Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1995-05-23
2002-06-25
Stucker, Jeffrey (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S389200, C530S388230, C424S085400, C424S085700
Reexamination Certificate
active
06410697
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the purification of human interferon, to purified form of human interferon and antibodies thereto, and to purification and preparative methods relevant thereto.
BACKGROUND OF THE INVENTION
As used herein, the term “protein” includes “glycoprotein”.
Many attempts have been made to purify human interferon. The objectives of such purification attempts have include a complete characterization of the interferon species for standardization purposes. To date, none of the attempts to purify human Le form interferon have been completely successful.
SUMMARY OF THE INVENTION
This invention is based on the discovery of purification methods which permit the preparation, for the first time, of all the components of human Le form interferon protein substantially free of inactive and otherwise undesirable impurities.
The Le form of interferon is defined in a paper by E. A. Havell, B. Berman, C. A. Ogburn, K. Berg, K. Paucker, and J. Vilcek, Proc. Nat. Acad. Sci. USA, 72, 2185-2187 (1975).
According to the invention, pure human leukocyte interferon proteins have been prepared from crude human leukocyte interferon through a number of special purification steps, and the pure human leukocyte interferon has been characterized by stained protein bands in SDS PAGE (sodium dodecylsulfate polyacrylamide gradient electrophoresis).
The particular experimental conditions used for the first preparation and characterization of the pure human leukocyte interferon proteins appear from the below sections “Materials and Methods” and “Experimental Section”. Some of the products and procedures involved in the preparation and characterization of the pure interferon proteins are novel per se and constitute aspects of the invention of general applicability within interferon technology and, in a broader sense, in protein purification technology. The pure human interferon proteins, and especially, pure human Le form interferon proteins, now made available and characterized according to the invention, are in themselves aspects of the invention and constitute the key to further new developments which are also aspects of the invention and which are explained and illustrated in the present specification.
In several repeated experiments, it has been established that under the SDS PAGE and staining conditions described in the section “Materials and Methods”, at a total interferon load of 0.9×10
6
IFU, pure human leukocyte interferon shows essentially only two sharp stained protein bands at 18,400±200 and 20,100±200 Daltons, respectively, and a minor stained protein band between 20,300±200 and 20,400±200 Daltons. As determined by the protein determination described below, the pure human leukocyte interferon has a specific activity of about 10
9
IFU per mg of protein; the specific activity found may vary to some extent depending upon the protein determination method employed, and the specific activity on a protein weight basis is judged to be 2×10
8
-2×10
9
IFU per mg of protein. The fact that the pure interferon shows two major distinct bands is in accordance with prior art findings using crude or partially purified interferon preparations which indicated that human leukocyte interferon comprises at least two major species. At a higher total interferon load, e.g., of 3.8×10
6
IFU, the above-mentioned SDS PAGE system has been found to be capable of showing a more differentiated protein pattern comprising six interferon protein bands, i.e. the two strongly stained bands at 18,410±200 Daltons and 20,180±200 Daltons, respectively, a medium-stained band at 20,420±200 Daltons (corresponding to the above-mentioned minor stained band) and just visible protein bands at 19,500±200 Daltons, 21,130±200 Daltons, and 23,440±200 Daltons, respectively. Each of the individual components in the above-mentioned bands of the SDS PAGE acrylamide gradient gel has been found to show biological interferon activities: antiviral activity, ability to neutralize only anti-human leukocyte interferon (but not anti-human fibroblast interferon), and anticellular activity, plus a variety of so-called non-viral activities, as exemplified by potentiation of Natural Killer cells, potentiation of MLC-CML, increase of HLA antigens, etc.
The complete purification of interferon proteins makes it possible, for the first time, to produce anti-interferon which is strictly specific to the active species simply by immunizing animals with the pure interferon preparation or one or more of its components. Such strictly monospecific anti-interferon is extremely useful for antibody affinity chromatography for purification of crude or partially purified interferon to obtain, in a simple and economic way, large amounts of pure interferon or highly purified interferon for clinical purposes, standardization, chemical studies, sequence studies, and as immunogen for repeated preparation of monospecific anti-interferon. It is within the scope of the present invention not only to purify human leukocyte interferon by means of the monospecific antibody raised against the pure human leukocyte interferon, but also to purify other interferon types which cross-react immunologically with the monospecific anti-interferon, e.g. “Namalva” interferon (human lymphoblastoid interferons; the Le form interferon constitutes about 85% of the biological activity of human lympho-blastoid or Namalva interferon, vide E. A. Havell, Y. K. Yip, and J. Vilcek, “Characterization of human lymphoblastoid (Namalva) interferon”, J. gen. Virol., 38, 51-59, (1977)), and interferon containing the Le form obtained by cultivation of a microorganism carrying DNA coding for the production of interferon proteins (or proteins having the significant biological interferon activity determinats).
The monospecific anti-interferon is also useful for establishing in a manner known per se a genetic engineering system for production of interferon protein: In accordance with known methods within genetic engineering, the first stage is the isolation of messenger RNA from interferon-producing cells in which the interferon synthesis has been triggered by means of an interferon inducer and has reached a degree of completion of the synthesis of interferon proteins at which the immunological determinants (or parts thereof) of the interferon have been expressed, while at the same time, the interferon is still attached to the ribosomes and the messenger RNA. A high clone producing Namalva cell suspension grown in the usual way or buffy coats (or lymphocytes isolated by Ficoll technique) is preferred as the interferon-producing cells. The messenger RNA is isolated from such cells by lysing the cells in a manner known per se and passing the lysate through an antibody affinity column where the antibody bound covalently is the monospecific anti-interferon. The antibody column selectively retains not only the interferon, but also the attached messenger RNA. By known methods, such as salt elution, the messenger RNA is isolated from the eluate from the column and is, also, by known methods, treated with reverse transcriptase to obtain the corresponding DNA. Alternatively, immunoprecipitation methods (known per se), possibly combined with double immunoprecipitation techniques, may be used. In accordance with known methods within genetic engineering techniques, such DNA coding for interferon or important parts thereof is incorporated in a suitable cloning vector, preferably a mini-plasmid and transformed into a microorganism, the culturing of which produces interferon and/or interferon derivatives released in the culturing medium, from which the interferon is obtained. The purification of such interferon obtained by cultivation of the microorganism can suitably be performed in the same manner as described above by passing the crude preparation through an antibody affinity column made by means of monospecific anti-interferon. Radiolabelled monospecific anti-interferon may be a valuable tool in
Lerner David Littenberg Krumholz & Mentlik LLP
Schering Corporation
Seharaseyon Jegatheesan
Stucker Jeffrey
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