HUT75678A - Non-denaturing potency assay for bovine somatotropin - Google Patents

Description

Procedure for the determination of the efficacy of bovine somatotropin by non-denaturing procedure

ELI LILLY AND COMPANY, INDIANAPOLIS, Indiana,

USA

Inventor:

CHANG Jen, P. INDIANAPOLIS, Indiana,

USA

Date of filing:

1994

priority:

1993/1/01

AMERICAN

26 (08 / 009,034),

UNITED STATES

International Application Number: PCT / US94 / 00850

International Publication Number: WO 94/17403 • · · ·

The present invention relates to bovine somatotropin (bST), more particularly, the present invention provides a non-denaturing method for the isolation of the biologically active bST protein fraction from bST, which is useful for testing the efficacy of bST.

Recombinant DNA technology has now made it possible to produce bST on a large scale. Recombinant microorganisms such as recombinant Escherichia coli produce bST in the form of insoluble granules in their plasma. Refractory or inclusion bodies contain aggregated, denatured bST.

Refractive bodies are recovered and usually treated with a denaturation such as guanidine hydrochloride, sodium dodecyl sulfate or urea. Denaturation unwinds and solubilizes the irregularly wound bST molecules. Later, the bST molecules are renatured to form the correctly folded, biologically active bST monomeric protein.

However, due to the imperfection of the denaturation and renaturation steps mentioned above, all irregularly wound, biologically inactive aggregates are also formed.

Thus, the high mass bST contains both a biologically active monomer and a biologically inactive bST aggregate. Therefore, it was important to develop a method for determining the effectiveness of bST in quality control.

The weight gain of the rat was previously used to determine the efficacy of bST. The essence of the method is to administer bST to the rat and monitor his weight gain, · · · ·

- 3 of which provides data that describes the efficacy of bST. However, for routine tests, this test procedure is not applicable to the accurate classification of bST. Radioreceptor assay (RRA) may also be used to determine the efficacy of bST, but this is time consuming. The RRA is also inaccurate, so more studies are usually performed and the average of the results gives the efficacy value of bST.

Reverse phase high performance liquid chromatography (RPHPLC) is used to determine proteins, however, most RPHPCL methods used are not applicable for measuring the potency of bST because they use acidic media and organic solvents in their mobile phase to denature bST.

Previously, size exclusion HPLC was used for the determination of bST, the mobile phase of which contained sodium dodecyl sulfate or guanidine hydrochloride. However, bioassay cannot be determined by this method as the mobile phase denatures bST.

Thus, there was a need for a suitable, accurate and reliable non-denaturing procedure for determining the biological efficacy of bST. The present invention seeks to meet this need.

A preferred embodiment of the present invention provides a method for determining the efficacy of a bovine somatotropin sample. The method consists of measuring the level of biologically active bovine somtotropin protein in a bovine somatotropin sample by size exclusion HPLC. For size exclusion HPLC, a hydrophilic, porous polymer gel with an average particle size of 5-15 μπι

- 4 is used as stationary phase; the mobile phase is a pH = 8-12 pH buffered aqueous solution which does not denature the bovine somatotropin sample. The determination of the potency of a bovine somatoropine sample is based on the measurement of the level of biologically active somatotropin protein in the sample.

Another preferred embodiment of the present invention provides a process for recovering a biologically active bovine somatotropin fraction from bovine somatotropin. The method uses size exclusion HPLC, wherein the stationary phase is a porous polymer having an average particle size of 5-15 μιη; the mobile phase is a pH = 8-12 pH buffered aqueous solution which does not denature bovine somototropin.

A further preferred embodiment of the present invention provides a process for the processing of bulk recombinant bovine somatotropin for the purpose of separating biologically active bovine somatotropin from a non-covalent soluble aggregate of biologically inactive bovine somatotropin.

