Board of Patent Appeals and Interferences
Patent and Trademark Office (P.T.O.)
*1 EX PARTE STEPHEN B. STORRS
Appeal No. 88-2568
November 25, 1988
Application for Patent filed February 22, 1985, Serial No. 06/704,341. Method of Somatotropin Solubilization.
Jon H. Beusen et al. for appellant
Supervisory Primary Examiner--John Kight
Examiner--Howard E. Schain
Before Seidleck, Steiner and W. Smith
This is an appeal from the final rejection of claims 1 through 12, all the claims remaining in the application. Claim 1 is illustrative of the subject matter on appeal and reads as follows:
1. A method for solubilization of somatotropin protein from refractile bodies of a host cell containing said protein which comprises contacting said bodies with an effective amount and concentration of urea at a pH effective to accomplish solubilization of said protein.
The references relied upon by the examiner are:
Emtage et al. (Emtage), "Synthesis of Calf Prochymosin (prorennin) in Escherichia Coli", Proc. Natl. Acad. Sci. U.S.A., Vol. 80, pp. 3671-3675 (June 1983).
Marston et al. (Marston), "Purification of Calf Prochymosin (Prorennin) Synthesized in Escherichia Coli", Biotechnology, pp. 800-804 (Sept.1984).
Two references relied upon by appellant are:
Builder et al. (Builder) 4,511,502 Apr. 16, 1985
Claims 1 through 12 stand rejected under 35 USC § 103 as unpatentable over Marston or Emtage. We reverse.
We have carefully considered the examiner's position as set forth in the Examiner's Answer and Supplemental Examiner's Answer, but find that we are in agreement with appellant that Marston or Emtage would not have suggested the claimed subject matter from a consideration of either reference alone.
Each of Marston and Emtage disclose that prochymosin synthesized in E. coli accumulates in the form of insoluble inclusion bodies. Therefore, prochymosin must be purified from these bacteria through the use of an appropriate method. Each of the references disclose that urea may be used to solubilize prochymosin from the inclusion bodies.
The examiner relies upon the disclosure of Marston which appears on page 803, right-hand column, third paragraph that the insoluble nature of recombinant prochymosin synthesized by E. coli facilitates its purification. This passage of Marston goes on to disclose that such a strategy could be useful in purifying any insoluble recombinant protein. The examiner concludes that this disclosure would have rendered prima facie obvious the use of the disclosed solubilization process of Marston to solubilize any insoluble protein synthesized by genetic engineering techniques, including the present somatotropin.
In our opinion, the above referenced disclosure in Marston is teaching one of ordinary skill in the art that insoluble recombinant proteins should be susceptible to the method of solubilization disclosed in Marston. However, Marston discloses in the last paragraph of the left-hand column of page 802 that somatotropin, e.g. human growth hormone, is not necessarily insoluble. Therefore, after reading Marston in its entirety, we do not find that this reference in and of itself fairly suggests the method of solubilizing somatotropin set forth in the claims on appeal.
*2 We reach a similar conclusion after considering Emtage in its entirety. The only disclosure of Emtage that the examiner references which would suggest that the method disclosed in that reference of solubilizing prochymosin using urea would be at all useful in solubilizing somatotropin is in the second full paragraph of the left-hand column of page 3671. However, this passage only indicates that direct expression of the chymosin gene would be expected to lead, by analogy with human growth hormone, to produce chymosin containing an additional methionine residue. It is not apparent how this passage teaches or suggests to one of ordinary skill in the art that the disclosed method of solubilizing prochymosin would be applicable to the solubilization of somatotropin.
Having found that the examiner has not established that either Emtage or Marston in and of itself would have rendered the subject matter on appeal prima facie obvious to one of ordinary skill in the art, the examiner's rejections are reversed.
Under the provisions of 37 CFR 1.196(b), we make the following new ground of rejection.
Claims 1 through 12 are rejected under 35 USC § 103 as unpatentable over the combined teachings of Marston and Builder.
Marston discloses a method of purifying prochymosin synthesized in E. coli from the inclusion bodies in which the protein is found. To this end, the inclusion bodies are isolated and treated with urea at pH 10.7. In the third paragraph of the right-hand column on page 801, Marston characterizes the denaturing conditions as extreme (8M urea or 6M guanidine hydrochloride). In the following paragraph, Marston discloses that the denaturing step must include urea as well as an alkaline pH in that neither alkaline pH nor urea alone produced an equivalent yield of solubilized protein. Optimal solubilization was obtained through the use of these two steps in succession.
As set forth above, while Marston does suggest on page 803 that a similar strategy can be used to purify any insoluble recombinant protein, Marston does not teach that somatotropin is such an insoluble protein.
Builder also discloses purification of proteins which have been obtained through recombinant techniques which are present in insoluble inclusion bodies in the recombinant bacteria through the use of a denaturing process. At column 2, lines 39-51, Builder teaches that somatotropin is only partially in included form but discloses several techniques which are to be used to completely insolubilize the partially soluble protein.
