FI69476B

Purified proteins and process therefor

Abstract

This record has no abstract on file.

FI69476B, drawing sheet 1 — Sheet 1 of 21

Term

Term ended

Expired 21 November 1999, 26.8 years ago.

16 claims: 2 independent, 14 dependent

1
Patenttivaatimukset 1. Menetelmä ihmisen leukosyytti-interferonin puhdistamiseksi, tunnettu siitä, että epäpuhtaan proteiinin vesipitoinen liuos viedään puskurilla tasapainoitetun pylvään läpi, jossa on syanopropyyli-, sykloheksyyli-, fenyyli-, oktyyli-, oktadekyyli- tai glyseryyli-ryhmillä modifioitu huokoinen SiC^-matriisi, HPLC-olosuhteissa, jolloin proteiini ensin adsorboituu ja sitten eluoidaan veden kanssa sekoittuvan liuottimen nousevalla tai laskevalla gradientilla, niin että se lopuksi saadaan puhtaammassa muodossa eluaatin tietyissä jakeissa.
2
Patenttivaatimuksen 1 mukainen menetelmä, tunnett u siitä, että käytetään puskurilla tasapainoitettua pylvästä, jossa on oktyyli- tai glyseryyliryhmillä modifioitu huokoinen SiO£-matriisi.
3
Menetelmä leukosyytti-interferonin valmistamiseksi homogeenisena proteiinina, tunnettu siitä, että A) epäpuhtaassa tilassa olevan leukosyytti-interferonin vesipitoinen liuos HPLC-olosuhteissa viedään puskurilla tasapainoitetun pylvään läpi, jossa on oktyyli-ryhmillä modifioitu SiC^matriisi, jolloin interferoni ensin adsorboituu ja sitten eluoidaan puskurissa olevan, veden kanssa sekoittuvan liuottimen nousevalla gradientilla, niin että se saadaan puhtaammassa muodossa eluaatin määrätyissä jakeissa;El nämä vaiheessa A) saadut määrätyt jakeet viedään puskurilla tasapainoitetun pylvään läpi, jossa on glyseryyli-ryhmillä modifioitu SiC^-matriisi, jolloin interferoni ensin adsorboituu ja sitten eluoidaan puskurissa olevan, veden kanssa sekoittuvan liuot-cimen laskevalla gradientilla niin, että se saadaan puhtaammassa muodossa selvinä päähuippuina määrätyissä jakeissa;C) nämä vaiheessa B) saadut jakeet, jotka vastaavat selvää huippua HPLC-olosuhteissa viedään puskurilla tasapainoitetun pylvään läpi, jossa on oktyyli-ryhm.illä modifioitu SiC^-matriisi, jolloin interferoni ensin adsorboituu ja sitten eluoidaan veden kanssa sekoittuvan liuottimen ja vesipitoisen puskurin seoksella, niin että se saadaan yhtenä ainoana selvänä huippuna eluaatin määrätyissä jakeissa homogeenisena proteiinina ja mahdollisesti toistetaan vaihe C) tuotteen äärimmäisen puhtauden saavuttamiseksi.
4
Patenttivaatimuksen 3 mukainen menetelmä, tunnett. u siitä, että veden kanssa sekoittuvana liuottimena käytetään alkanolia.
5
Patenttivaatimuksen 3 mukainen menetelmä, tunnett u siitä, että veden kanssa sekoittuvana liuottimena käytetään n-propanolia, vaiheessa A) puskuria, jonka pH on n. 7,5, ja vaiheessa B) puskuria, jonka pH on n. 4,0.
6
Patenttivaatimuksen 5 mukainen menetelmä, tunnett u siitä, että vaiheessa A) käytettynä puskurina on 1 M natriumasetaatti/etikkahappo ja n-propanoli-gradientti nousee 0:sta 40 %:iin (tilav./tilav.), vaiheessa B) käytettynä puskurina on noin 0,IM natriumasetaatti ja n-propanoli-gradientti laskee 72,5:stä 50 %:iin (tilav./tilav.) ja että vaiheessa C) käytettynä puskurina on IM pyridiini/2M muurahaishappo ja n-propanoli-gradientti asetetaan nousemaan 20:stä 40 %:iin (tilav./tilav.).
7
Patenttivaatimuksen 6 mukainen menetelmä, tunnett u siitä, että määrätyt, selville päähuipuille kuuluviksi katsottavat jakeet yhdistetään, n-propanoli poistetaan uuttamalla n-heksaanilla ja ennen vaihetta C jäljelle jäänyt n-heksaani poistetaan vesipitoisesta faasista. 24 694 7 6 15. Jonkin patenttivaatimuksen 4-7 mukainen menetelmä, tunnettu siitä, että saadaan interferoni 16. Jonkin patenttivaatimuksen 4-7 mukainen menetelmä, tunn ettu siitä, että saadaan interferoni

