CA2137784A1 - Tumor marker control - Google Patents

Abstract

The present invention provides a stable control serum or plasma for tumor diagnosis, wherein the control contains the tumor markers relevant for the diagnosis of tumors. The serum or plasma has a reduced lipid content. The present invention also provides for a method of making the control.

Description

Tumor Marker Control Field of the Invention This invention relates generally to the field of stable human serum based controls for use in in 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.

Back~round 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 ~roteins 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 radioi."~ l.,oassay, enzyme immunoassay, fluorescence immunoassays and other clinical analysis techniques.
Controlling and monitoring the accuracy, precision, and reliability of these assay systems is critical to ensure that the patient receives the correct treatment and that the results of the assays are medically relevant.
Currently, some human serum based controls are commercially available. Controls, generally, are in levels represen~ing 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, SU8STITUTE SHEET (RULE 26) 2~3~

however, lack many of the tumor markers that are required by the clinical oncologists. Moreover, the cLIllelltly 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 (i.e. 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 tili7ed 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 Tr~dçm~rks of Centocor Diagnostics, a division of Centocor Inc.), CA 549, Carcinoembryonic Antigen (CEA), Ferritin, Sl)BSTITUTE SHEET (RVLE 2~

213778g 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-l. A preservative system should also be include in the control.
The preservative system should include a preservative that is stable both prior to lyophili7~tion 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 microor~nicmc. 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 lyophili7~tion, however, it is useful in controlling microbial growth during the m~nllf~cturing process. The gell~a",icin sulfate and cycloheximide are used to control growth of microorg~nicmc after reconctitntion. 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 ~refeiably 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 elimin~te the PAP from the - control solution. If the antibodies are successfully removed or their effects elimin~ted 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 triglycerides in the human serum or plasma is less than SUBSTITUTE SHEET (RULE 2~

~3~8~

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 dçn~tnred or oxidized is increased when the lipid content is reduced.
This is because when the lipids break down, they form oxidation by-products 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 elimin~ted. The reconstitution time of the lyophilized control is delayed by between about 15 to 30 minutes if serum or plasma cont~ining normal amounts of lipids are ~ltili7~d Third, high levels of lipids can cause interference in measuling 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 ltili7e-l 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 cont~min~ted with other markers or co~t~ ted 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-con~.nil-~tion. In addition, the tumor markers that are SUBSTITUTE SHEE~ tRULE 26) wo 94/24s69 21 3 7 7 8 4 PCT/US94/03884 to be added to the base material should be assayed to detelmine the presence of cross-cont~min~nts 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 rli~filtered into an a~,pr(~ iate buffer and concentrated. The B2M, a ~roteil~, 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 cont~ining 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 immlmoassay. 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 illlpu~ilies of immllnoglobulins 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 co~ tion 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 sc~ ed. Both of these markers are shed into the serum during turnor growth and due to the similarities of these markers the separation of them is diffficult. 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 rem~ining 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 (RVLE 26~

WO 94t24569 PCT/US94/03884 2~3~a~
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 lg/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 glycoproteill 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 ga~lrointestinal 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 20 times. The concentrated supem~t~nt 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 SUBSTITUTE SHEET (RULE 26~

WO 94/24569 2 1 3 7 7 8 ~ PCT/US94/03884 tre~tm~nt 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 conce~ ted~ buffer exchanged supçrn~t~nt is applied onto a Phenyl Sepharose column. As described for the B2M purification, the TPA
binds 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 detelmilled 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, C.H.J., et al. Immunoadsorbent Purification of Carcinoembryonic Anti~en usin~ A Monoclonal Antibody: A Direct Comparison with a Conventional Method. Tumor Biol. Vol. 8: pages 241-250 (1987), a colurnn is prepared which contains a media that has an antibody specific to CEA attached to the chromatography media. This colurnn 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 this method.
Since the CEA is not required to be obtained from this method, it is ~le~led to combine the fractions from the Phenyl Sepharose column and then wash the Phenyl Sepharose column with a phosphate buffer, prefe,~bly 50 mM phosphate at pH 7.2, cont~inin~ 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 column is next eluted with the same buffer but also cont~ining about 6 M urea. The TPA is eluted and collected.
The CA l9-9/CEA cont~ining 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 SUBSTITUTE StlEET (RULE 26~

