WO1994017403A1 - Non-denaturing potency assay for bovine somatotropin - Google Patents

Non-denaturing potency assay for bovine somatotropin Download PDF

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Publication number
WO1994017403A1
WO1994017403A1 PCT/US1994/000850 US9400850W WO9417403A1 WO 1994017403 A1 WO1994017403 A1 WO 1994017403A1 US 9400850 W US9400850 W US 9400850W WO 9417403 A1 WO9417403 A1 WO 9417403A1
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Prior art keywords
bst
bovine somatotropin
polymer gel
porous polymer
size exclusion
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PCT/US1994/000850
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French (fr)
Inventor
Jen P. Chang
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Eli Lilly And Company
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Application filed by Eli Lilly And Company filed Critical Eli Lilly And Company
Priority to EP94907307A priority Critical patent/EP0681696A1/en
Priority to KR1019950703075A priority patent/KR960700454A/en
Priority to AU60947/94A priority patent/AU6094794A/en
Priority to JP6517256A priority patent/JPH08509288A/en
Priority to BR9405672A priority patent/BR9405672A/en
Publication of WO1994017403A1 publication Critical patent/WO1994017403A1/en
Priority to NO952948A priority patent/NO952948L/en

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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
    • G01N30/26—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
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • 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

Definitions

  • 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.
  • bST bovine somatotropin
  • 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.
  • a denaturant such as guanadine hydrochloride, sodium dodecyl sulfate or urea.
  • the denaturant unfolds and solubilizes the improperly folded bST molecules.
  • the bST molecules are renatured to form the properly folded, biologically active bST monomeiic protein.
  • the bST bulk material obtained contains both the biologically active bST monomer and the biologically inactive bST aggregates. It is therefore i ⁇ iportant to establish a method for determining the 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 is monitored, and from this data a value representing the potency of the bST is obtained.
  • this assay cannot be employed to accurately quantify bST in routine analysis.
  • bST potency has also been determined by radio receptor assay (RRA) .
  • RRA radio receptor assay
  • 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.
  • RPHPLC Reversed-phase high performance liquid chromatography
  • One preferred embodiment of the present invention provides a method for determining the potency of a bovine somatotropin sample.
  • This method comprises 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 15 ⁇ 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 separating a biologically active bovine somatotropin fraction from bovine somatotropin by size exclusion HPLC employing as a solid phase a hydrophilic porous polymer gel 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 non-denaturing 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 somatotropin non-covalent soluble aggregates contained in the bulk recombinant bovine somatotropin.
  • the method comprises the step of subjecting the bulk recombinant bovine somatotropin to size exclusion HPLC employing as a solid phase a hydrophilic porous polymer gel 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 non-denaturing to bovine somatotropin, so as to separate the biologically active bovine somatotropin from the biologically inactive bovine somatotropin non-covalent soluble aggregates.
  • size exclusion HPLC employing as a solid phase a hydrophilic porous polymer gel 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 non-denaturing to bovine somatotropin, so as to separate the biologically active bovine somatotropin from the biologically inactive bovine somatotropin
  • 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.
  • Figure IA is a size exclusion HPLC chromatogram of a biologically active bST reference standard using the methodology described in the Examples, infra.
  • Figures IB through IE are size exclusion HPLC chromatograms of bulk recombinant bST materials using the methodology 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 the biologically active bST protein fraction using the methodology described in the Examples, infra.
  • Figure 2B is a graph showing the effect of bicarbonate buffer concentration on the size exclusion HPLC peak area response of the biologically 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 bST protein fraction using the methodology described in the Examples, infra.
  • Figure 3B a graph showing is the effect of mobile phase pH variation on the size exclusion HPLC peak area response of the biologically active bST protein fraction using the methodology described in the Examples, infra.