The method consists in subjecting the bulk recombinant bovine somatotropin to size exclusion HPLC, a solid phase hydrophilic, porous polymer gel having an average particle size of 5-15 μιη; using pH 8-12 as a mobile phase buffered aqueous solution which does not denature bovine somatotropin. Chromatography is performed to separate the biologically active bovine somatotropin from non-covalent soluble aggregates of the biologically inactive bovine somatotropin.

The present invention provides methods for beneficially separating the biologically active bST protein from the biologically inactive bST form. The methods are well suited for the beneficial, accurate and reliable determination of the efficacy of bST. Furthermore, the inventive methods were the first to separate and thoroughly characterize the non-covalent soluble aggregate of bST.

Further objects, features and advantages of the invention will be apparent from the description.

Description of the figures:

Figure 1A - Standard size exclusion HPLC chromatogram of biologically active bST using the method described in the Examples below.

Figures 1B-1E are size exclusion HPLC chromatograms of high volume recombinant bST materials using the methods described in the Examples. The chromatograms show the presence of soluble aggregates of bST in the bulk materials.

Figure 2A - Effect of the concentration of bicarbonate buffer on size exclusion HPLC retention time of biologically active bST protein fraction using the procedure described in the Examples.

Figure 2B - Effect of the concentration of bicarbonate buffer on the size exclusion HPLC peak area of the biologically active bST protein fraction using the procedure described in the Examples.

Figure 3A - Effect of mobile phase chemistry on size exclusion HPLC retention time of biologically active bST protein fraction using the procedure described in the Examples.

Figure 3B - Effect of mobile phase chemistry on the size exclusion HPLC peak of the biologically active bST protein fraction using the procedure described in the Examples.

Figure 4 - Correlation between potency data (JU / mg) of biologically active bST protein with radioreceptor assay and concentration data by size exclusion HPLC for some of the different recombinant bSTs using the procedures described in the examples.

Figure 5 - Relationship between hanging molecular weight and size exclusion HPLC elution time, calibration curve with standard proteins, and data for biologically active bST protein (B / A bST) and bST soluble aggregate (Agg). The measurement conditions are given in the examples below.

Figure 6 - Effect of ammonium bicarbonate concentration on peak area obtained by size exclusion HPLC for biologically active bST protein fraction (B / A bST) and fraction of bST soluble aggregate (Agg); the assay conditions are described in the examples below.

Figure 7 - Effect of mobile phase chemistry on peak area obtained by size exclusion HPLC for biologically active fraction of bST protein and fraction containing soluble aggregate of bST (Agg); the assay conditions are given in the examples below.

Figure 8 - Effect of centrifugation on peak area by size exclusion HPLC for biologically active bST protein fraction (B / A bST) and fraction containing bST soluble aggregate (Agg); the assay conditions are given in the examples below.

For a better understanding of the principles of the invention, reference will be made to a preferred embodiment of the invention and a special expression will be used to describe it.

It is, however, to be understood that this does not limit the scope of the invention, and changes, further modifications, and application of the principles of the invention illustrated herein will be apparent to those skilled in the art.

As noted above, a preferred embodiment of the present invention is directed to the separation of the biologically active bovine somatotropin fraction from bovine somatotropin. The term bovine somatotropin refers to a substance of natural or synthetic origin which exhibits the properties of natural bovine somatotropin. Bovine somatotropin may be extracted from appropriate bovine tissues such as the pituitary gland; or, by a well-established technique, bovine somatotropin and the like can be produced by genetically modified microorganisms such as bacteria. Often, methods that produce modified bovine somatotropin, that is, somatotropins whose structure is different from that of naturally occurring growth hormone but retains the biological activity of the natural hormone, are often suitable or even more preferred. For example, a modified bovine somatotropin may have one or more amino acids at one or both ends of the polypeptide chain. Further modifications are known to those skilled in the art. Thus, in the present invention, the term bovine somatotropin is used to refer to naturally occurring bovine somatotropin as well as to virtually produced bovine somatotropin having the same biological property of naturally occurring bovine somatotropin as ropin. or it may be different.