WO 94/24569 ; PCTIUS94/03884 ~3~4 aliquots of the concentrate for the presence of CEA by immunoassay techniques. The approximate recovery can be up to 100%.
~ lt~rn~tively, the fractions cont~inin~ the CEA/CA 19-9 can be discarded. Then, only the fractions cont~inin~ 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%. Cross-co..~ tion is dete~ ed using immunoassay techniques.
The CEA can be obtained as described above using the monoclonal antibody method or it may be obtained from other comrnercially available sources. The CEA should be tested for cross-cont~min~tion with immunoassay methods prior to use in a control. If col~ tion 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 frorn fresh or freshly frozen human brain.
Purified NSE may be obtained comrnercially. The ~le~,led method for purification is accomplished by preparing a homogenate of the brain, centrifuging the homogenate and collecting the supern~t~nt 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 conce~ ed. The concentrate is chromatographed on DE-52 and eluted with a 0.15 M -0.35 M NaCl gradient. The peak cont~inin~ 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 m~ n~nt hepatoma and in some cases of ovarian and testicular cancers The best source of this antigen is human cord serum collected at the tirne of birth. This serum contains high levels of AFP (about 60,000 ng/mL) and contains only one cont~min~ting tumor marker, Prolactin.

SUBSTITUTE SHEE~ (RULE 26) WO 94/24569 213 7 7 8 4 PCTtUS94tO3884 There are methods for purifying AFP described in the art. For instance, Chudy D. and Zizkovsky V., A simple and rapid method for the isolation of human alpha-fetoprotein from human cord serum, Neoplasma 34 (4) 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 lmg/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 cont~min~nt is not a problem since the serum or plasma based material contains albumin. The purified AFP can be tested using immlmoassay procedures.
The other tumor markers such as ACTH, aldosterone, hCG, beta-hCG, CA 15-3, CA 549, Calcitonin, Ferritin, Gastrin, PAP, PSA, Prolactin and LD-l 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, cross-cont~min~tion can be assessed by immlmoassay techniques. The LD-1 is added as a component of LDH by delellllillillg the amount of LD-l 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 SUBSTITUTE SHEET (RULE 26) 2~3`1'1 a4 Table IV, then adding the a~propliate 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-l9-9, 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, ~mm~ 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 lyophili7e~ using standard methods. The bottles cont~ining the lyophilized controls are sealed under vacuum and then stored at about 4C. The controls are reconstituted with water or other appropliate liquids such as buffers.
For ACTH, lyophilization studies are required to delellnine 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 specific post lyophili7~tion level of ACTH.
The stability of all of the markers in the lyophilized control was detell,~ ed 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 markers except ACTH, 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 -20C.

SlJBSTITUTE SHEET (RULE 2 wo s4/24s69 21 3 7 7 8 9 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 -20C.
See, Table VIII.
The following Examples are given for the purpose of illustrating the present invention:

Example 1 Purification of B2M
A urine concentrate wa$ pre~a~ed 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 50 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 lJloleills. Fractions cont~ining the B2M were combined, pooled and concentrated to about 1 g/dL.
Purification has also been accomplished on Superdex 75 (Ph~ cia). 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 SWl 116, 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 SUBSTITUTE SHEET (RULE 26) 2~3~a~

washed with the buffer and fractions were collected. The fractions were evaluated for CEA and CA 19-9 activity using an immunoassay.
Fractions co~ g CEA were pooled and concentrated and fractions CO..~ g CA 19-9 were pooled and concentrated. A buffer cont~ining about 50 mM phosphate at pH 7.2 with increasing amounts of urea was applied to the column. The TPA was eluted with urea at about 6M. The TPA cont~ining 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 cont~min~tes 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 12S, CA 549, calcitonin, CEA, Ferritin, gastrin, NSE, PAP, PSA, Prolactin, TPA, and LD-l using immllnoassay techniques.
Each of the markers were obtained through purifications as described herein or were obtained commercially. The amount of each - marker was detellnilled. An amount of each marker necessary to reach the values given in 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 SUBSTITUTE S~IEET (RULE 2~) WO 94/24569 21 3 7 7 8 ~ PCT/US94/03884 milliliter aliquots from Pool 2 (Level 3) were 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.

SUBSTITUTE SHEET (RULE 26) 2~,3rl ~
CLASSIFICATION OF TUMOR MARKERS

1. Oncofetal Antigens AFP, CEA
Produced during fetal development and low levels in adults. Tumors cause re-expression 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 85% carbohydrate.