Therefore, at the time the present invention was made, one of ordinary skill in the art would have recognized from the combined disclosures of these references, that to the extent Marston discloses somatotropin is not an insoluble protein, and thus may or may not be amenable to the purification techniques disclosed in that reference, complete insolubilization of somatotropin according to the teachings of Builder would allow the use of the solubilization technique disclosed by Marston in recovering somatotropin from its inclusion bodies. Therefore, we find that it would have been prima facie obvious to one of ordinary skill in the art at the time the present invention was made to solubilize somatotropin from the inclusion bodies in which it is present as a result of its manufacture from genetic engineering techniques by using a denaturing procedure which comprises treatment with urea at a high alkaline pH as disclosed by Marston. One of ordinary skill in the art would embark upon such an endeavor with the requisite reasonable expectation of success. In re O'Farrell, 853 F.2d 894, 7 USPQ2d 1673 (Fed.Cir.1988).
*3 In response to previous rejections of the claims on appeal over Builder and the corresponding Jones patent, appellant argued that these two references teach away from the present invention. On page 4 of his Answer, the examiner withdrew rejections under 35 USC § 102 and § 103 over Builder or Jones stating that he agrees with appellant's conclusion that Builder or Jones do not disclose solubilization of somatotropin by urea. In responding to these arguments, we will focus our attention upon the Builder reference as being representative of the two as appellant did.
As set forth above, Builder discloses methods for solubilizing proteins which are produced through recombinant techniques which appear in the transformed hosts in inclusion bodies. Builder characterizes guanidine as a strong denaturant and urea as a weak denaturant. See column 7, line 24-column 8, line 25. The working examples of Builder solubilize somatotropin using strong denaturing compositions comprising guanidine. Appellant concludes that Builder is teaching one of ordinary skill in the art that a weak denaturant such as urea will not solubilize somatotropins and thus, teaches away from its use as in the presently claimed invention. We note, however, that Builder defines a weak denaturing solution at column 8, lines 19-25 in terms of a functional definition, i.e., a weak denaturing solution is one which permits refolding from whatever contorted conformation the protein has to a conformation which is capable of exhibiting biological activity. At column 16, lines 58-65, Builder discloses that a weakly denaturing medium which would provide a route to proper refolding can comprise urea at pH 5-9, preferably 6-8. Thus, what Builder is disclosing is that for the purposes of allowing the protein to refold, urea at pH 5-9 would be considered for the purposes of that reference as a weak denaturant.
In contrast, Marston characterizes 8M urea as an extreme denaturant, equivalent to 6M guanidine. As disclosed by Marston, 8M urea, in and of itself, while an extreme denaturant, is not as effective as 8M urea used at a pH of 10.7. It is apparent then, that Marston is teaching that 8M urea whether or not it is used at a pH of 10.7 is considered to be a strong denaturant.
When the disclosures of Builder and Marston are considered together in their entireties, it is not seen that Builder is teaching away from the claimed subject matter or is in fact in conflict with the disclosure of Marston, but rather is only teaching that if urea is used at a pH of 7-9, it is a relatively weak denaturant which permits refolding of the protein. Marston discloses that if urea is used at a higher alkaline pH it is considered a strong denaturant and will solubilize proteins from inclusion bodies. Therefore, one of ordinary skill in the art having these two references before him would have found it obvious to solubilize somatotropin from the inclusion bodies in which it is present using urea at an appropriately chosen high alkaline pH.
*4 Appellant argues on page 10 of the Appeal Brief that Builder clearly implies that weak denaturants such as urea are not suitable for solubilization of somatotropin refractile bodies as well as explicitly teaching that weak denaturants are unsuitable for solubilization of these bodies. However, as set forth above, the purpose of the weak denaturant composition in Builder is to allow the protein to refold. For that purpose, the characterization of urea as a weak denaturant in that reference is necessarily accompanied by the limitation that it is at a pH of 7-9. Further, appellant's analysis of the disclosure of Builder does not take into account the specific teaching of Marston that urea at such a low pH is considered to be a relatively strong denaturant for the purposes of that reference.
We are cognizant that Marston prefers a two-step procedure for the optimal solubilization of prochymosin. However, claim 1 on appeal does not exclude such a two-step process, nor is there any evidence of record which would establish that a one-step process produces any results that could be termed unexpected over the preferred two-step method of Marston. By disclosing a preference for a two-step method, Marston is inferring that a one-step process is less preferred.
Any request for reconsideration or modification of this decision by the Board of Patent Appeals and Interferences based upon the same record must be filed within one month from the date of the decision (37 CFR 1.197). Should appellant elect to have further prosecution before the examiner in response to the new rejection under 37 CFR 1.196(b) by way of amendment or showing of facts, or both, not previously of record, a shortened statutory period for making such response is hereby set to expire two months from the date of this decision.
37 CFR 1.136(a) does not apply to the times for taking any subsequent action in connection with this appeal.
REVERSED 37 CFR 1.196(b)
BOARD OF PATENT APPEALS AND INTERFERENCES
James A. Seidleck
Arthur A. Steiner
William F. Smith