The present invention is advantageously applicable to bST (recombinant bST) produced by recombinant DNA technology. Typically, recombinant bST is obtained by isolating, denaturing and dissolving the recombinant bST-containing inclusions. The bST is then renatured into the biologically active bST protein. The denaturation / renaturation steps produce biologically inactive bST non-covalent aggregates with the desired biologically active bST protein. Colloids and certain very high molecular weight impurities can be removed by ultrafiltration. A substantial amount of the biologically inactive bST non-covalent aggregates, however, passes through the ultrafilter together with the biologically active bST protein. As a result, the recombinant bST final product contains both a biologically active bST protein and a biologically inactive bST soluble aggregate.

The method of the present invention can be used to process this high mass recombinant bST with the aim of separating the biologically active bST protein from the biologically inactive bST non-covalent aggregates. This method further provides a method for determining the efficacy, reliability, and accuracy of high mass bST.

Two examples of recombinant bSTs are known as somidobore and sometribove. The present invention is particularly advantageous for the use of the two recombinant bSTs for the purpose of the present invention. Rates are relative.

The method of the present invention utilizes size exclusion HPLC using a hydrophilic porous polymer gel as the solid phase. The porous gel useful in the present invention preferably has an average particle diameter of 5 to 15, more preferably 10 to 15 µm. The gel has an average pore size of 100-200 Å, and 150 Å is preferred for the present invention. In another aspect, the porous gel used in the present invention has an exclusion limit on average greater than the molecular weight of the non-covalent soluble aggregate of bST, for example 10 ⁶ or more, more preferably 10 ⁷ or more.

The mobile phase used in the present invention is an aqueous buffer solution having a pH of 8-12 which does not denature the bST sample. In the mobile phase, any concentration up to the ionic strength that does not salt the protein from the bST sample can generally be used. A preferred mobile phase is, for example, an aqueous solution of aqueous bicarbonate (HCO3) buffer, such as ammonium bicarbonate. The concentration of the bicarbonate buffer should be desirable below 0.7M, preferably from 0.1 to 0.4M. Most preferably, the pH of the mobile phase is between pH 9-11 and most preferably between pH 9-10.

The solid phase is loaded onto a standard HPLC column that is part of a standard size exclusion HPLC apparatus. For example, a size exclusion HPLC apparatus typically comprises pumps, aqueous buffer tanks, an ultraviolet (UV) absorption detector, and a fraction collector. The bST sample processed by the method of the present invention can preferably be prepared in the same aqueous buffer solution used as mobile phase. The desired concentration of bST in the sample is 0.05 to 2 milligrams per milliliter. More preferably, the sample has a bST concentration of 0.1-1 mg / ml.

The pressure applied to the size exclusion HPLC column provides the appropriate flow rate for the separation according to the invention. The pressure required for a sufficient flow rate is, of course, determined by the particle size of the stationary phase. In a preferred method of the invention, the pressure and flow rate used for size exclusion HPLC completely elutes the biologically active bST protein fraction within 30 minutes, providing a suitable assay opportunity. In this respect, a typical column pressure is 500-1500 psi, more preferably 800-1000 psi.

Alternatively, the non-denaturing size exclusion HPLC method described above can be used to isolate and characterize the high molecular weight soluble aggregate of bST. Figures 1B and IC show approx. At 9 minutes, peaks containing these high molecular weight soluble bST aggregates begin. These soluble bST aggregates have a molecular weight greater than 660,000 as determined by size exclusion HPLC (Figure 5). As demonstrated by the radio receptor assay, the bST soluble aggregates are biologically inactive.

