CA2154794A1 - Non-denaturing potency assay for bovine somatotropin - Google Patents
Non-denaturing potency assay for bovine somatotropinInfo
- Publication number
- CA2154794A1 CA2154794A1 CA002154794A CA2154794A CA2154794A1 CA 2154794 A1 CA2154794 A1 CA 2154794A1 CA 002154794 A CA002154794 A CA 002154794A CA 2154794 A CA2154794 A CA 2154794A CA 2154794 A1 CA2154794 A1 CA 2154794A1
- Authority
- CA
- Canada
- Prior art keywords
- bovine somatotropin
- biologically active
- bst
- sample
- size exclusion
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108010006025 bovine growth hormone Proteins 0.000 title claims abstract description 210
- 238000003556 assay Methods 0.000 title description 14
- 238000000034 method Methods 0.000 claims abstract description 59
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 50
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 50
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 claims abstract description 43
- 229920000642 polymer Polymers 0.000 claims abstract description 13
- 239000002245 particle Substances 0.000 claims abstract description 12
- 239000007864 aqueous solution Substances 0.000 claims abstract description 8
- 241000283690 Bos taurus Species 0.000 claims abstract description 7
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 27
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 11
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 11
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 11
- 239000001099 ammonium carbonate Substances 0.000 claims description 11
- 238000000926 separation method Methods 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 10
- 239000012062 aqueous buffer Substances 0.000 claims description 7
- 239000011148 porous material Substances 0.000 claims description 6
- 238000011088 calibration curve Methods 0.000 claims description 2
- 108010051696 Growth Hormone Proteins 0.000 abstract description 6
- 102000018997 Growth Hormone Human genes 0.000 abstract description 6
- 239000012071 phase Substances 0.000 description 30
- 230000007717 exclusion Effects 0.000 description 16
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- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 10
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- 238000010828 elution Methods 0.000 description 9
- 239000000499 gel Substances 0.000 description 9
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 6
- 239000000463 material Substances 0.000 description 6
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- 238000004128 high performance liquid chromatography Methods 0.000 description 5
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- 239000000126 substance Substances 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
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- 239000003398 denaturant Substances 0.000 description 2
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- 239000000122 growth hormone Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
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- 241000894006 Bacteria Species 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
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- 150000001413 amino acids Chemical class 0.000 description 1
- -1 aromatic amino acids Chemical class 0.000 description 1
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- 244000309464 bull Species 0.000 description 1
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- 150000001875 compounds Chemical class 0.000 description 1
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- 238000004925 denaturation Methods 0.000 description 1
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- 230000003292 diminished effect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000762 glandular Effects 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- 210000003635 pituitary gland Anatomy 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
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- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/262—Synthetic macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
- B01J20/267—Cross-linked polymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28004—Sorbent size or size distribution, e.g. particle size
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
- B01J20/285—Porous sorbents based on polymers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/291—Gel sorbents
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N2030/524—Physical parameters structural properties
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- 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
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- 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
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Abstract
Described is a method for determining the potency of bovine somatrotopin. The level of biologically active bovine somatotropin protein in the bovine somatotropin sample is measured by size exclusion HPLC employing as the stationary phase a hydrophilic porous polymer gel having an average particle diameter of about 5 µm to about 15 µm and as a mobile phase a buffered aqueous solution having a pH of about 8 to about 12 and which is non-denaturing to the boving somatotropin sample. The potency of the bovine somatotropin sample is determined based upon the level of biologically active bovine somatotropin protein so measured. The method provides an expedient, precise and accurate measure of the potency of a bST sample.
Description
~ WO94/17~3 21 S 47 9 ~ PCT~S94/00850 NON-DENA~JRING POT~NCY ASSAY FOR
~OVINE SOMATOTROPIN
BACKGROUND
The present invention generally relates to bovine somatotropin (bST). More particularly, the present invention relates to a non-denaturing method for separating a biologically active bST protein fraction from bST in a manner suitable to provide a bST potency assay.
The production of bST in large scale has recently been fostered by recombinant DNA technology. Recombinant microorganisms such as recombinant Escherichia coli produce insoluble granules of bST in their cytoplasm.
These granules, known as refractile bodies or inclusion bodies, contain aggregated denatured bST. The refractile bodies are recovered and usually treated with a denaturant such as guanadine hydrochloride, sodium dodecyl sulfate or urea. The denaturant unfolds and solubilizes the improperly folded bST molecules.
Afterward, the bST molecules are renatured to form the properly folded, biologically active bST monome~ic protein.
Due to inefficiencies of these denaturing and renaturing steps, however, some aggreyates of improperly folded, biologically inactive bST aggregates are also formed. Thus, the bST bulk material obtained contains both the biologically active bST monomer and the biologically inactive bST aggregates. It is therefore WO94/17~3 PCT~S94/00850 ~1~47~ ~
important to establish a method for determining t~le potency of bST in quality control.
The potency of bST has previously been estimated by a rat weight gain method. Essentially, the weight gain of rats to which bST samples are administered i~ monitored, and from this data a value representing the potency of the bST is obtained. However, this assay cannot be employed to accurately quantify bST in routine analysis.
bST potency has also been determined by radio receptor assay (RRA). However, RRA is time consuming. Also, RRA
is inaccurate, and thus several tests are usually performed and the results averaged to provide a bST
potency value.
Reversed-phase high performance liquid chromatography (RPHPLC) has been employed to determine proteins.
However, most RPHPLC methods which have been used are not appropriate candidates for measuring the potency of bST
because they have employed acidic mediums and organic solvents in the mobile phase, which denature bST.
Size exclusion high performance liquid chromatography ("size exclusion HPLC") using a mobile phase containing sodium dodecyl sulfate or guanadine hydrochloride has previously been employed to determine bST. However, such methods are not bio-potency assays because their mobile phases are denaturing to bST.
What is tllerefore needed is an expedient, precise and accurate non-denaturing assay for determining the bio-potency of bST. The present invention addresses these needs.
~ WO94/17~3 215 4 7 9 4 PCT~S94100850 SUMMARY OF THE INVENTION
One preferred embodiment oE the present invention provides a method for determining the potency oE a bovine somatotropin sample. This method cornprises measuring the level of biologically active bovine somatotropin protein in the bovine somatotropin sample by size exclusion HPLC
employing as a solid phase a hydrophilic porous polymer gel having an average particle diameter of about 5 ~m to about l5 ~m and as a mobile phase a buffered aqueous solution having a pH of about 8 to about 12 and which is non-denaturing to the bovine somatotropin sample. The potency of the bovine somatotropin sample is determined based upon the level of biologically active bovine somatotropin protein measured in the sample.
Another preferred embodiment of the present invention provides a method for separating a biologically active bovine somatotropin fraction from bovine somatotropin.
The method comprises separatiny a biologically active bovine somatotropin fraction from bovine somatotropin by size exclusion EIPLC employing as a solid phase a hydrophilic porous polymer yel having an average particle size of about 5 ~m to about 15 ~m and as a mobile phase a buffered aqueous solution having a pH of about 8 to about 12 and which is no11-denatl1ring to the bovine somatotropin.
Another preferred embodiment of the invention provides a method for treating bulk recombinant bovine somatotropin to separate biologically active bovine somatotropin protein from biologically inactive bovine 30 somatotropin non-covalent soluble agyregates contained in the bulk recombinant bovine somatotLopin. The method comprises the step of subjecting the bulk recombinant bovine somatotropin to size exclusion H~LC employing as a solid phase a hydrophilic porous polymer gel havin~ an average particle size of about 5 ~m to about 15 ~m W O 94/17403 21 S ~ ~ 9 4 PCT~US94/00850 and as a mobile phase a buffered aqueous solution having a pH of about 8 to about 12 and which is non-denaturing to bovine somatotropin, so as to separate the bioloyically active bovine somatotropin from the biologically inactive bovine somatotropin non-covalent soluble aggregates.
The invention provides methods that achieve advantageous separations of biologically active bST
protein from biologically inactive bST forms. The methods are well adapted to serve in expedient, precise and accurate assays for determining of the potency of bST
samples. Additionally, for the first time, the separation and extensive characterization of bST
non-covalent soluble aggregates have been achieved using the inventive methods. Additional objects, features and advantages of the invention will be apparent from the description herein.
~ WO94/17~3 21~ ~ 7 9 4 PCT~S94/00850 DESCRIPTION OF THE FIGUR~S
Figure lA is a size exclusion HPLC chrolnatograln of a biologically active bST reference standard using the methodology described in ~he Examples, infra.