Independent claims7

483 paragraphs in 17 sections, as filed

Protein purification has long been a problem in peptide chemistry. Methods used for this purpose include precipitation, gel filtration, ion exchange chromatography, gel electrophoresis, affinity chromatography, and many others. Methods for isolating naturally occurring, high molecular weight proteins present in extremely low concentrations in biological material include the above-mentioned procedures. In many cases, very large amounts of raw material must be used and processed, taking into account the large losses throughout the process. As a result, purification methods for such proteins are very laborious and expensive. A good example in this respect is various attempts to isolate and characterize interferon. Since the discovery of interferon by Isaacs and Lindenmann in 1957, this protein, derived from both white blood cells and fibroblasts, has successfully resisted attempts by scientists around the world over two decades to isolate it as a homogeneous peptide in quantities that allow its specific biological and chemical properties to be characterized and determined. .

In U.S. Patent No. 3,699,222, which deals with the first interferon studies by Isaacs and Lindenmann, purification of the active ingredient involves only ammonium sulfate precipitation followed by dialysis. Because such a method is relatively non-specific, the product obtained is still very impure.

U.S. Patent 3,414,651 describes a multi-step method for combining interferon comprising the steps of: selective adsorption on alumina-silicate, elution with iodine or thiocyanate solution, further precipitation of undesired proteins with aqueous HCl and aqueous NaOH, precipitation of interferon with a water-miscible solvent such as methanol, ethanol or acetone, such as 2-diethylaminoethylcellulose11a. With this purification method, the specific activity of the interferon could be increased to a factor of about 6000. Specific interferons are mentioned in this U.S. patent as chick and monkey interferon.

Another purification method is described in U.S. Patent 3,975,344, which discloses the purification of crude human fibroblast interferon by a density gradient ultracentrifugation method. It was shown that this method achieves a higher yield and purity than the method using column chromatography on Sephadex G-100.

The following publications also cover the purification and characterization of interferon:

Kningth. E., Proc. Natl. Acad. Sei. USA 73, 520-3 (1976); Törmä, ET et al., J. Biol. Chem. 251, 4810-6 (1976); Bridgen, PJ et al., J. Biol. Chem. 252, 6585-7 (1977); DeMaeyer-Guignard, J. et al., Nature 271, 622-5 (1978); Kawakita, M. et al., J. Biol. Chem. 253, 598-692 (1978); Berthold, W. et al., J. Biol. Chem. 253, 5206-11 (1978); Jankowski, WJ et al., J. Virology 16, 1124-30 (1975);

Davey MW et al., J. Biol. Chera. 251, 7620-5 (1976);

Chadha, KC et al., Biochemistry 17, 196-200 (1978).

Although many of the above publications claim that mouse or human interferon could be purified to homogeneity, no classical way to demonstrate the homogeneity of proteins has been presented, nor has the properties of the compounds thought to be pure described.

The use of high pressure liquid chromatography (HPLC) for the purification of proteins is well known in the art, in particular ion exchange and separation chromatography. e.g., Regier, FE et al., J. Chromatog. Sei. 14, 316-20 (1976) and Chang. S.-H. et al., Anal. Chem. 48, 1839-45 (1976) 7.

For example, Lichrosorb RP-18 (columns with octadecyl-modified SiO 2) was successfully used in reverse phase chromatography to purify peptides such as M-endorphin / see. e.g., Rubinstein, M. et al., Proc. Natl. Acad. Sci., USA 74, 4969-72 (1977) 7.

Finally, Cabrer, B. et al., J. Biol. Chem. 254, 3681-4 (1979) described the partial characterization of three types of interferon (MW - 33'000, 26'000 and 20'000) obtained from mouse Ehrlich Ascites tumor cells.

The invention relates to an improved process for the purification of human leukocyte interferon to give a highly soluble product in good yields on a preparative scale.

The process is characterized in that an aqueous solution of the crude protein is passed through a buffer-equilibrated column based on porous SiO modified with cyanopropyl, cyclohexyl, phenyl, octyl, octadecyl or glyceryl groups.2-to the matrix, under HPLC conditions, in which the protein is first adsorbed and then eluted with an ascending or descending gradient of a water-miscible solvent so that it is finally obtained in a purer form in certain fractions of the eluate. These columns, which can be used sequentially and under different conditions in terms of pH and organic solvents, offer the possibility of purifying interferon, which is present in natural material in extremely small amounts, up to homogeneity.