The behavior of the bST soluble aggregate in solution was also determined. The soluble aggregate bST is significantly soluble in the non-denaturing aqueous buffer solution having a pH greater than pH 8.5 and a pH lower than pH 12 (ID. 1E and Fig. 7). Outside this range, the soluble aggregate precipitates out of the non-denaturing aqueous buffer. Salt concentration may also result in precipitation of the soluble aggregate bST (Figure 6). For example, at concentrations above 0.7M hydrogen carbonate, salting of the soluble aggregate of bST is observed. In addition, the bST soluble aggregate remains in solution in 0.2M bicarbonate buffer (pH 9) when centrifuged at 16000 g for 30 minutes or longer (Figure 8). Denaturing agents such as sodium dodecyl sulfate or urea denature the bST soluble aggregate to monomeric bST. These and other properties of the bST soluble aggregate are further discussed in the examples below.

The following are examples to illustrate what has been said. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. Variations and modifications not affecting the principle of the invention are also within the scope of the invention.

Examples

Compounds and Reagents: All were of analytical purity and were used without further purification. The water was obtained from the Millipore Milli-Q Water Purifier. Reference substance bST and high-mass recombinant bST (somidobove) were obtained from Eli Lilly and Company.

Size exclusion HPLC chromatographic conditions: The brittle, porous polymer gel used in the Examples contains a number of hydrophilic groups (the gel is crosslinked with hydroxylated polyether) and has an average particle size of 10 μτη (Beckman Sepharogel TSK 3000 PW column,

600x7.5mm diameter). Unless otherwise stated, the separations described herein are used as a mobile phase in 0.2M ammonium bicarbonate solution adjusted to pH 9 with sodium hydroxide. All separations were performed at room temperature and at a flow rate of 0.5 ml / min unless otherwise indicated (pressure reached 800-1000 psi). The volume of the injected sample is 20 μΐ. Most separations are performed on a Waters 625 LC instrument equipped with a 911 photodiode array detector and WISP 721 automatic sample dispenser. However, accurate and linearity tests are performed on a Beckman System-Gold HPLC, which consists of a Model 126 solvent supply system, a Model 116 variable wavelength detector and a Model 507 automatic sample dispenser.

Preparation of the sample: prepare samples and a solution of standard materials in the mobile phase. Unless otherwise stated, all bST samples are at a concentration of 1 mg / ml. To reduce the risk of incomplete dissolution, one exception of the mobile phase is added to the bST and the sample solution is allowed to stand at room temperature for 30 minutes and then the sample solution is gently stirred for 5-10 minutes. Filter the sample through an Acrodisc 0.45 μιη filter before injection.

Results of size exclusion HPLC chromatography of bST samples:

Figure 1A is a size exclusion HPLC chromatogram of a reference standard for a biologically active recombinant bST protein, with only one major peak containing the biologically active bST protein.

The chromatogram shown in Figure 1A was obtained according to the procedure described above, except that the flow rate was 1 ml / min. Figures 1B and 1C show size exclusion HPLC chromatograms of various bST materials obtained in bulk. Two strong peaks are observed between them with excellent baseline support. The first eluting peak (λ- 9 min) contains the high molecular weight bST soluble aggregate, the second peak 13 min) contains the biologically active bST protein.

Figures 1D and 1E show size exclusion chromatograms of samples from the same recombinant bST material used to record the chromatogram of Figure IC. The unique chromatogram of Figure ID was obtained with a pH of 11.3 mobile phase. The stacked chromatograms of Figure 1E were recorded using mobile phases between pH 7.9 and 12.2. The area under the peak decreases at the lower and higher pH values tested, demonstrating the decreasing solubility of the bST soluble aggregate. It is preferred to use the mobile phase of the invention in the process of the invention with a higher solubility of the soluble aggregate of bST, for example in the range of pH 9 to 12.

Additional size exclusion chromatograms were recorded between 1B and ···· · with 0.05M borate

- the same conditions as those used for Figure 14 IC except that another non-denaturing mobile phase was used as a buffer. The chromatograms were similar to those in Figures 1B and IC, where two peaks were observed with good baseline support. Thus, other non-denaturing aqueous buffers can be readily used for size exclusion HPLC separation.