  • Figure 4 is a graph showing the correlation between radio-receptor assay polency data (IU/ g) and the size exclusion HPLC- easured level of biologically 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 graph 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 the effect of mobile phase pH on the size exclusion HPLC peak area for the biologically active bST protein fraction ("B/A bST”) and 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 of 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. DESCRIPTION OF THE PREFERRED EMBODIMENT
  • bovine somatotropin denotes a substance, of natural or synthetic origin, which exhibits 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 synthesize bovine somatotropin and other such substances by the use of genetically-modified microorganisms such as bacteria.
  • a modified bovine somatotropin that is, a substance that differs as to its structure from the naturally occurring growth hormone, but which retains the biological activity of the naturally occurring growth hormone.
  • a modified bovine somatotropin may contain one or more additional amino acids, at one or both ends of the polypeptide chain. Additional modifications, will be understood by those skilled in the art. Therefore, the term bovine somatotropin is used throughout this document to refer to both naturally occurring bovine somatotropin as well as synthetically produced bovine somatotropin which shares the biological properties of naturally occurring bovine somatotropin, and which may be identical or which may vary as to structure.
  • the present invention is preferred for use with bST produced by recombinant DNA technology ("recombinant bST").
  • recombinant bST is obtained by isolating and denaturing and solubilizing inclusion bodies which contain the recombinant bST.
  • the bST is then renatured to form the biologically active bST protein.
  • biologically inactive bST non-covalent aggregates are formed along with the desired biologically active bST protein. Colloids and some very high molecular weight impurities can be removed from the solubilized inclusion bodies by ultrafiltration.
  • the finally-obtained bulk recombinant bST contains biologically active bST 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.
  • recombinant bSTs Two examples are the compounds known as somidobove and sometribove.
  • the present invention is especially preferred for application to these two recombinant bSTs.
  • the method of the invention employs size exclusion HPLC using as the solid phase a hydrophilic porous polymeric gel.
  • Preferred porous gels for use in the invention have an average particle diameter of about 5 ⁇ m to about 15 ⁇ m, and more preferably about lO ⁇ m to about
  • the average pore diameter of the gel will be about 100 to about 200 angstroms, and for the present invention a gel having an average pore diameter of about 150 angstroms is preferred.
  • the porous gel used in the invention preferably has an exclusion limit average molecular weight greater than the molecular weight of the bST non-covalent soluble aggregate, e.g. about 10 or greater, and more
  • the mobile phase for use in the present invention is an aqueous buffer solution having a pH of about 8 to about 12 and which does not denature the bST sample.
  • the buffer may be included in the mobile 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 (HCOl) buffer solution 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 about 0.1 to about 0.4 M.
  • the pH of the mobile phase is more preferably about 9 to about 11, and most preferably about 9 to about 10.
  • size exclusion HPLC set-ups typically include pumps, reservoirs for the aqueous buffer solution, an ultra violet (UV) absorption detector and a fraction collection means.
  • UV ultra violet
  • the bST sample to be treated by the method of the invention is preferably reconstituted in an aqueous buffer solution identical to that used as the mobile phase.
  • concentration of bST in the sample is desirably about 0.05 to about 2 milligrams per milliliter ( g/niL) . More preferably, the bST concentration in the sample is about 0.1 mg/mL to about 1 nig/mL.
  • the pressure on the size exclusion HPLC column provides a suitable flow rate for separations performed in accordance with the invention. The pressure necessary to achieve advantageous flow rates will of course be dependent upon the particle size of the stationary phase.
  • 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.
  • typical column pressures are about 500 to about 1500 psi, and more typically about 800 to about 1000 psi.
  • the behavior of this bST soluble aggregate in solution has also been characterized.
  • the bST soluble aggregate is substantially soluble in non-denaturing aqueous buffer solutions having pH's greater than about 8.5 and less than about 12 ( Figures ID, IE and 7). At pH'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 6). For instance, salting out of the bST soluble aggregate is observed at bicarbonate concentrations above about 0.7 M.
