relevant pa.vages [Relevant to claim No. CLINICAL CHEMISTRY., vol.28, no.4, April 1982, WINSTON US pages 709 716 C. B. REIMER ET AL. 'The U.S. national reference preparation for aipha-fetoprotein in mid-pregnancy maternal serum' see the whole document US,A,4 489 167 OCHI ET AL.) 18 December 1984 see abstract WO,A,92 08976 (ONCOMEDICS B. 29 May 1992 see claims 34-36 EP,A,0 351 117 (BAXTER INTERNATIONAL INC.) 17 January 1990 see abstract 1,2 1,2 1,2 1,2 1 rorm PC TISAJ210 (CantinuaitiOn Of kecard £lieei) (July 1992) page 2 of 2 IN IIRNAAIt..fNAL bLAKI.t..1 iA.JeUIk international application No. .nfrmion on patn family membecrs PCT/US 94/03884 Patcnt. document Publication Patent family Publication cited in search report date member(s) Idate EP-A-0040058 18-11-81 CA-A- 1166957 08-05-84 JP-B- 1021909 24-04-89 ~JP-C- 1538808 16-01-90 Z e -4 S Z) JP-A- 57006364 13-01-82 WO-A-9116632 31-10-91 US-A- 5188964 23-02-93 AU-A- 7785091 11-11-91 CA-A- 2079345 13-10-91 EP-A- 0S25115 03-02-93 US-A-4489167 18-12-84 JP-C- 1747022 25-03-93 ~JP-A- 2238000 20-09-90 JP-B- 4028718 15-05-92 JP-C- 1616363 30-08-91 JP-B- 2044030 02-10-90 JIP-A- 58000758 05-01-83 WO-A-9208976 29-05-92 US-A- 5296346 22-03-94 AU-A- 9022791 11-06-92 EP-A-0351117 17-01-90 US-A- 4994375 19-02-91 rPortn PCTIISAJ2IO (patent (amily Wit~) (July 192)