Figures l~ through lE are size exclusion 11PLC
chromatograms of bulk recombinant bST materials usi11g the metllodology described in the Examples, infra. and demonstrating the presence of bST soluble aggregates in the bulk materials.
Figure 2A is a graph showing the effect of bicarbonate buffer concentration on the size exclusion HPLC retention time of t11e biologically active ~ST
protein fraction usiny the methodology described in the Examples, in~ra.
Figure 2B is a graph showing the effect of bicarbonate buffer concentration on the size exclusion HPLC peak area response of the bioloyically active bST
protein fraction under conditions described in the Examples, infra.
Figure 3A is a graph showing the effect of mobile phase pH variation on the size exclusion HPLC elution time of the biologically active ~ST protein fraction using the methodology described in the Exam~les, infra.
Figure 3B a graph showing is the effect of mobile phase pH variation on t~1e size exclusion HPLC peak area response of ~he biologically active b~T protein fraction using the methodology descri~ed in the Examples, infra.
W094/17~3 ~ 2lS 47 9 ~ PCT~S94/00850 ~
Figure 4 is a graph showing the correlation between radio-receptor assay ~o~ency data (IU/mg) and the size exclusion HPLC-nleasured level of ~iologically active bST
protein for several different lots of bulk recombinant bST under the conditions described in the Examples, infra.
Figure 5 is a size exclusion HPLC calibration curve of log MW vs. elution time for standard proteins and the biologically active bST protein ("B/A bST~) and bST
soluble aggregate ("Agg.") under conditions described in the Examples, infra.
Figure 6 is a ~raph showing the effect of ammonium bicarbonate concentration on the size exclusion HPLC peak area response for the biologically active bST protein fraction ("B/A bST") and for the fraction including the bST soluble aggregate ("Agg.") under conditions described in the Examples, infra.
Figure 7 is a graph showing t11e effect of mobile phase pH on the size exclusion HPLC peak area for the biologically active bST protein fraction ("B/A bST") an~
for the fraction including the bST soluble aggregate ("Agg.) under the conditions described in the Examples, infra.
Figure 8 is a graph showing the effect of centrifuging on the size exclusion HPLC peak area oE the biologically active bST protein fraction ("B/A bST") and of the fraction including the bST soluble aggregate ("Agg.") under the conditions described in the Examples, infra.
~ WO94117~3 21~ 4 7 9 ~ PCT~S94/00850 DESCRIPTION OF THE PR~FERRED EMRODIMENT
For the purposes of promoti11y an understanding oL the principles of the invention, reEerence will now be made to a preferred embodiment thereof and specific lanyl1age will be used to describe the same. lt will nevert11eless be understood that no limitation of the scope of the invention is thereby intended, such alterations, further modifications and applications of tlle principles of the invention as illustrated herein being contemplated as would normally occur to one skilled in the art to which the invention relates.
As indicated above, a preferred en1bodiment of the present invention relates to a method for separating a biologically active bovine somatotropin fraction from bovine somatotropin. As used herein, "bovine somatotropin" denotes a substance, of natural or synthetic origin, which exllibits the properties of natural bovine somatotropin. The bovine somatotropin can be extracted from appropriate glandular tissues of bovines, e.g. pituitary glands; or, it is now well established practice to syn~hesize bovine somatotropin and otller such substances by the use of genetically-modified microorganisms such as bacteria. It is oftentimes convenient or even preferred that such processes yield a modified bovine somatotropin, that is, a substance that differs as to its structure from the naturally occurring growth hormone, but which retaillS the biological activity of the naturally occurring growth hormone. For example, a modified bovine somatotropin may contain one or more additional amino acids, at one or both ends of the polypeptide chai1l. Additional modifications will be understood by those skilled in the art. Therefore, the term bovine somatotropin is used throughout this document to ~-efer to both naturally WO94/17~3 PCT~S94/00850 ~
~OVINE SOMATOTROPIN
BACKGROUND
The present invention generally relates to bovine somatotropin (bST). More particularly, the present invention relates to a non-denaturing method for separating a biologically active bST protein fraction from bST in a manner suitable to provide a bST potency assay.
The production of bST in large scale has recently been fostered by recombinant DNA technology. Recombinant microorganisms such as recombinant Escherichia coli produce insoluble granules of bST in their cytoplasm.
These granules, known as refractile bodies or inclusion bodies, contain aggregated denatured bST. The refractile bodies are recovered and usually treated with a denaturant such as guanadine hydrochloride, sodium dodecyl sulfate or urea. The denaturant unfolds and solubilizes the improperly folded bST molecules.
Afterward, the bST molecules are renatured to form the properly folded, biologically active bST monome~ic protein.
Due to inefficiencies of these denaturing and renaturing steps, however, some aggreyates of improperly folded, biologically inactive bST aggregates are also formed. Thus, the bST bulk material obtained contains both the biologically active bST monomer and the biologically inactive bST aggregates. It is therefore WO94/17~3 PCT~S94/00850 ~1~47~ ~
important to establish a method for determining t~le potency of bST in quality control.
The potency of bST has previously been estimated by a rat weight gain method. Essentially, the weight gain of rats to which bST samples are administered i~ monitored, and from this data a value representing the potency of the bST is obtained. However, this assay cannot be employed to accurately quantify bST in routine analysis.
bST potency has also been determined by radio receptor assay (RRA). However, RRA is time consuming. Also, RRA
is inaccurate, and thus several tests are usually performed and the results averaged to provide a bST
potency value.
Reversed-phase high performance liquid chromatography (RPHPLC) has been employed to determine proteins.
However, most RPHPLC methods which have been used are not appropriate candidates for measuring the potency of bST
because they have employed acidic mediums and organic solvents in the mobile phase, which denature bST.
Size exclusion high performance liquid chromatography ("size exclusion HPLC") using a mobile phase containing sodium dodecyl sulfate or guanadine hydrochloride has previously been employed to determine bST. However, such methods are not bio-potency assays because their mobile phases are denaturing to bST.
What is tllerefore needed is an expedient, precise and accurate non-denaturing assay for determining the bio-potency of bST. The present invention addresses these needs.
~ WO94/17~3 215 4 7 9 4 PCT~S94100850 SUMMARY OF THE INVENTION
One preferred embodiment oE the present invention provides a method for determining the potency oE a bovine somatotropin sample. This method cornprises measuring the level of biologically active bovine somatotropin protein in the bovine somatotropin sample by size exclusion HPLC
employing as a solid phase a hydrophilic porous polymer gel having an average particle diameter of about 5 ~m to about l5 ~m and as a mobile phase a buffered aqueous solution having a pH of about 8 to about 12 and which is non-denaturing to the bovine somatotropin sample. The potency of the bovine somatotropin sample is determined based upon the level of biologically active bovine somatotropin protein measured in the sample.
Another preferred embodiment of the present invention provides a method for separating a biologically active bovine somatotropin fraction from bovine somatotropin.
The method comprises separatiny a biologically active bovine somatotropin fraction from bovine somatotropin by size exclusion EIPLC employing as a solid phase a hydrophilic porous polymer yel having an average particle size of about 5 ~m to about 15 ~m and as a mobile phase a buffered aqueous solution having a pH of about 8 to about 12 and which is no11-denatl1ring to the bovine somatotropin.
Another preferred embodiment of the invention provides a method for treating bulk recombinant bovine somatotropin to separate biologically active bovine somatotropin protein from biologically inactive bovine 30 somatotropin non-covalent soluble agyregates contained in the bulk recombinant bovine somatotLopin. The method comprises the step of subjecting the bulk recombinant bovine somatotropin to size exclusion H~LC employing as a solid phase a hydrophilic porous polymer gel havin~ an average particle size of about 5 ~m to about 15 ~m W O 94/17403 21 S ~ ~ 9 4 PCT~US94/00850 and as a mobile phase a buffered aqueous solution having a pH of about 8 to about 12 and which is non-denaturing to bovine somatotropin, so as to separate the bioloyically active bovine somatotropin from the biologically inactive bovine somatotropin non-covalent soluble aggregates.
The invention provides methods that achieve advantageous separations of biologically active bST
protein from biologically inactive bST forms. The methods are well adapted to serve in expedient, precise and accurate assays for determining of the potency of bST
samples. Additionally, for the first time, the separation and extensive characterization of bST
non-covalent soluble aggregates have been achieved using the inventive methods. Additional objects, features and advantages of the invention will be apparent from the description herein.
~ WO94/17~3 21~ ~ 7 9 4 PCT~S94/00850 DESCRIPTION OF THE FIGUR~S
Figure lA is a size exclusion HPLC chrolnatograln of a biologically active bST reference standard using the methodology described in ~he Examples, infra.