  • the bST soluble aggregate 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) . Denaturing agents such 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 the Examples, infra.
  • a rigid porous polymeric gel having a plurality of hydrophilic groups (the gel was a crosslinked hydroxylated polyether) and having an average particle size of about 10 ⁇ m (tradename, 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 pressure in the range of about 800 to 1000 psi) unless otherwise indicated.
  • the sample injection volume was 20 ⁇ L.
  • 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.
  • Figure IA shows a size exclusion HPLC chromatogram of a biologically active recombinant bST protein reference standard in which only a single main peak containing biologically active bST protein was found.
  • the chromatogram of Figure IA was obtained using the methodology described above, except the flow rate was 1 mL/min.
  • Figures IB and IC show size exclusion HPLC chromatograms of different lots of recombinant bST bulk material. Two strong peaks with excellent baseline resolution between them were observed.
  • the chromatographic first eluting peak ( ⁇ 9 min) contains the high molecular weight bST soluble aggregates, and the second eluting peak (-13 min) is biologically active bST protein.
  • Figures ID and IE show size exclusion chromatographs of samples from the same bulk recombinant bST used in preparing the chromatogram of Figure IC.
  • the lone chromatogram shown in Figure ID was taken with a mobile phase having a pH of 11.3.
  • Two strong peaks were again observed as in Figure IB.
  • the superimposed chromatograms of Figure IE were developed using mobile phases having pH's ranging from 7.9 to 12.2. Diminished peak area response at the lower and higher pH's which were studied evidenced the diminished solubility of the bST soluble aggregate.
  • More advantageous methods of the invention are performed using mobile phase pH's where higher solubility of the bST soluble aggregate exists, e.g. in these runs at pH's between about 9 and about 12.
  • Varying Size Exclusion HPLC conditions The effect of varying ammonium 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 solution.
  • a slight change 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 when the concentration of ammonium bicarbonate ranged above about 0.4 M.
  • the peak area of bST significantly decreased when increasing salt (ammonium bicarbonate ) concentration to higher than about 0.7 M ( Figure 2B) .
  • Preferred size exclusion HPLC methods of the invention are thus performed using a bicarbonate salt concentration in the mobile phase of less than about 0.7 M.
  • the biologically active bST protein fraction in the reference standard (see Figure IA) was collected and its biological activity measured by RRA.
  • a strong correlation was demonstrated between the level of biologically active bST protein measured by the inventive size exclusion HPLC method and the potency data (I.U./mg) measured by RRA.
  • a linear regression plot of the data from the size exclusion HPLC method and RRA is shown in Figure 4. The correlation coefficient for the data was 0.845.
  • inventive size exclusion HPLC method can be used in a determination of the potency value of bST bulk materials. That is, the inventive size exclusion HPLC method can be used to measure 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 be 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 other methods, for example the well known rat weight gain method. Thus, a similar correlation of the size exclusion HPLC results can be made to data obtained by other known bST bio-potency assays.
  • the linearity of the inventive size exclusion HPLC 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/ ⁇ iL treated by size exclusion HPLC in triplicate and a linear regression analysis of the data performed. The reproducibility of the linearity obtained using three different 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%.
  • bST Soluble 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 IB-IE) .
  • a UV spectrum taken by photodiode array detector in the size exclusion HPLC process demonstrated that the UV profiles of the biologically active bST protein fraction and bST soluble aggregate fraction were similar.
  • a UV 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 weight of bST soluble aggregates was measured by a calibration 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 higher than 660,000 (MW of thyroglobumin) .
  • 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 HC1 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 UV-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.
  • SDS sodium dodecyl sulfate
  • the stability of the soluble aggregates in bicarbonate solution was also studied.
  • the isolated bST soluble aggregate solution is stable for two days at 4°C, and for more than 9 hours at room temperature (as determined by measuring size exclusion HPLC peak area over time) .