3. Hormones hCG, ACTH, Calcitonin, Prolactin, Aldosterone, Gastrin 4. Serum Proteins beta2-microglobulin, ferritin 5. Enzymes PAP, NSE, LD1 S4BSTITUTE SHEFr (RULE 26 WO 94/24569 21 3 7 7 8 ~ PCT/US94/03884 TUMOR MARKER CONTROL
CLINICAL MARKERS

TUMOR MARKER SITE(S) Adenocortitropic Hormone Lung IACTH) Alphafetoprotein (AFP) Testicular, Liver Aldosterone Kidney Beta 2 Microglobulin Bone Marrow Beta Human Chorionic Gynecological, Gonadotropin testicular CA 15-3 / CA 549 Breast CA 19-9 Pancreas, Colorectal, Stomach CA 125 Ovarian Carcinoembryonic Antigen Colorectal, Breast, Lung, (CEA) Stomach, Pancreas Table ll SUBSTITUTE SHEE~ (RULE 26) 2~3rl1~4 TUMOR MARKER CONTROL
CLINICAL MARKERS

TUMOR MARKER SITE(S) Ferritin Liver Gamma Enolase Lung, Brain Gastrin Pancreas Human Chorionic Testicular Gonadotropin (hCG) Lactate Dehydrogenase Brain Isoenzyme (LD-1) Prolactin Pituitary Prostatis Acid Phosphatase Prostate (PAP) Prostate Specific Antigen Prostate (PSA) Tissue Polypeptide Antigen Bladder, Prostate, (TPA) Gynecological, Lung Table ll (cont.) SUBSTITUTE SHEET (RIJLE 26 wo 94/24569 2 1 3 7 7 8 ~ PCr/USs4/03884 TUMOR MARKER CONTROL

ANTIGENS SOURCE

B2 Microglobulin Renal failure urine CA 15-3 / CA 549 Breast ascites, Pleurol fluid, Hybritech mouse tumor CA 19-9 SW1 1 16 Supernate CEA SW1116 Supernate TPA SW1116 Supernate Ovarian cancer ascites Pleural fluids CA 125 Ovarian cancer ascites Pleural fluid - breast Prolactin Human cord serum Alpha-fetoprotein Cord serum Table lll SlJBSTlTVTE SHEET (RVLE 261 g~ 94/24569 PCT/US94/03884 9 ~3~rl f ~ ~ ~ ~ _ _ _ _ _ a~ O O O O O O O O O
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ACCELERATED STABILITY STUDIES
Four weeks ~37C

Analyte Level I Level I Level ll Level ll fresh 37C fresh 37C
AFP 10.2 10.3 293 293 Aldos 79 74 800 780 B2M 1.0 0.97 4.2 4.2 Gastrin 77 72 289 275 Calcitonin 38 35 270 275 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 CA549 8.9 8.6 27 30 beta hCG 2.7 2.8 475 446 hCG 2.7 2.7 460 439 CEA 2.9 2.9 57 57 Table V

45SUBST~TUTE SHEET (RULE 26) 213778 i WO 94/24569 ^ PCTtUS94/03884 RECONSTITUTED STABILITY STUDIES
Fourteen days ~i) 2-8C

Analyte Level I Level I Level ll Level ll fresh 14 Davs fresh 14 Days AFP 10.2 10.6 293 294 B2M 1.1 1.0 4.2 4.4 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 CA549 8.9 8.9 27 30 beta hCG 2.7 2.6 475 446 hCG 2.7 2.7 422 414 CEA 2.9 2.6 57 56 LD1 47.7 49.8 51 51 Table Vl SUBSTITUTE SHEET (RULE 26 2~3~

FROZEN STABILITY STUDIES
Thirtv Days ~ -20C

Analyte Level I Level I Level ll Level ll Aldos 76 78 840 834 B2M 0.98 0.97 4.7 4.8 Ferritin 31 28 726 686 PAP 2.2 2.1 20 20 PSA 2.6 2.6 33 33 Prolactin 4.2 4.4 125 133 CA549 9.1 9.9 36 35 beta hCG 2.6 2.6 426 438 CEA 2.6 2.6 60 61 LD-1 48% 48% 51 % 51 %

Table Vll SUBSTITUTE SHEET (RULE 26 FROZEN STABILITY STUDIES
Seven Days ~D -20C

Analyte Level I Level I Level ll Level ll Gastrin 101 98 322 313 Calcitonin 123 110 342 353 FROZEN STABILITY STUDIES
Twentv Four Hours ~ -20C