Figures l~ through lE are size exclusion 11PLC
chromatograms of bulk recombinant bST materials usi11g the metllodology described in the Examples, infra. and demonstrating the presence of bST soluble aggregates in the bulk materials.
Figure 2A is a graph showing the effect of bicarbonate buffer concentration on the size exclusion HPLC retention time of t11e biologically active ~ST
protein fraction usiny the methodology described in the Examples, in~ra.
Figure 2B is a graph showing the effect of bicarbonate buffer concentration on the size exclusion HPLC peak area response of the bioloyically active bST
protein fraction under conditions described in the Examples, infra.
Figure 3A is a graph showing the effect of mobile phase pH variation on the size exclusion HPLC elution time of the biologically active ~ST protein fraction using the methodology described in the Exam~les, infra.
Figure 3B a graph showing is the effect of mobile phase pH variation on t~1e size exclusion HPLC peak area response of ~he biologically active b~T protein fraction using the methodology descri~ed in the Examples, infra.
W094/17~3 ~ 2lS 47 9 ~ PCT~S94/00850 ~
Figure 4 is a graph showing the correlation between radio-receptor assay ~o~ency data (IU/mg) and the size exclusion HPLC-nleasured level of ~iologically active bST
protein for several different lots of bulk recombinant bST under the conditions described in the Examples, infra.
Figure 5 is a size exclusion HPLC calibration curve of log MW vs. elution time for standard proteins and the biologically active bST protein ("B/A bST~) and bST
soluble aggregate ("Agg.") under conditions described in the Examples, infra.
Figure 6 is a ~raph showing the effect of ammonium bicarbonate concentration on the size exclusion HPLC peak area response for the biologically active bST protein fraction ("B/A bST") and for the fraction including the bST soluble aggregate ("Agg.") under conditions described in the Examples, infra.
Figure 7 is a graph showing t11e effect of mobile phase pH on the size exclusion HPLC peak area for the biologically active bST protein fraction ("B/A bST") an~
for the fraction including the bST soluble aggregate ("Agg.) under the conditions described in the Examples, infra.
Figure 8 is a graph showing the effect of centrifuging on the size exclusion HPLC peak area oE the biologically active bST protein fraction ("B/A bST") and of the fraction including the bST soluble aggregate ("Agg.") under the conditions described in the Examples, infra.
~ WO94117~3 21~ 4 7 9 ~ PCT~S94/00850 DESCRIPTION OF THE PR~FERRED EMRODIMENT
For the purposes of promoti11y an understanding oL the principles of the invention, reEerence will now be made to a preferred embodiment thereof and specific lanyl1age will be used to describe the same. lt will nevert11eless be understood that no limitation of the scope of the invention is thereby intended, such alterations, further modifications and applications of tlle principles of the invention as illustrated herein being contemplated as would normally occur to one skilled in the art to which the invention relates.
As indicated above, a preferred en1bodiment of the present invention relates to a method for separating a biologically active bovine somatotropin fraction from bovine somatotropin. As used herein, "bovine somatotropin" denotes a substance, of natural or synthetic origin, which exllibits the properties of natural bovine somatotropin. The bovine somatotropin can be extracted from appropriate glandular tissues of bovines, e.g. pituitary glands; or, it is now well established practice to syn~hesize bovine somatotropin and otller such substances by the use of genetically-modified microorganisms such as bacteria. It is oftentimes convenient or even preferred that such processes yield a modified bovine somatotropin, that is, a substance that differs as to its structure from the naturally occurring growth hormone, but which retaillS the biological activity of the naturally occurring growth hormone. For example, a modified bovine somatotropin may contain one or more additional amino acids, at one or both ends of the polypeptide chai1l. Additional modifications will be understood by those skilled in the art. Therefore, the term bovine somatotropin is used throughout this document to ~-efer to both naturally WO94/17~3 PCT~S94/00850 ~
2~s~794 -occurring bovine somatotropin as well as synthetically produced bovine somatotropin which s~lares the bioloyical properties of naturally occurring ~ovine somatotropin, and which may be identical or which may vary as to structure.
The present invention is preferred for use wi~ll bs~r produced by recombinant DNA technology ("recombinant bST"). Typically, the recombinant bST is obtained by isolating and denaturinc3 and solubilizing inclusion bodies which contain the recombinant bST. The bST is then renatured to form the biologically active bST
protein. During the denaturing/renaturing steps, biologically inactive bST non-covalent aggregates are formed along with the desired biologically active bST
protein. Colloids and some very ~ligh molecular weiyht impurities can be removed from the solubilized inclusion bodies by ultrafiltration. However, substantial quantities of biologically inactive bST non-covalent aggregates usually pass throug~l the ultrafilter along with the biologically active bST protein. As a result, the finally-obtained bulk recombinant bST contains ~iologically active ~ST protein as well as biologically inactive bST soluble aggregates. The method of the present invention can be used to treat this bulk recombinant bST to separate the biologically active bST
protein from the biologically inactive bST non-covalent soluble aggregates. Further, this separation is achieved in a manner which can be used to provide an expedient, precise and accurate assay for the potency of the bulk bST.
Two examples of recombinant bSTs are the compounds known as somidobove and sometribove. The present inven~ion is especially preferred for application to these two recombinant bSTs.
l'he method of the invention employs size exclusion HPLC usin~ as the solid phase a hydrophilic porous ~ WO94/17~3 2I ~ 4 79 ~ PCT~S94/00850 _g_ polymeric gel. Preferred porous gels for use in the inventioll have an average particle diameter of about 5~m to about 15~m, and more preferably a~out lO~m to about 15~m. Typically, the average pore diameter of the gel will be about lOU to about 200 angstroms, and for the present inventiorl a gel haviny an average pore diameter of about 150 angstromS is preferred. In another aspect, the porous gel used in the invention preferably has an exclusion limit average molecular weight ~reater than the molecular weight of the bST non-covalent soluble aggregate, e.g. about 106 or greater, and more preferably about 10 or greater.
The mobile phase for use in t}le present invention is an aqueous buffer solution having a pH of about 8 to about 12 and which does not denature the bST sample.
Generally, the buffer may be included in tlle mo~ile phase at any effective concentration so long as the ionic strength of the mobile phase remains sufficiently low to avoid salting out of protein from the bST sample. An aqueous bicarbonate (EICO3) bufer solu~ion, for example provided as an aqueous solution of ammonium bicarbonate, provides a preferred mobile phase. The bicarbonate buffer is desirably at a concentration of less than about 0.7 M, and more preferably in the range of a~out 0.1 to about 0.4 M. The pE~ of the mobile phase is more pre~erably about 9 to about 11, and most preferably about 9 to about lO.
The solid phase is packed in a convelltional HPLC
column, which is a component of a conventiorlal size exclusion HPLC set-up. For example, size exclusion HPl.C
set-ups typically include ~umps, reservoir.s for the aqueous buffer solution, an ultra violet ( W) absorption detector and a fraction collection means.
The bST sample to be treated hy the method of the invention is preferably reconstituted in an aqueous buffer solution identical to that used as the mobiLe WO94/17~3 PCT~S94/00850 ~
21~4~9~
phase. The concentration of bST in the sample is desirably about 0.05 to about 2 milligrams per milliliter (mg/mL). More preferably, the bST concentratiol1 in the sample is about O.l mg~mL to about l mg/mL.
The pressure on the size exclusion HPLC column provides a suitable 1OW rate for separations performe~
in accordance with the invention. The pressure necessary to achieve advantageous flow rates will of course be depelldent UpOIl the particle size of the stationary phase. In preferred methods of the invention, the pressure and the resulting flow rate used in the size exclusion HPLC will completely elute the biologically active bST protein fraction within about 30 minutes so as to provide an expedient assay. In this regard, typical columll pressures are about 500 to about 1500 psi, and more typically about 800 to about lO00 psi.
In another aspect of the applicant's work, using the above-described non-denaturing size exclusion HPLC
method, a bST soluble aggregate of high molecular weight has been isolated and extensively characterized. In Figures lB and lC, the peak excursions beginning at about 9 minutes contain these high molecular weight bST soluble aggregates. The molecular weight of these bST soluble aggregates is greater than 660,000 as determined by size exclusion ~IPLC (Figure 5). The bST soluble aggregates are biologically inactive as determined by radio receptor assay.