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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 νm to about 15 νm 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

NON-DENATIJRING POTENCY ASSAY FOR BOVINE 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 monomeiic protein.
Due to inefficiencies of these denaturing and renaturing steps, however, some aggregates 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 iπiportant to establish a method for determining the 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 is 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 therefore needed is an expedient, precise and accurate non-denaturing assay for determining the bio-potency of bST. The present invention addresses these needs. SUMiiARY OF THE INVENTION
One preferred embodiment of the present invention provides a method for determining the potency of a bovine somatotropin sample. This method comprises 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 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 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 separating a biologically active bovine somatotropin fraction from bovine somatotropin by size exclusion HPLC employing as a solid phase a hydrophilic porous polymer gel having an average particle size 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 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 somatotropin non-covalent soluble aggregates contained in the bulk recombinant bovine somatotropin. The method comprises the step of subjecting the bulk recombinant bovine somatotropin to size exclusion HPLC employing as a solid phase a hydrophilic porous polymer gel having an average particle size 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 bovine somatotropin, so as to separate the biologically 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.
DESCRIPTION OF THE FIGURES
Figure IA is a size exclusion HPLC chromatogram of a biologically active bST reference standard using the methodology described in the Examples, infra.
Figures IB through IE are size exclusion HPLC chromatograms of bulk recombinant bST materials using the methodology 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 the biologically active bST protein fraction using the methodology described in the Examples, infra.
Figure 2B is a graph showing the effect of bicarbonate buffer concentration on the size exclusion HPLC peak area response of the biologically 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 bST protein fraction using the methodology described in the Examples, infra.
Figure 3B a graph showing is the effect of mobile phase pH variation on the size exclusion HPLC peak area response of the biologically active bST protein fraction using the methodology described in the Examples, infra. Figure 4 is a graph showing the correlation between radio-receptor assay polency data (IU/ g) and the size exclusion HPLC- easured level of biologically 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 graph 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 the effect of mobile phase pH on the size exclusion HPLC peak area for the biologically active bST protein fraction ("B/A bST") and 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 of 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. DESCRIPTION OF THE PREFERRED EMBODIMENT
For the purposes of pro otiny an understanding of the principles of the invention, reference will now be made to a preferred embodiment thereof and specific lanyuage will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations, further modifications and applications of the 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 embodiment 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 exhibits 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 synthesize bovine somatotropin and other 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 retains 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 chain. Additional modifications, will be understood by those skilled in the art. Therefore, the term bovine somatotropin is used throughout this document to refer to both naturally occurring bovine somatotropin as well as synthetically produced bovine somatotropin which shares the biological properties of naturally occurring bovine somatotropin, and which may be identical or which may vary as to structure.
The present invention is preferred for use with bST produced by recombinant DNA technology ("recombinant bST"). Typically, the recombinant bST is obtained by isolating and denaturing 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 high molecular weight impurities can be removed from the solubilized inclusion bodies by ultrafiltration. However, substantial quantities of biologically inactive bST non-covalent aggregates usually pass through the ultrafilter along with the biologically active bST protein. As a result, the finally-obtained bulk recombinant bST contains biologically active bST 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 invention is especially preferred for application to these two recombinant bSTs. The method of the invention employs size exclusion HPLC using as the solid phase a hydrophilic porous polymeric gel. Preferred porous gels for use in the invention have an average particle diameter of about 5μm to about 15μm, and more preferably about lOμm to about
15μm. Typically, the average pore diameter of the gel will be about 100 to about 200 angstroms, and for the present invention a gel having 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 greater than the molecular weight of the bST non-covalent soluble aggregate, e.g. about 10 or greater, and more
7 preferably about 10 or greater.
The mobile phase for use in the 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 the mobile 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 (HCOl) buffer solution, 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 about 0.1 to about 0.4 M. The pH of the mobile phase is more preferably about 9 to about 11, and most preferably about 9 to about 10.