Changes in size exclusion HPLC chromatographic conditions:

The effect of different concentrations of ammonium bicarbonate on the elution time of the biologically active bST protein fraction was investigated. A series of experiments demonstrate that the biologically active bST protein, by varying the ammonium bicarbonate solution, gives a typical size exclusion HPLC curve (Figure 2A). A slight change in the elution time is observed between 0.1M and 0.4M ammonium bicarbonate. The elution time increases faster when the concentration of ammonium bicarbonate rises above 0.4M. The peak area of bST is significantly reduced when the salt concentration (ammonium bicarbonate) is higher than 0.7M (Figure 2B). Thus, for the preferred size exclusion HPLC used in the present invention, the concentration of hydrogen carbonate salt in the mobile phase is less than 0.7M.

We also studied the effect of the mobile phase on the elution time and the peak area of the biologically active bST protein fraction. Increasing the pH of the 0.2M ammonium bicarbonate mobile phase at pH 7.5 and pH 10.5 has no effect on the elution time of bST; however, the area under the peak slightly increases in the same pH range (Figures 3A, 3B).

The most preferred flow rate for size exclusion HPLC in our assays was 0.5 ml / min. A flow rate of 0.3 ml / min is applicable and has the same efficiency, but the runtime is nearly doubled. The bioactive bST protein peak and the blank peak overlap at a flow rate of 1 ml / min.

To demonstrate that the method of the invention does not denature bST, a fraction of the reference standard biologically active bST protein was collected and its biological activity (Figure IA) was measured by the RRA method. The results show that the biologically active bST protein component collected in the mobile phase retained the biological activity of the stock solution prior to size exclusion HPLC.

Examination of several different recombinant bST samples shows a strong correlation between the level of activity of the biologically active bST protein according to the invention by size exclusion HPLC and the efficacy data (IU / mg) measured by the RRA method. A linear regression graph of data obtained by size exclusion HPLC and RRA is shown in Figure 4. The correlation coefficient of the data is 0.845. These data clearly demonstrate that the size exclusion HPLC of the present invention is a useful method for determining the efficiency of bST samples. That is, the size exclusion HPLC method of the present invention provides

- 16 - .........

can be used to determine the level of biologically active bST protein (e.g., as a percentage of the peak area) of a bST sample of unknown efficiency. The level of biologically active bST in the sample can then be plotted against a calibration graph similar to Figure 4, consisting of bST controls obtained by RRA and size exclusion HPLC. The RRA data are also well in line with the bioactivity data obtained by other methods, such as the well-known rat weight gain method. A similar correlation can thus be found between data obtained by size exclusion HPLC and other known bST bioavailability assays.

Linearity and Accuracy of the Method: To determine the linearity of the size exclusion HPLC method of the invention, a concentration series is prepared by solubilizing the mobile phase with a biologically active reference bST. Standard bST solutions of 0.1-1 mg / ml are assayed in triplicate by size exclusion HPLC and linear regression analysis of the data is performed. The reproducibility of the linearity determined on three different columns and on different days is shown in Table I. The correlation coefficient is in the range 0.9998-1.000 and the mean relative standard deviation (R.S.D.) is 1.36%

- 17 · ♦ ·· ··

TABLE I

steepness 306600 301 200 311 400 R.S.D. % 0.60 1.51 0.77 axis of y ordinate -3 940 -2 050 -3 270 correlation coefficient 1.0000 .9998 0.9999

The precision of the size exclusion HPLC of the present invention was determined over three days with two different HPLC systems, three different serial columns, and three different bST samples. II. The results presented in Table II illustrate that the biologically active bST protein has an established R.S.D of less than 2.8% and a retention time reproducibility of less than 0.18% of R.S.D. value.

····· · ♦ ·· ······················································································•

II. SPREADSHEET

Beckman system Waters system

sample Sun n bST% RSD% R.I. RSD% bST% RSD% R.I. 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 2.33 849 0.18 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 0.10 003 1 3 44.9 0.47 853 0.08 44.5 2.40 827 0.10 2 3 46.3 1.48 837 0.13 46.9 1.21 854 0.17 3 3 46.8 0.75 829 0.10

n = number of repetitions

R.I. = retention time (seconds)

RSD = relative standard deviation

The bST recovery assays were performed by adding the biologically active bST protein reference standard at two different concentrations to two different bST samples. The recovery values obtained in these tests are in the range of 101-104% (Table III).