The behavior oE this bST soluble aggregate in solution has also been characterized. The bST soluble aggregate is substantially soluble in non-denaturing aqueous bufEer solutions having pH's greater than about 8.5 and less than about 12 (Figures lD, lE and 7). At p~I's outside this range, the soluble aggregate precipitates from non-denaturing aqueous buffer solutions. High salt concentration will also cause the bST soluble aggregate to precipitate (Figure ~). For 21~479~
WO94tl7~3 ~ PCT~S94/00850 instance, salting out of the ~ST soluble aggregate is observed at bicarbonate concentrations above a~out 0 7 M. Moreover, under centrifuging at 16,000 times the force of gravity ("16,000 g"), the bST soluble aggregate remains in 0.2 M bicarbonate buffer solution (pll 9) for 30 minutes or more (Figure 8). ~enaturing agents SUCll as sodium dodecyl sulfate and urea will denature the bST
soluble aggregate to monomeric bST. These and other characteristics of the bST soluble aggregate are discussed further in tlle Examples, infra.
In order to promote a further understanding of the invention, the following specific Examples are provided.
It will be understood that these Examples are illustrative and not limiting in nature.
~x~MPr.
Chemicals and Reagents All reagents were of analytical-reagent grade and were used without fur~ller purificaLion. Water was obtained from a Millipore Milli-Q water purification system. The bST reference standard and recombinant bST
bulk materials (somidobove) were from Eli Lilly and Company.
Con~itions o Size E~clusion HPLC Chromatography A rigid porous polymeric gel having a plurality of hydrophilic groups (the gel was a crosslinked hydroxylated polyether) and havirlg an average particle size of about 10 ~m (tradellame, Beckman Spherogel TSK
3000 PW column (600 x 7.5 mm, I.D.)) was employed in these Examples. The mobile phase for separations reported herein was 0.2 M ammonium bicarbonate adjusted to pH 9 with NaOH, unless otherwise indicated. All separations were achieved at room temperature and at a flow rate of 0.5 mL/min. (generating a corresponding WO94117~3 PCT~S94/00~50 ~
i~5~79~
pressure in the range of about 800 to 1000 psi) unless otherwise indicated. The sample injection volume was 20 ~L. Most separations were performed using the Waters 625 LC system with a 911+ photodiode array detector and a WISP 721 autosampler. However, the precision and linearity studies were carried out on a Beckman System-Gold HPLC system collsisting of a Model 126 solvent delivery system, a Model 166 variable wavelength detector, and a Model 507 autosampler.
Sample Preparation Samples and standards were prepared in the mobile phase. All bST samples were prepared at a concentration of 1 mg/mL bST unless otherwise indicated. To minimize incomplete dissolution, an aliquot of mobile phase was added to the bST and the sample solution was allowed to stand at room temperature for 30 minutes. Thereafter, the sample solution was gently shaken for 5 to 10 minutes. Sample solutions were then filtered through an Acrodisc 0.45 ~m filter prior to injection.
Results of Size E~clusion ~iPLC of bST Samples Figure lA shows a size exclusion HPLC chromatogram of a biologically active recornbinant bST protein reference standard in which only a single main peak containing biologically active bST protein was found. The chromatograrn of Figure lA was obtained using the methodology described above, except the ~low rate was 1 mL/min. Figures lB and lC show size exclusion HPLC
chromatograms of diferent lots of recombinant bST bulk material. Two strong peaks with excellent baseline resolution between them were observed. The chromatographic first eluting peak (-9 rnin) contains the high molecular weight bST soluble aggregates, and the second e]uting peak (~13 min) is biologically active bST
protein .
~ WO94/17~3 215 4 7 9 4 PCT~S94/00850 Figures 1~ and lE sllow size exclusion chromato~rap~ls of samples from the same bull~ recombinant bST used in preparing the chromatogram oE Figure lC. The lone chromatogram shown in Fiyure lD was taken with a mobile phase having a p~ of 11.3. Two strony peaks were again observed as in Figure lB. Tlle superimposed chromatograms of Figure lE were developed using mobile phases llaviny pH's ranging from 7.9 to 12.2. ~iminished peak area response at the lower and higher pH's which were studied evidenced the diminished solubility of the bST soluble aygregate. More advantageous methods of the invention are performed using mobile phase pH's where higher solubility of the bST soluble aggrega~e exists, e.y. in these runs at pH's between a~out 9 and about 12.
Additional size exclusion HPLC chromatograms of the lots shown in Figures lB and lC were taken under the same conditions except usiny a di~ferent non-denaturing mobile phase (0.05 M borate buffer solution). The chromatograms obtained were similar to t~lose shown in Figures lB and lC, having two strong peaks and good baseline resolution between them. Thus other non-denaturing aqueous buffer solutions may be readily employed in size exclusion HPLC
separations according to tlle invention.
Varying Size E~clusion HPLC c~n~itions The effect of varying a-nmollium bicarbonate concentration on elution time of the biologically active bST protein fraction in size exclusion HPLC was investigated. A series of experiments demonstrated that biologically active bST protein gave a typical size exclusion HPLC curve (Figure 2A) upon varying the ammonium bicarbonate solutiorl. A slight chanye in elution time was observed as the ammonium bicarbonate concentration ranged from about 0.1 M to about 0.4 M.
Elution time increased more rapidly ~lleII the concentration of amrnonium bicarbonate ranged above about WO94/17~3 PCT~S94/00850 ~
21~94 O . 4 M. Further, the peak area of bST significantly decreased when increasing salt (ammonium bicarbonate ) concentratioll to higller than abou~ 0.7 M (Figure 2R).
Preferred size exclusion HPLC methods of the invention are ~hus perforrned using a bicarbonate saI~ concentration in the mobile phase of less than about ~.~ M.
The effect of var~ing pE~ of the mobile phase on size exclusion llPLC elution time and peak area response of the biologically active bST protein fraction was also investigated. Increasing the pH of the 0.2 M ammonium bicarbonate mobile phase in the range of a~out 7.5 to about lO.5 did not affect ttle elution time of ~ST;
however, peak area response was slightly increased over the same pl~ range (Figures 3A, 3B).
The most advan~ageous size exclusion HPLC flow rate in this study was found to be about 0.5 mL/min. A flow rate of 0.3 mL~min. is usable and results in e~uivalent efficiency, but nearly doubles the run time. The biologically active bST protein peak overlapped with the void peak at l mL/min. flow rate.
Non-denaturation of bST Sample In order to confirm that the inventive method is non-denaturing to the bST, the biologically active bST
protein fraction in the reference standard (see Figure lA) was collected and its biological activity measured by RRA. Tlle results indicated that the collected biologically active bST proteiII component in mobile phase retains equal biological activity to the parent solution before treatment by the size exclusion EIPLC.
ln testing of several different lots of recombinant bulk bST, a strong correlation was demonstrated between the level of biologically active bs~r protein measured by the inventive size exclusion HPLC method and tlle potency data (I.U./mg) measured by RRA. A linear regression ~lot of the data from the size exclusion HPLC method and RRA
~ WO94/17~3 215 ~ 7 9 ~ PCT~S94/00850 is shown in Figure 4. The correlation coefficient for the data was 0.845. l'llese results clearly demons~rate t~lat the inventive size exclusion I~PLC metllod can be used in a determination of the potency value of bST ~ulk materials. That is, the inventive size exclusion HPLC
method can be used to measllre the level (e.g. in peak area percent) of biologically active bST protein in a bST
sample of unknown potency. The level of biologically active bST protein measured in the sample can then ~e plotted on a calibrated curve similar to that in Figure 4, including data from RRA and size exclusion HPLC assays on bST controls. RRA data correlates well to bio-potency data obtained by still otller me~hods, for example the well known rat weig~Jt gain me~hod. Tllus, a similar correlation of the size exclusion ~lPLC results can be made to data obtained by other known bST bio-potency assays.
Linearity And rrecision of Method The linearity of the inventive size exclusion IIPLC
method was measured by preparing varying concentrations of biologically active bST protein reference standard in mobile phase. Standard solutions in the bST
concentration range of 0.1 to 1 mg/nlL treated by size exclusion HPLC in triplica~e and a linear regression analysis of the data performed. The re~roducibility of the linearity obtained using three differellt columns in three different days is shown in Table 1. The correlation coefficient ranged from 0.9998 to 1.000, and the average relative standard deviation (R.S.D.) was 1.36%.
Slope 306600 301200 311~00 R.S.I~. % 0.60 1.51 0.77 Y-Intercept -39~0 -2050 -3270 35 Correlation 1.0000 0.999~ 0.9999 coefficient WO94/17~3 PCT~S94/00850 ~
2~794 The precision of tlle inventive size exclusion HPLC met~lod was evaluated over three days while using two different HPLC
systems, three columns with different series numbers, and three different lots of bST bulk materials. The resulting data, shown in Table 2, de~nonstrate that tlle R.S.D. of biologically active bST protein measured is less tllan 2.8 %, and L~le reproducibility of elution time is less tllan 0.18%
R.S.D.