The solid phase is packed in a conventional HPLC column, which is a component of a conventional size exclusion HPLC set-up. For example, size exclusion HPLC set-ups typically include pumps, reservoirs for the aqueous buffer solution, an ultra violet (UV) absorption detector and a fraction collection means.
The bST sample to be treated by the method of the invention is preferably reconstituted in an aqueous buffer solution identical to that used as the mobile phase. The concentration of bST in the sample is desirably about 0.05 to about 2 milligrams per milliliter ( g/niL) . More preferably, the bST concentration in the sample is about 0.1 mg/mL to about 1 nig/mL. The pressure on the size exclusion HPLC column provides a suitable flow rate for separations performed in accordance with the invention. The pressure necessary to achieve advantageous flow rates will of course be dependent upon 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 column pressures are about 500 to about 1500 psi, and more typically about 800 to about 1000 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 IB and IC, 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 HPLC (Figure 5) . The bST soluble aggregates are biologically inactive as determined by radio receptor assay.
The behavior of this bST soluble aggregate in solution has also been characterized. The bST soluble aggregate is substantially soluble in non-denaturing aqueous buffer solutions having pH's greater than about 8.5 and less than about 12 (Figures ID, IE and 7). At pH'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 6). For instance, salting out of the bST soluble aggregate is observed at bicarbonate concentrations above about 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) . Denaturing agents such 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 the 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.
EXAMPLES
Chemicals and Reagents
All reagents were of analytical-reagent grade and were used without further purification. 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.
Conditions of Size Exclusion HPLC Chromatography
A rigid porous polymeric gel having a plurality of hydrophilic groups (the gel was a crosslinked hydroxylated polyether) and having an average particle size of about 10 μm (tradename, 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 pressure in the range of about 800 to 1000 psi) unless otherwise indicated. The sample injection volume was 20 μL. 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 consisting 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 μm filter prior to injection.
Results of Size Exclusion HPLC of bST Samples
Figure IA shows a size exclusion HPLC chromatogram of a biologically active recombinant bST protein reference standard in which only a single main peak containing biologically active bST protein was found. The chromatogram of Figure IA was obtained using the methodology described above, except the flow rate was 1 mL/min. Figures IB and IC show size exclusion HPLC chromatograms of different lots of recombinant bST bulk material. Two strong peaks with excellent baseline resolution between them were observed. The chromatographic first eluting peak (~9 min) contains the high molecular weight bST soluble aggregates, and the second eluting peak (-13 min) is biologically active bST protein. Figures ID and IE show size exclusion chromatographs of samples from the same bulk recombinant bST used in preparing the chromatogram of Figure IC. The lone chromatogram shown in Figure ID was taken with a mobile phase having a pH of 11.3. Two strong peaks were again observed as in Figure IB. The superimposed chromatograms of Figure IE were developed using mobile phases having pH's ranging from 7.9 to 12.2. Diminished peak area response at the lower and higher pH's which were studied evidenced the diminished solubility of the bST soluble aggregate. More advantageous methods of the invention are performed using mobile phase pH's where higher solubility of the bST soluble aggregate exists, e.g. in these runs at pH's between about 9 and about 12. Additional size exclusion HPLC chromatograms of the lots shown in Figures IB and IC were taken under the same conditions except using a different non-denaturing mobile phase (0.05 M borate buffer solution). The chromatograms obtained were similar to those shown in Figures IB and IC, 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 the invention.
Varying Size Exclusion HPLC conditions The effect of varying ammonium 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 solution. A slight change 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 when the concentration of ammonium bicarbonate ranged above about 0.4 M. Further, the peak area of bST significantly decreased when increasing salt (ammonium bicarbonate ) concentration to higher than about 0.7 M (Figure 2B) . Preferred size exclusion HPLC methods of the invention are thus performed using a bicarbonate salt concentration in the mobile phase of less than about 0.7 M.