- 19 - • · · · * · · • * · · · · · • ·· · · «* III. T A B L A Z A T sample added refe- measured value n recovery RSD rencia standard (mg) (Mg) (%) (%) 003A 0.186 0.191 3 102.5% 0.34 003A 0.465 0.471 3 101.2% 0.58 003B 0.186 0.193 3 103.8% 0.90 003B 0.465 0.472 3 101.4% 0.72

η = number of repetitions

RSD = relative standard deviation

Characterization of the bst soluble aggregate: As noted above, the peak at 13 minutes represents the biologically active bST protein fraction and the peak at 9 minutes represents the fraction of the bST soluble aggregate (Figures 1B-1E). UV spectra of a size exclusion HPLC photodiode array detector indicate that the biologically active bST protein fraction and the bST soluble aggregate fraction have similar UV spectra. The UV spectrum of the soluble aggregate of bST exhibits a difference in the range of 250-270 nm, which may be due to the alteration of the aromatic amino acid environment after aggregation.

The molecular weights of the bST aggregates were determined using a calibration curve using size exclusion HPLC. The results are shown in Figure 5. The soluble aggregate bST elutes at the column exclusion volume and has a molecular weight of more than 660000 (thyroglobulin molecular weight).

• ·

To further characterize the bST soluble aggregate, the bST sample was dissolved in 0.4 M ammonium bicarbonate solution (pH 9) at a concentration of 5 mg / ml. After centrifugation at 16000 g, the supernatant was subjected to standard size exclusion HPLC as described above, except that the mobile phase was 0.4M ammonium bicarbonate. Separation yields two components, the biologically active bST protein and the bST soluble aggregate, which are collected in separate fractions.

Each collected fraction was re-chromatographed to confirm their retention time and purity. The results show that the bST aggregate remains in solution after centrifugation.

Attempts were made to confirm that the bST soluble aggregate is non-covalently bound. If the bST soluble aggregate is a non-covalently bound protein, it must be dissociated into a monomer in the presence of denaturants such as detergents, high concentrations of urea and guanidine-HCl.

To determine this, a 1: 1 mixture of soluble aggregate fraction of bST and 2% sodium dodecyl sulfate (SDS) was collected by chromatography on a duPont 250 column; mobile phase: 0.4M hydrogen carbonate solution containing 1% SDS. As expected, the chromatographic profile and UV spectrum of the dissociated protein were identical to that of the SDS monomer obtained under the same conditions. Similar studies demonstrate that the bST soluble aggregate may also be dissociated in 3M urea.

Further characteristics of the soluble aggregate of bST in solution were also determined. The solubility of the bST soluble aggregate can be strongly influenced by the salt concentration of the solution. By increasing the concentration of bicarbonate, the solubility of the soluble aggregate is sharply reduced due to the protein salinity phenomenon, as demonstrated by size exclusion HPLC. The assays were performed using the HPLC conditions described above, with the difference that the concentration of bicarbonate in the sample and in the mobile phase was changed as shown in Figure 6. Below pH 8.5 and above pH 12.5, the bST solution snow aggregate precipitates in 0.2M bicarbonate solution. Between pH 8.5 and pH 9.5, the solubility of the bST soluble aggregate increases with increasing pH (Figure 7).

The stability of the soluble aggregate in bicarbonate solution was studied. The bST soluble aggregate solution is stable at 4 ° C for two days at room temperature for more than 9 hours (determined by the peak area of the size exclusion HPLC chromatogram).

Furthermore, centrifugation of the bST solubilizing aggregate at 16,000 g will remain in solution for at least 30 minutes (Figure 8).