Beckman System Waters System Lot Day n %bST %RSD te %RSD %bST %RSD te %RSD
001 1 3 83.5 0.70 851 0.12 83.8 2.67 827 0.07 2 3 81.7 1.27 831 0.09 83.7 0.91 846 0.07 3 3 82.7 0.75 822 0.10 002 1 3 87.1 2.23 849 0.]8 86.1 2.80 824 0.15 2 3 86.1 2.72 830 0.06 86.7 2.56 850 0.09 3 3 88.9 1.34 824 O.lU
003 1 3 44.9 ().47 853 0.0~ 44.5 2.40 8~7 0.l0 2 3 46.3 1.14 837 0.13 46.9 1.32 854 ~.17 3 3 46.8 0.75 829 0.10 n = Replicate Number te . Retention Time (in secon~s) RSD = Relative Starldard Deviatiorl A bST recovery study was carried out by the addition of biologically active bST protein reference standard in two different concentrations to two different to bulk bST. The recoveries obtained in this experiment ranged from 101 to 104 % (Table 3).
~ WO94/17~3 215 ~ 7 9 4 PCT~S94/00850 TA~LE 3 Reference Sample Standard Found n Recovery RSD
Added (mg) (mg) (%) (%) 5 003A 0.]~6 0.191 3102.5% 0.34 003A 0.465 0.471 3101.2% 0.58 003B 0.186 0.193 3103.8% O.9U
003B 0.465 0.472 3101.4% 0.72 n = Replicate Number RSV = Relative Standard Deviation The above results demonstrate that the inventive size exclusion HPLC method provides for the accurate and precise determination of bST potency in bulk bST materials.
bST Solub]e Aggregate Characterization As indicated above, a peak at about 13 min. represents the biologically active bST protein fraction and a peak at about 9 min. represents a bST soluble aggregate fraction (Figures lB-lE). In further work, a W spectrum taken by photodiode array detector in the size exclusion HPLC process demonstrated that the W profiles of the biologically active bST protein fraction and bST soluble aggregate fraction were similar. A W spectral change of the bST soluble aggregate was noted at 250-270 nm, and may be due to a change in the environment of the aromatic amino acids after aggregation.
The molecular weiyht of bST soluble aggregates was measured by a cali~ration curve using size exclusion HPLC.
The results are shown in Figure 6. The bST soluble aggregate eluted at the exclusion volume of the column and its molecular weight (MW) was hig~ler than 660,000 (MW of tllyroglobumin).
To ~urther characterize the bST soluble aggregate, bulk bST was dissolved in 0.4 M a~ onium bicarbonate (pH 9) to WO94/17~3 PCT~S94/00850 ~
2~ ~7 9 ~
make a 5 mg/mL solution. After centrifugation at 16,000 g, the supernatant was treated under the standard size exclusion IIPLC conditions described above except the mobile phase was a 0.4 M ammonium bicarbonate solution. Two components obtained in this separation, biologically active bST protein and bST
soluble aggregates, were collected i~ separate fractions.
Each collected fraction was re-chromatographed to confirm its elution time and purity. The results demonstrated that the bST aggregate remained in solution even after the centrifugation.
Experiments were performed to confirm that the bST
soluble aggregate is non-covalently bonded. If the bST
soluble aggregate were a non-covalent bonded protein, it would dissociate into monomer in the presence of denaturing agents such as detergents, high concentration urea and guanadine HCl in solution. To confirm this, a 1:1 mixture of the collected bST soluble aggregate fraction with 2% sodium dodecyl sulfate (SDS) was chromatographed in a system including a du Pont 250 column and a 0.4 M bicarbonate mobile phase containing 1 % SDS. As expected, the chromatographic profile and W -spectrum of the dissociated proteins was identical to that of SDS-monomer obtained under the same experimental conditions. In similar experimentation it was demonstrated that the bST soluble aggregates can also be dissociated by 3 M urea.
Additional behaviors of bST soluble aggregates in solution have been investigated. The solubility of the bST
solu~le aggregates was strongly affected by sa]t concentration in solution. Increasing bicarbonate concentration sharply decreases the solubility of soluble aggregate due to salting-out of the proteins as shown by size exclusion EIP~C under the above-noted conditions except varying the concentration of ammonium bicarbonate in the prepared sample and mobile phase, as indicated in Figure 6.
Silllilarly, the eEfect of the pH of the bicarbonate buffer solutions on tlle solubility of the bST soluble aggregate was ~ WO94/17~3 21~ 4 79 ~ PCT~S94/00850 investigated. At pH less than 8.5 and plI greater than 12.5, bST soluble aggregate preciE7itates out of the 0.2 M
bicarbonate solution. Solubility of the bST soluble ag~regate increased with increasinc~ pH starting from pH 8.5, and reached a maximum at pH 9.5 (Figure 7).
The stability of the soluble aggregates in bicarbonate solution was also studied. The isolated bST soluble aggregate solution is stable for two days at 4C, and for more than 9 hours at room temperature (as determined by measuring size exclusion HPLC peak area over time).
Additionally, when centrifuging at 16,000 g, the bST soluble aggregate remains in solution for at least 30 minutes (Figure 8).
While the invention has been illustrated and described in detail in the foregoing description, tlle same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been described and that all changes and modifications that come within the spirit of the invention are desired to be protected.
The present invention is preferred for use wi~ll bs~r produced by recombinant DNA technology ("recombinant bST"). Typically, the recombinant bST is obtained by isolating and denaturinc3 and solubilizing inclusion bodies which contain the recombinant bST. The bST is then renatured to form the biologically active bST
protein. During the denaturing/renaturing steps, biologically inactive bST non-covalent aggregates are formed along with the desired biologically active bST
protein. Colloids and some very ~ligh molecular weiyht impurities can be removed from the solubilized inclusion bodies by ultrafiltration. However, substantial quantities of biologically inactive bST non-covalent aggregates usually pass throug~l the ultrafilter along with the biologically active bST protein. As a result, the finally-obtained bulk recombinant bST contains ~iologically active ~ST protein as well as biologically inactive bST soluble aggregates. The method of the present invention can be used to treat this bulk recombinant bST to separate the biologically active bST
protein from the biologically inactive bST non-covalent soluble aggregates. Further, this separation is achieved in a manner which can be used to provide an expedient, precise and accurate assay for the potency of the bulk bST.
Two examples of recombinant bSTs are the compounds known as somidobove and sometribove. The present inven~ion is especially preferred for application to these two recombinant bSTs.
l'he method of the invention employs size exclusion HPLC usin~ as the solid phase a hydrophilic porous ~ WO94/17~3 2I ~ 4 79 ~ PCT~S94/00850 _g_ polymeric gel. Preferred porous gels for use in the inventioll have an average particle diameter of about 5~m to about 15~m, and more preferably a~out lO~m to about 15~m. Typically, the average pore diameter of the gel will be about lOU to about 200 angstroms, and for the present inventiorl a gel haviny an average pore diameter of about 150 angstromS is preferred. In another aspect, the porous gel used in the invention preferably has an exclusion limit average molecular weight ~reater than the molecular weight of the bST non-covalent soluble aggregate, e.g. about 106 or greater, and more preferably about 10 or greater.
The mobile phase for use in t}le present invention is an aqueous buffer solution having a pH of about 8 to about 12 and which does not denature the bST sample.
Generally, the buffer may be included in tlle mo~ile phase at any effective concentration so long as the ionic strength of the mobile phase remains sufficiently low to avoid salting out of protein from the bST sample. An aqueous bicarbonate (EICO3) bufer solu~ion, for example provided as an aqueous solution of ammonium bicarbonate, provides a preferred mobile phase. The bicarbonate buffer is desirably at a concentration of less than about 0.7 M, and more preferably in the range of a~out 0.1 to about 0.4 M. The pE~ of the mobile phase is more pre~erably about 9 to about 11, and most preferably about 9 to about lO.
The solid phase is packed in a convelltional HPLC
column, which is a component of a conventiorlal size exclusion HPLC set-up. For example, size exclusion HPl.C
set-ups typically include ~umps, reservoir.s for the aqueous buffer solution, an ultra violet ( W) absorption detector and a fraction collection means.