The effect of varying pH of the mobile phase on size exclusion HPLC 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 about 7.5 to about 10.5 did not affect the elution time of bST; however, peak area response was slightly increased over the same pH range (Figures 3A, 3B) . The most advantageous size exclusion HPLC flow rate in this study was found to be about 0.5 πiL/min. A flow rate of 0.3 mL/min. is usable and results in equivalent efficiency, but nearly doubles the run time. The biologically active bST protein peak overlapped with the void peak at 1 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 IA) was collected and its biological activity measured by RRA. The results indicated that the collected biologically active bST protein component in mobile phase retains equal biological activity to the parent solution before treatment by the size exclusion HPLC. In testing of several different lots of recombinant bulk bST, a strong correlation was demonstrated between the level of biologically active bST protein measured by the inventive size exclusion HPLC method and the potency data (I.U./mg) measured by RRA. A linear regression plot of the data from the size exclusion HPLC method and RRA is shown in Figure 4. The correlation coefficient for the data was 0.845. These results clearly demonstrate that the inventive size exclusion HPLC method can be used in a determination of the potency value of bST bulk materials. That is, the inventive size exclusion HPLC method can be used to measure 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 be 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 other methods, for example the well known rat weight gain method. Thus, a similar correlation of the size exclusion HPLC results can be made to data obtained by other known bST bio-potency assays.
Linearity And Precision of Method
The linearity of the inventive size exclusion HPLC 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/πiL treated by size exclusion HPLC in triplicate and a linear regression analysis of the data performed. The reproducibility of the linearity obtained using three different 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%.
Figure imgf000017_0001
The precision of the inventive size exclusion HPLC method 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, demonstrate that the R.S.D. of biologically active bST protein measured is less than 2.8 %, and the reproducibility of elution time is less than 0.18% R.S.D.
TABLE 2
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
2 3 81.7 1.27 831 0.09
3 3 82.7 0.75 822 0.10 002 1 3 87.1 2.23 849 0.18
2 3 86.1 2.72 830 0.06
3 3 88.9 1.34 824 0.10
003 1 3 44.9 0.47 853 0.08
2 3 46.3 1.14 837 0.13
Figure imgf000018_0001
3 3 46.8 0.75 829 0.10 n = Replicate Number te = Retention Time (in seconds)
RSD = Relative Standard Deviation
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) . S amp le n Recovery RSD (%) (%)
003A 3 102.5% 0.34 003A 3 101.2% 0.58 003B 3 103.8% 0.90 003B 3 101.4% 0.72
n = Replicate Number RSD = 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 Soluble 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 IB-IE) . In further work, a UV spectrum taken by photodiode array detector in the size exclusion HPLC process demonstrated that the UV profiles of the biologically active bST protein fraction and bST soluble aggregate fraction were similar. A UV 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 weight of bST soluble aggregates was measured by a calibration 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 higher than 660,000 (MW of thyroglobumin) .
To further characterize the bST soluble aggregate, bulk bST was dissolved in 0.4 M ammonium bicarbonate (pH 9) to make a 5 mg/πiL solution. After centrifugation at 16,000 g, the supernatant was treated under the standard size exclusion HPLC 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 in 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 HC1 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 UV-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 soluble aggregates was strongly affected by saϋt 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 HPLC under the above-noted conditions except varying the concentration of ammonium bicarbonate in the prepared sample and mobile phase, as indicated in Figure 6. Similarly, the effect of the pH of the bicarbonate buffer solutions on the solubility of the bST soluble aggregate was investigated. At pH less than 8.5 and pH greater than 12.5, bST soluble aggregate precipitates out of the 0.2 M bicarbonate solution. Solubility of the bST soluble agqregate increased with increasing 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 4°C, 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, the 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

What is claimed is:
1. A method for determining the potency of bovine somatotropin, comprising: measuring the level of biologically active bovine 5 somatotropin protein in the 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 to 0 about 12 and which is 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.