Claims (30)

1. A method for determining the efficacy of bovine somatotropin by measuring the biological activity of bovine somatotropin protein by size exclusion HPLC, 5-15 μπι average particle size, hydrophilic, porous, chemically, using a non-denaturing buffered aqueous solution of the bovine somatotropin sample; and determining the efficacy of the bovine somatotropin sample based on the measured level of biologically active bovine somatotropin protein. The process of claim 1, wherein said hydrophilic porous polymer gel has an average particle size of 10-15 µπι. The process of claim 1, wherein said buffered aqueous solution is a bicarbonate buffer solution. The process of claim 3, wherein said hydrophilic porous polymer gel has an average particle size of 10-15 µιη. The method of claim 4, wherein said determination means that the measured level of the biologically active bST protein is plotted against a calibration curve obtained from size exclusion HPLC and correlated by RRA. The process according to claim 4, wherein said hydrophilic porous polymer gel has an average particle size of about. 10 μτη. • · ··· · «· · •, · · · · · · · · · · · · · · · »·· · * The process of claim 4, having an average pore diameter of a porous polymer gel The process of claim 6, wherein said hydrophilic is 150 Å. wherein the pH of the mobile phase is 9-11. The process of claim 8, wherein said hydrophilic porous polymer gel has an average pore diameter of 150 Å. The method of claim 6, wherein said size exclusion HPLC is conducted at a pressure of 800-1000 psi. The process of claim 6, wherein the mobile phase is an ammonium bicarbonate buffer solution at a concentration of less than 0.7M. The process of claim 11, wherein said size exclusion HPLC is conducted at a pressure of 800-1000 psi. The process of claim 12, wherein said hydrophilic porous polymer gel has an average pore diameter of 150 Å. 14. A method for separating a biologically active bovine somatotropin fraction from a biologically inactive somatropin fraction in a bovine somatotropin sample, wherein the biologically active bovine somatotropin fraction is used to separate the biologically inactive bovine somatotropin fraction from -15 μτη. average particle size hydrophilic porous polymer gel and the mobile phase is a pH 8 to 12 buffered aqueous solution of non-denatured bovine somatotropin. The process of claim 14, wherein said buffered aqueous solution is a bicarbonate solution. · · · · · · · · · · · · · · · · · · · · · · · · ··· The process of claim 14, wherein said hydrophilic porous polymer gel has an average particle size of 10-15 µτη. The process of claim 14, wherein said hydrophilic porous polymer gel has an average pore diameter of 150 Å. The method of claim 16, wherein said size exclusion HPLC is conducted at a pressure of 800-1000 psi. The process of claim 16, wherein the mobile phase has a pH of 9-11. The process of claim 16, wherein the mobile phase is a solution of ammonium bicarbonate at a concentration of less than 0.7M. The process of claim 18, wherein said hydrophilic porous polymer gel has an average pore diameter of 150 Å. The method of claim 20, wherein said size exclusion HPLC is conducted at a pressure of 800-1000 psi. The method of claim 22, wherein said hydrophilic porous polymer has an average porosity of 150 A. 24. A process for processing high-weight recombinant bovine somatotropin to separate biologically active bovine somatotropin from a biologically inactive recombinant bovine somatotropin, - Somatotropin is subjected to size exclusion HPLC with 5-15 μπι average particle size, hydrophilic, porous polymer gel as the stationary phase, pH 8-12 as the mobile phase, bovine matotropin a non-denaturing, buffered aqueous solution is used. The process of claim 24, wherein said buffered aqueous solution is a bicarbonate solution. The process of claim 24, wherein said hydrophilic porous ppolymer gel has an average particle size of 10-15 µιη. The method of claim 26, wherein said size is by exclusion HPLC at 800-1000 psi. The method of claim 26, wherein the mobile phase has a pH of 9-11. The method of claim 26, wherein the mobile phase is a solution of ammonium bicarbonate at a concentration of less than 0.7M. The process of claim 27, wherein said hydrophilic porous polymer gel has an average pore diameter of 150 Å.