The bST sample to be treated hy the method of the invention is preferably reconstituted in an aqueous buffer solution identical to that used as the mobiLe WO94/17~3 PCT~S94/00850 ~
21~4~9~
phase. The concentration of bST in the sample is desirably about 0.05 to about 2 milligrams per milliliter (mg/mL). More preferably, the bST concentratiol1 in the sample is about O.l mg~mL to about l mg/mL.
The pressure on the size exclusion HPLC column provides a suitable 1OW rate for separations performe~
in accordance with the invention. The pressure necessary to achieve advantageous flow rates will of course be depelldent UpOIl the particle size of the stationary phase. In preferred methods of the invention, the pressure and the resulting flow rate used in the size exclusion HPLC will completely elute the biologically active bST protein fraction within about 30 minutes so as to provide an expedient assay. In this regard, typical columll pressures are about 500 to about 1500 psi, and more typically about 800 to about lO00 psi.
In another aspect of the applicant's work, using the above-described non-denaturing size exclusion HPLC
method, a bST soluble aggregate of high molecular weight has been isolated and extensively characterized. In Figures lB and lC, the peak excursions beginning at about 9 minutes contain these high molecular weight bST soluble aggregates. The molecular weight of these bST soluble aggregates is greater than 660,000 as determined by size exclusion ~IPLC (Figure 5). The bST soluble aggregates are biologically inactive as determined by radio receptor assay.
The behavior oE this bST soluble aggregate in solution has also been characterized. The bST soluble aggregate is substantially soluble in non-denaturing aqueous bufEer solutions having pH's greater than about 8.5 and less than about 12 (Figures lD, lE and 7). At p~I's outside this range, the soluble aggregate precipitates from non-denaturing aqueous buffer solutions. High salt concentration will also cause the bST soluble aggregate to precipitate (Figure ~). For 21~479~
WO94tl7~3 ~ PCT~S94/00850 instance, salting out of the ~ST soluble aggregate is observed at bicarbonate concentrations above a~out 0 7 M. Moreover, under centrifuging at 16,000 times the force of gravity ("16,000 g"), the bST soluble aggregate remains in 0.2 M bicarbonate buffer solution (pll 9) for 30 minutes or more (Figure 8). ~enaturing agents SUCll as sodium dodecyl sulfate and urea will denature the bST
soluble aggregate to monomeric bST. These and other characteristics of the bST soluble aggregate are discussed further in tlle Examples, infra.
In order to promote a further understanding of the invention, the following specific Examples are provided.
It will be understood that these Examples are illustrative and not limiting in nature.
~x~MPr.
Chemicals and Reagents All reagents were of analytical-reagent grade and were used without fur~ller purificaLion. Water was obtained from a Millipore Milli-Q water purification system. The bST reference standard and recombinant bST
bulk materials (somidobove) were from Eli Lilly and Company.
Con~itions o Size E~clusion HPLC Chromatography A rigid porous polymeric gel having a plurality of hydrophilic groups (the gel was a crosslinked hydroxylated polyether) and havirlg an average particle size of about 10 ~m (tradellame, Beckman Spherogel TSK
3000 PW column (600 x 7.5 mm, I.D.)) was employed in these Examples. The mobile phase for separations reported herein was 0.2 M ammonium bicarbonate adjusted to pH 9 with NaOH, unless otherwise indicated. All separations were achieved at room temperature and at a flow rate of 0.5 mL/min. (generating a corresponding WO94117~3 PCT~S94/00~50 ~
i~5~79~
pressure in the range of about 800 to 1000 psi) unless otherwise indicated. The sample injection volume was 20 ~L. Most separations were performed using the Waters 625 LC system with a 911+ photodiode array detector and a WISP 721 autosampler. However, the precision and linearity studies were carried out on a Beckman System-Gold HPLC system collsisting of a Model 126 solvent delivery system, a Model 166 variable wavelength detector, and a Model 507 autosampler.
Sample Preparation Samples and standards were prepared in the mobile phase. All bST samples were prepared at a concentration of 1 mg/mL bST unless otherwise indicated. To minimize incomplete dissolution, an aliquot of mobile phase was added to the bST and the sample solution was allowed to stand at room temperature for 30 minutes. Thereafter, the sample solution was gently shaken for 5 to 10 minutes. Sample solutions were then filtered through an Acrodisc 0.45 ~m filter prior to injection.
Results of Size E~clusion ~iPLC of bST Samples Figure lA shows a size exclusion HPLC chromatogram of a biologically active recornbinant bST protein reference standard in which only a single main peak containing biologically active bST protein was found. The chromatograrn of Figure lA was obtained using the methodology described above, except the ~low rate was 1 mL/min. Figures lB and lC show size exclusion HPLC
chromatograms of diferent lots of recombinant bST bulk material. Two strong peaks with excellent baseline resolution between them were observed. The chromatographic first eluting peak (-9 rnin) contains the high molecular weight bST soluble aggregates, and the second e]uting peak (~13 min) is biologically active bST
protein .
~ WO94/17~3 215 4 7 9 4 PCT~S94/00850 Figures 1~ and lE sllow size exclusion chromato~rap~ls of samples from the same bull~ recombinant bST used in preparing the chromatogram oE Figure lC. The lone chromatogram shown in Fiyure lD was taken with a mobile phase having a p~ of 11.3. Two strony peaks were again observed as in Figure lB. Tlle superimposed chromatograms of Figure lE were developed using mobile phases llaviny pH's ranging from 7.9 to 12.2. ~iminished peak area response at the lower and higher pH's which were studied evidenced the diminished solubility of the bST soluble aygregate. More advantageous methods of the invention are performed using mobile phase pH's where higher solubility of the bST soluble aggrega~e exists, e.y. in these runs at pH's between a~out 9 and about 12.
Additional size exclusion HPLC chromatograms of the lots shown in Figures lB and lC were taken under the same conditions except usiny a di~ferent non-denaturing mobile phase (0.05 M borate buffer solution). The chromatograms obtained were similar to t~lose shown in Figures lB and lC, having two strong peaks and good baseline resolution between them. Thus other non-denaturing aqueous buffer solutions may be readily employed in size exclusion HPLC
separations according to tlle invention.
Varying Size E~clusion HPLC c~n~itions The effect of varying a-nmollium bicarbonate concentration on elution time of the biologically active bST protein fraction in size exclusion HPLC was investigated. A series of experiments demonstrated that biologically active bST protein gave a typical size exclusion HPLC curve (Figure 2A) upon varying the ammonium bicarbonate solutiorl. A slight chanye in elution time was observed as the ammonium bicarbonate concentration ranged from about 0.1 M to about 0.4 M.
Elution time increased more rapidly ~lleII the concentration of amrnonium bicarbonate ranged above about WO94/17~3 PCT~S94/00850 ~
21~94 O . 4 M. Further, the peak area of bST significantly decreased when increasing salt (ammonium bicarbonate ) concentratioll to higller than abou~ 0.7 M (Figure 2R).
Preferred size exclusion HPLC methods of the invention are ~hus perforrned using a bicarbonate saI~ concentration in the mobile phase of less than about ~.~ M.
The effect of var~ing pE~ of the mobile phase on size exclusion llPLC elution time and peak area response of the biologically active bST protein fraction was also investigated. Increasing the pH of the 0.2 M ammonium bicarbonate mobile phase in the range of a~out 7.5 to about lO.5 did not affect ttle elution time of ~ST;
however, peak area response was slightly increased over the same pl~ range (Figures 3A, 3B).
The most advan~ageous size exclusion HPLC flow rate in this study was found to be about 0.5 mL/min. A flow rate of 0.3 mL~min. is usable and results in e~uivalent efficiency, but nearly doubles the run time. The biologically active bST protein peak overlapped with the void peak at l mL/min. flow rate.
Non-denaturation of bST Sample In order to confirm that the inventive method is non-denaturing to the bST, the biologically active bST
protein fraction in the reference standard (see Figure lA) was collected and its biological activity measured by RRA. Tlle results indicated that the collected biologically active bST proteiII component in mobile phase retains equal biological activity to the parent solution before treatment by the size exclusion EIPLC.
ln testing of several different lots of recombinant bulk bST, a strong correlation was demonstrated between the level of biologically active bs~r protein measured by the inventive size exclusion HPLC method and tlle potency data (I.U./mg) measured by RRA. A linear regression ~lot of the data from the size exclusion HPLC method and RRA
~ WO94/17~3 215 ~ 7 9 ~ PCT~S94/00850 is shown in Figure 4. The correlation coefficient for the data was 0.845. l'llese results clearly demons~rate t~lat the inventive size exclusion I~PLC metllod can be used in a determination of the potency value of bST ~ulk materials. That is, the inventive size exclusion HPLC
method can be used to measllre the level (e.g. in peak area percent) of biologically active bST protein in a bST
sample of unknown potency. The level of biologically active bST protein measured in the sample can then ~e plotted on a calibrated curve similar to that in Figure 4, including data from RRA and size exclusion HPLC assays on bST controls. RRA data correlates well to bio-potency data obtained by still otller me~hods, for example the well known rat weig~Jt gain me~hod. Tllus, a similar correlation of the size exclusion ~lPLC results can be made to data obtained by other known bST bio-potency assays.