5 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.
o 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 lovel of biologically active 5 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 Mm-
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.
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 separating a biologically active bovine somatotropin fraction from bovine somatotropin, comprising: separating a biologically active bovine somatotropin fraction from bovine somatotropin by size exclusion HPLC employing as a stationary phase a hydrophilic porous polymer gel having an average particle size 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 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 μ .
17. The method of claim 14 wherein said hydrophilic porous polymer gel has an average pore diameter of about 150 angstroms.
18. The method of claim 16 wherein said size exclusion HPLC is conducted at pressure of about 800 to about 1000 psi.
19. The method of claim 16 wherein the pH of the mobile phase is about 9 to about 11.
20. 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.
21. The method of claim 18 wherein said hydrophilic porous polymer gel has an average pore diameter of about 150 angstroms.
22. The method of claim 20 wherein said size exclusion HPLC is conducted at a pressure of about 800 to about 1000 psi.
23. The method of claim 22 wherein said hydrophilic porous polymer gel has an average pore diameter of about 150 angstroms.
24. A method for treating bulk recombinant bovine somatotropin to separate biologically active bovine somatotropin protein from biologically inactive bovine somatotropin non-covalent soluble aggregates contained in the bulk recombinant bovine somatotropin, comprising: subjecting the bulk recombinant bovine somatotropin to size exclusion HPLC employing as a stationary phase a hydrophilic porous polymer gel having an average particle size 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 bovine somatotropin, so as to separate the biologically active bovine somatotropin from the biologically inactive bovine somatotropin non-covalent soluble aggregates.
25. The method of claim 24 wherein said buffered aqueous solution is a bicarbonate buffer solution.
26. The method of claim 24 wherein said hydrophilic porous polymer gel has an average particle size of about 10 μm to about 15 μ .
27. The method of claim 26 wherein said size exclusion HPLC is conducted at pressure of about 800 to about 1000 psi.
28. The method of claim 26 wherein the pH of the mobile phase is about 9 to about 11.
29. The method of claim 26 wherein the mobile phase is an ammonium bicarbonate buffer solution at a concentration of less than about 0.7 M.
30. The method of claim 27 wherein said hydrophilic porous polymer gel has an average pore diameter of about 150 angstroms.
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CHEMICAL ABSTRACTS, Vol. 108, No. 13, issued 1988, SEAMAN, W.J., "The Lack of a Growth-Promoting Effect of Orally Administered Bovine Somatotropin in the Rat Body-Weight Gain Bioassay", page 105, column 1, Abstract Number 106843r, Fundam. Appl. Toxicol., 10 (2), 287-294, entire document. *
Journal of Chromatography, Vol. 326, issued 1985, ANDERSSON, T., "Agarose-Based Media for High-Resolution Gel Filtration of Biopolymers", pages 33-44 (1985), entire document. *
Journal of Chromatography, Vol. 435, issued 1988, RIGGIN, R.M., "High-Performance Size-Exclusion Chromatographic Determination of the Potency of Biosynthetic Human Growth Hormone Products", pages 307-318, entire document. *
See also references of EP0681696A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0747698A1 (en) * 1995-06-06 1996-12-11 Eli Lilly And Company Non-denaturing potency assay for somatotropin

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HU9502233D0 (en) 1995-09-28
HUT75678A (en) 1997-05-28
ZA94169B (en) 1994-09-07
EP0681696A1 (en) 1995-11-15
JPH08509288A (en) 1996-10-01
CZ193595A3 (en) 1996-05-15
NO952948D0 (en) 1995-07-25
NO952948L (en) 1995-09-18
KR960700454A (en) 1996-01-20
EP0681696A4 (en) 1995-11-22
CN1119042A (en) 1996-03-20
CA2154794A1 (en) 1994-01-24
PL310053A1 (en) 1995-11-13
AU6094794A (en) 1994-08-15
BR9405672A (en) 1995-11-14

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