Analyte Level I Level I Level ll Level ll Table Vlll SUBSTITUTE SHEET (RVLE 26) ~311~

INSTRUMENT/METHOD COMPARISON

AnalYte Method Units Level 1 Level 3 ACTH Diagnostic Products pg/mL 33 461 Incstar RIA pg/mL 14 466 Nichols Alegro RIA pmol/L 14 69 Clinical Assays pg/mL 20 466 Aldosterone Diagnostics Products pg/mL 79 875 AFP Clinical Assays ng/mL 2.9 263 Diagnostics Products ng/mL 5.9 238 Hybritech Stratus ng/mL 8.5 324 Hybritech Tandem E ng/mL 9.0 284 Amerlex-M AFP RIA ng/mL 10.5 286 Beta-2-microglobulin Abbott IMX mg/L 1.0 4.2 Pharmacia mg/L 1.1 4.4 CA 15-3* Byk Sangtec RIA U/mL 30 146 CIS ELSA U/mL 41 260 Sorin Gammadab U/mL 30 178 CA 19-9* Abbott IMX U/mL 45 344 Byk Sangtec RIA U/mL 26 171 Centocor ER U/mL 29 221 CIS ELSA U/mL 51 289 CA 125* Centocor U/mL 22 377 CA 549 Hytritech Tandem-R u/mL 10 30 CEA Abbott IMX ng/mL 4.9 108 Abbott RIA ng/mL 3.9 110 Hybritech Stratus ng/mL 2.7 56 Hybritech Tandem E ng/mL 2.4 60 Roche EIA ng/mL 3.5 94 Table IX

SUBSTITUTE SHEET (RULE 2 WO 94/24569 21 3 7 7 8 ~ PCT/US94/03884 INSTRUMENT/METHOD COMPARISON

Analyte Method Units Level 1Level 3 rer,ilin Abbott IMX ng/mL 23 760 Clinical Assays (GC) ng/mL 23 600 Clinical Assays (GD) ng/mL 23 548 Diagnostics Products ng/mL 25 623 Gastrin Clinical Assays pg/mL 172 460 Diagnostics Products pg/mL 56 347 hCG Abbott IMX mlU/mL 2.4 387 Clinical Assays mlU/mL 8.4 456 Diagnostics Products mlU/mL 2.9 385 Diagnostics Products (DA) 11 94 Stratus Immunoassay mlU/mL 5.0 460 Serono mlU/mL 4.2 434 beta hCG Abbott IMX mlU/mL 3.6 453 Hybritech Tandem-R mlU/mL 2.2 341 Stratus Immunoassay mlU/mL 2.8 433 Medgenix RIA 100 ng/mL 1.0 3.5 Gamma Enolase Byk-Sangtec ug/L 14 65 Prolactin CIS HPRLK-PR mlU/L 70 4546 CIS ELSA ng/mL 3.9 54 Clinical Assays ng/mL below range90 Diagnostics Products ng/mL 3.5 141 Hybritech Tandem-E ng/mL 7.1 152 Stratus Immunoassay ng/mL 4.0 152 Table IX (cont.) SUBSTITUTE SHEET (RULE 2 z~3~

INSTRUMENT/METHOD COMPARISON

AnalYte Method Units Level 1 Level 3 PAP Clinical Assays ng/mL 1.1 16 Hybritech Tandem-E ng/mL 2.1 19 Hybritech Tandem-R ng/mL 2.6 24 Hybritech Stratus ng/mL 2.6 17 PSA Abbott IMX ng/mL 4.6 59 Hybritech Stratus ng/mL 8.1 106 Hybritech Tandem-R ng/mL 2.4 35 TPA Byk-Sangtec ng/mL 54 978 Calcitonin Diagnostic Products pg/mL 61 173 Incstar, ll RIA pg/mL 129 367 * CA 15-3, Ca 19-9, CA 125 are trademarks of Centocor Diagnostics, a division of Centocor Table IX (cont.) SUBSTITUTE SHEE~ (RULE 26

Claims (2)

We claim:

1. A control for the determination of tumor markers comprising a mixture of :
(a) a base material comprising human serum or plasma having reduced lipids wherein the serum or plasma is essentially free of antibodies to PAP;
(b) a plurality of tumor markers.

2. The control of claim I wherein said control is lyophilized.