Linearity And rrecision of Method The linearity of the inventive size exclusion IIPLC
method was measured by preparing varying concentrations of biologically active bST protein reference standard in mobile phase. Standard solutions in the bST
concentration range of 0.1 to 1 mg/nlL treated by size exclusion HPLC in triplica~e and a linear regression analysis of the data performed. The re~roducibility of the linearity obtained using three differellt columns in three different days is shown in Table 1. The correlation coefficient ranged from 0.9998 to 1.000, and the average relative standard deviation (R.S.D.) was 1.36%.
Slope 306600 301200 311~00 R.S.I~. % 0.60 1.51 0.77 Y-Intercept -39~0 -2050 -3270 35 Correlation 1.0000 0.999~ 0.9999 coefficient WO94/17~3 PCT~S94/00850 ~
2~794 The precision of tlle inventive size exclusion HPLC met~lod was evaluated over three days while using two different HPLC
systems, three columns with different series numbers, and three different lots of bST bulk materials. The resulting data, shown in Table 2, de~nonstrate that tlle R.S.D. of biologically active bST protein measured is less tllan 2.8 %, and L~le reproducibility of elution time is less tllan 0.18%
R.S.D.
Beckman System Waters System Lot Day n %bST %RSD te %RSD %bST %RSD te %RSD
001 1 3 83.5 0.70 851 0.12 83.8 2.67 827 0.07 2 3 81.7 1.27 831 0.09 83.7 0.91 846 0.07 3 3 82.7 0.75 822 0.10 002 1 3 87.1 2.23 849 0.]8 86.1 2.80 824 0.15 2 3 86.1 2.72 830 0.06 86.7 2.56 850 0.09 3 3 88.9 1.34 824 O.lU
003 1 3 44.9 ().47 853 0.0~ 44.5 2.40 8~7 0.l0 2 3 46.3 1.14 837 0.13 46.9 1.32 854 ~.17 3 3 46.8 0.75 829 0.10 n = Replicate Number te . Retention Time (in secon~s) RSD = Relative Starldard Deviatiorl A bST recovery study was carried out by the addition of biologically active bST protein reference standard in two different concentrations to two different to bulk bST. The recoveries obtained in this experiment ranged from 101 to 104 % (Table 3).
~ WO94/17~3 215 ~ 7 9 4 PCT~S94/00850 TA~LE 3 Reference Sample Standard Found n Recovery RSD
Added (mg) (mg) (%) (%) 5 003A 0.]~6 0.191 3102.5% 0.34 003A 0.465 0.471 3101.2% 0.58 003B 0.186 0.193 3103.8% O.9U
003B 0.465 0.472 3101.4% 0.72 n = Replicate Number RSV = Relative Standard Deviation The above results demonstrate that the inventive size exclusion HPLC method provides for the accurate and precise determination of bST potency in bulk bST materials.
bST Solub]e Aggregate Characterization As indicated above, a peak at about 13 min. represents the biologically active bST protein fraction and a peak at about 9 min. represents a bST soluble aggregate fraction (Figures lB-lE). In further work, a W spectrum taken by photodiode array detector in the size exclusion HPLC process demonstrated that the W profiles of the biologically active bST protein fraction and bST soluble aggregate fraction were similar. A W spectral change of the bST soluble aggregate was noted at 250-270 nm, and may be due to a change in the environment of the aromatic amino acids after aggregation.
The molecular weiyht of bST soluble aggregates was measured by a cali~ration curve using size exclusion HPLC.
The results are shown in Figure 6. The bST soluble aggregate eluted at the exclusion volume of the column and its molecular weight (MW) was hig~ler than 660,000 (MW of tllyroglobumin).
To ~urther characterize the bST soluble aggregate, bulk bST was dissolved in 0.4 M a~ onium bicarbonate (pH 9) to WO94/17~3 PCT~S94/00850 ~
2~ ~7 9 ~
make a 5 mg/mL solution. After centrifugation at 16,000 g, the supernatant was treated under the standard size exclusion IIPLC conditions described above except the mobile phase was a 0.4 M ammonium bicarbonate solution. Two components obtained in this separation, biologically active bST protein and bST
soluble aggregates, were collected i~ separate fractions.
Each collected fraction was re-chromatographed to confirm its elution time and purity. The results demonstrated that the bST aggregate remained in solution even after the centrifugation.
Experiments were performed to confirm that the bST
soluble aggregate is non-covalently bonded. If the bST
soluble aggregate were a non-covalent bonded protein, it would dissociate into monomer in the presence of denaturing agents such as detergents, high concentration urea and guanadine HCl in solution. To confirm this, a 1:1 mixture of the collected bST soluble aggregate fraction with 2% sodium dodecyl sulfate (SDS) was chromatographed in a system including a du Pont 250 column and a 0.4 M bicarbonate mobile phase containing 1 % SDS. As expected, the chromatographic profile and W -spectrum of the dissociated proteins was identical to that of SDS-monomer obtained under the same experimental conditions. In similar experimentation it was demonstrated that the bST soluble aggregates can also be dissociated by 3 M urea.
Additional behaviors of bST soluble aggregates in solution have been investigated. The solubility of the bST
solu~le aggregates was strongly affected by sa]t concentration in solution. Increasing bicarbonate concentration sharply decreases the solubility of soluble aggregate due to salting-out of the proteins as shown by size exclusion EIP~C under the above-noted conditions except varying the concentration of ammonium bicarbonate in the prepared sample and mobile phase, as indicated in Figure 6.
Silllilarly, the eEfect of the pH of the bicarbonate buffer solutions on tlle solubility of the bST soluble aggregate was ~ WO94/17~3 21~ 4 79 ~ PCT~S94/00850 investigated. At pH less than 8.5 and plI greater than 12.5, bST soluble aggregate preciE7itates out of the 0.2 M
bicarbonate solution. Solubility of the bST soluble ag~regate increased with increasinc~ pH starting from pH 8.5, and reached a maximum at pH 9.5 (Figure 7).
The stability of the soluble aggregates in bicarbonate solution was also studied. The isolated bST soluble aggregate solution is stable for two days at 4C, and for more than 9 hours at room temperature (as determined by measuring size exclusion HPLC peak area over time).
Additionally, when centrifuging at 16,000 g, the bST soluble aggregate remains in solution for at least 30 minutes (Figure 8).
While the invention has been illustrated and described in detail in the foregoing description, tlle same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been described and that all changes and modifications that come within the spirit of the invention are desired to be protected.
Claims (21)
1. A method for determining the potency of bovine somatotropin, comprising:
measuring the level of biologically active bovine somatotropin protein in a bovine somatotropin sample by size exclusion HPLC employing as a stationary phase a hydrophilic porous polymer gel having an average particle diameter of about 5 µm to about 15 µm and as a mobile phase a buffered aqueous solution having a pH of about 8.5 to about 12 which maintains biologically active bovine somatotropin protein and biologically inactive bovine somatrotropin soluble aggregates contained in said sample in solution, said measuring further being conducted under conditions which are non-denaturing to the bovine somatotropin sample; and, determining the potency of the bovine somatotropin sample based upon the measured level of biologically active bovine somatotropin protein.
measuring the level of biologically active bovine somatotropin protein in a bovine somatotropin sample by size exclusion HPLC employing as a stationary phase a hydrophilic porous polymer gel having an average particle diameter of about 5 µm to about 15 µm and as a mobile phase a buffered aqueous solution having a pH of about 8.5 to about 12 which maintains biologically active bovine somatotropin protein and biologically inactive bovine somatrotropin soluble aggregates contained in said sample in solution, said measuring further being conducted under conditions which are non-denaturing to the bovine somatotropin sample; and, determining the potency of the bovine somatotropin sample based upon the measured level of biologically active bovine somatotropin protein.
2. The method of claim 1 wherein said hydrophilic porous polymer gel has an average particle size of about 10 µm to about 15 µm.
3. The method of claim 1 wherein said buffered aqueous solution is a bicarbonate buffer solution.
4. The method of claim 3 wherein said hydrophilic porous polymer gel has an average particle size of about 10 µm to about 15 µm.
5. The method of claim 4 wherein said determining includes plotting said measured level of biologically active bST protein on a calibration curve correlating RRA potency values with size exclusion HPLC data on bovine somatotropin control samples.
6. The method of claim 4 wherein said hydrophilic porous polymer gel has an average particle size of about 10 µm.
7. The method of claim 4 wherein said hydrophilic porous polymer gel has an average pore diameter of about 150 angstroms.
8. The method of claim 6 wherein the pH of the mobile phase is about 9 to about 11.
9. The method of claim 8 wherein said hydrophilic porous polymer gel has an average pore diameter of about 150 angstroms.
10. The method of claim 6 wherein said size exclusion HPLC
is conducted at a pressure of about 800 to about 1000 psi.
is conducted at a pressure of about 800 to about 1000 psi.
11. The method of claim 6 wherein the mobile phase is an ammonium bicarbonate buffer solution at a concentration of less than about 0.7 M.
12. The method of claim 11 wherein said size exclusion HPLC is conducted at a pressure of about 800 to about 1000 psi.
13. The method of claim 12 wherein said hydrophilic porous polymer gel has an average pore diameter of about 150 angstroms.
14. A method for determining the potency of a sample of bulk recombinant bovine somatotropin containing biologically active bovine somatotropin and biologically inactive bovine somatotropin non-covalent soluble aggregates, comprising:
dissolving the sample of bovine somatotropin in a non-denaturing aqueous buffer solution having a pH greater than about 8.5 and less than about 12 which maintains the biologically active bovine somatotropin and the biologically inactive bovine somatotropin non-covalent soluble aggregates in solution;
measuring the level of said biologically active bovine somatrotropin in said sample by size exclusion HPLC in a mobile phase which is non-denaturing to said biologically active bovine somatotropin and under conditions which are effective to achieve separation of said biologically active bovine somatotropin from said biologically-inactive soluble aggregates of bovine somatotropin; and, determining the potency of the sample based upon the measured level of biologically active bovine somatotropin.
dissolving the sample of bovine somatotropin in a non-denaturing aqueous buffer solution having a pH greater than about 8.5 and less than about 12 which maintains the biologically active bovine somatotropin and the biologically inactive bovine somatotropin non-covalent soluble aggregates in solution;
measuring the level of said biologically active bovine somatrotropin in said sample by size exclusion HPLC in a mobile phase which is non-denaturing to said biologically active bovine somatotropin and under conditions which are effective to achieve separation of said biologically active bovine somatotropin from said biologically-inactive soluble aggregates of bovine somatotropin; and, determining the potency of the sample based upon the measured level of biologically active bovine somatotropin.
15. The method of claim 14 wherein said buffered aqueous solution is a bicarbonate buffer solution.
16. The method of claim 14 wherein said hydrophilic porous polymer gel has an average particle size of about 10 µm to about 15 µm.
17. The method of claim 16 wherein said size exclusion HPLC is conducted at pressure of about 800 to about 1000 psi.
18. The method of claim 16 wherein the pH of the mobile phase is about 9 to about 11.
19. The method of claim 16 wherein the mobile phase is an ammonium bicarbonate buffer solution at a concentration of less than about 0.7 M.
20. The method of claim 17 wherein said hydrophilic porous polymer gel has an average pore diameter of about 150 angstroms.
21. A method for determining the potency of a sample of bulk recombinant bovine somatotropin containing biologically active bovine somatotropin and biologically inactive bovine somatotropin non-covalent soluble aggregates, comprising dissolving the sample of bovine somatotropin in a non-denaturing aqueous buffer solution having a pH greater than about 8.5 and less than about 12 which maintains the biologically active bovine somatotropin and the biologically inactive bovine somatotropin non-covalent soluble aggregates in solution, said non-covalent soluble aggregates having a molecular weight of greater than about 660,000;
measuring the level of said biologically active bovine somatrotropin in said sample by size exclusion HPLC in a mobile phase which is non-denaturing to said biologically active bovine somatotropin and under conditions which are effective to achieve separation of said biologically active bovine somatotropin from said biologically-inactive soluble aggregates of bovine somatotropin having a molecular weight above about 660,000; and, determining the potency of the sample based upon the measured level of biologically active bovine somatotropin.
measuring the level of said biologically active bovine somatrotropin in said sample by size exclusion HPLC in a mobile phase which is non-denaturing to said biologically active bovine somatotropin and under conditions which are effective to achieve separation of said biologically active bovine somatotropin from said biologically-inactive soluble aggregates of bovine somatotropin having a molecular weight above about 660,000; and, determining the potency of the sample based upon the measured level of biologically active bovine somatotropin.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US903493A | 1993-01-26 | 1993-01-26 | |
| US08/009,034 | 1993-01-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2154794A1 true CA2154794A1 (en) | 1994-01-24 |
Family
ID=21735207
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002154794A Abandoned CA2154794A1 (en) | 1993-01-26 | 1994-01-24 | Non-denaturing potency assay for bovine somatotropin |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP0681696A1 (en) |
| JP (1) | JPH08509288A (en) |
| KR (1) | KR960700454A (en) |
| CN (1) | CN1119042A (en) |
| AU (1) | AU6094794A (en) |
| BR (1) | BR9405672A (en) |
| CA (1) | CA2154794A1 (en) |
| CZ (1) | CZ193595A3 (en) |
| HU (1) | HUT75678A (en) |
| NO (1) | NO952948L (en) |
| PL (1) | PL310053A1 (en) |
| WO (1) | WO1994017403A1 (en) |
| ZA (1) | ZA94169B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5567620A (en) * | 1993-01-26 | 1996-10-22 | Eli Lilly And Company | Non-denaturing potency assay for somatotropin |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4511502A (en) * | 1982-12-22 | 1985-04-16 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
| BG49718A3 (en) * | 1983-07-15 | 1992-01-15 | Bio Technology General Corp | Method for preparing of polypeptid with superoxiddismutasne activitty |
| US5079230A (en) * | 1988-09-12 | 1992-01-07 | Pitman-Moore, Inc. | Stable bioactive somatotropins |
| US5064943A (en) * | 1988-12-16 | 1991-11-12 | American Cyanamid Company | Method for solubilization and naturation of somatotropin |
| US4975529A (en) * | 1989-08-18 | 1990-12-04 | Monsanto Company | Method of folding somatotropins |
| US5047511A (en) * | 1989-08-28 | 1991-09-10 | Pitman-Moore, Inc. | Method for recovering recombinant proteins |
-
1994
- 1994-01-11 ZA ZA94169A patent/ZA94169B/en unknown
- 1994-01-24 CN CN94191402A patent/CN1119042A/en active Pending
- 1994-01-24 AU AU60947/94A patent/AU6094794A/en not_active Abandoned
- 1994-01-24 PL PL94310053A patent/PL310053A1/en unknown
- 1994-01-24 CZ CZ951935A patent/CZ193595A3/en unknown
- 1994-01-24 WO PCT/US1994/000850 patent/WO1994017403A1/en not_active Application Discontinuation
- 1994-01-24 KR KR1019950703075A patent/KR960700454A/en not_active Application Discontinuation
- 1994-01-24 BR BR9405672A patent/BR9405672A/en not_active Application Discontinuation
- 1994-01-24 EP EP94907307A patent/EP0681696A1/en not_active Withdrawn
- 1994-01-24 CA CA002154794A patent/CA2154794A1/en not_active Abandoned
- 1994-01-24 HU HU9502233A patent/HUT75678A/en unknown
- 1994-01-24 JP JP6517256A patent/JPH08509288A/en active Pending
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1995
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Also Published As
| Publication number | Publication date |
|---|---|
| HUT75678A (en) | 1997-05-28 |
| KR960700454A (en) | 1996-01-20 |
| PL310053A1 (en) | 1995-11-13 |
| CN1119042A (en) | 1996-03-20 |
| EP0681696A4 (en) | 1995-11-22 |
| ZA94169B (en) | 1994-09-07 |
| EP0681696A1 (en) | 1995-11-15 |
| CZ193595A3 (en) | 1996-05-15 |
| HU9502233D0 (en) | 1995-09-28 |
| JPH08509288A (en) | 1996-10-01 |
| NO952948D0 (en) | 1995-07-25 |
| AU6094794A (en) | 1994-08-15 |
| BR9405672A (en) | 1995-11-14 |
| WO1994017403A1 (en) | 1994-08-04 |
| NO952948L (en) | 1995-09-18 |
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