In a preferred embodiment, the invention comprises a method for purifying human leukocyte interferon into a homogeneous product and even in amounts sufficient to allow accurate characterization of this medically valuable substance for the first time. The ability to chemically characterize interferon constitutes a significant advance in the preparation of this substance, as this is a prerequisite for synthesizing the substance, whether by conventional peptide syntheses, or by genetic engineering with the aid of suitable organisms, preferably bacteria.

The process according to the invention for the production of human leukocyte interferon as a homogeneous protein is characterized in that

A) an aqueous solution of impure human leukocyte interferon under HPLC conditions is passed through a buffer equilibrated column with an octyl group modified SiO 2 matrix, whereby the interferon is first adsorbed and then eluted with a rising gradient of water-miscible solvent in the buffer, in the fractions is obtained in a purer form;

B) certain fractions obtained in step A) are passed through a buffer-equilibrated column with glyceryl-modified SiO 2 -natrize, whereby the interferon is first adsorbed and then eluted with a decreasing gradient of the water-miscible solution in the buffer so that it clears in certain eluate fractions. is obtained in a purer form;

C) These fractions obtained in step B), which correspond to a clear main peak, are passed under HPLC conditions through a buffer-equilibrated column with an octyl-modified SiO 2 matrix, whereby the interferon is first adsorbed and then eluted with a mixture of a water-miscible solvent and an aqueous buffer, so that it is obtained in a single, at a clear peak in certain fractions of the eluate as a homogeneous protein and possibly repeating step C) to obtain the extreme purity of the product.

HPLC columns based on a porous SiO 2 matrix modified with octyl or glyceryl groups (particle size = 10 μm average pore size = 100 Å) and used in the practice of this invention are commercial products produced by e.g. EM

Laboratories of Elmsford, NY USA, under the trade name Lichrosorb

RP-8 and Lichrosorb-Diol. Equivalent octyl-modified porous SiO2-columns (called Chromegabond C-8) are produced by ES

Industries, Marlton, NJ, USA).

A suitable HPLC system in which the above columns can be used is described in U.S. Patent 4,116,046.

According to the invention, a solution of impure interferon is introduced, preferably in the presence of an aqueous buffer. at a pH appropriate for the protein SiO2-through the column. Normally this occurs under pressure, preferably between about 50-5000 psi (3.4-340 atm). The protein adsorbed on the column sample is then eluted stepwise and selectively using a water-miscible solvent gradient. Suitable solvents for this purpose are, for example, alkanols, such as n-propanol, 2-propanol, ethanol, methanol, tert-butanol. The fractionation of the eluate takes place in a manner known per se, in which case the interferon content of the individual fractions is determined on very sensitive monitors. One suitable system for this purpose is described by Bohlen et al., Anal. Biochem. 67, 438 (1975). It is also recommended to monitor the presence of interferon by an appropriate bioassay.

The decision as to which of the two types of columns (column for normal fractionation chromatography or column for reverse phase chromatography) and in which order the columns are used depends largely on the nature of the protein to be purified. It was found that in the case of human leukocyte interferon, the best results are obtained when the impure interferon solution is first applied to a column with octyl-modified SiO2matrix (reverse phase chromatography) using a buffer having a pH of about 7.5 (preferably 1 M sodium acetate / acetic acid) and eluting with an increasing gradient of n-propanol, then the collected active fractions are passed through a column modified with glyceryl groups. SiO2~ matrix in 0.1 M sodium acetate buffer and eluted with a decreasing n-propanol gradient and finally the separated interferon components are applied to a column with octyl-modified SiO2~ matrix using a buffer with a pH of about 4.0, preferably 7— 1M pyridine / 2M formic acid, and eluted with an ascending n-propanol gradient. In this way, each of the three separated human leukocyte interferons (and P) can be further separated, resulting in sharply distinct peaks in the chromatogram that describe these homogeneous proteins. Throughout the purification method, from the incubation medium to chromatography with another octyl group modified SiO2matrix gave a coefficient of purity increase of 60,000-80000.

In a particular embodiment of the method for purifying human leukocyte interferon, the fractions obtained in step B) corresponding to the main peaks are combined, extracted with n-hexane to remove n-propanol and the remaining n-hexane is removed before carrying out step C.

Homogeneous human leukocyte interferon species prepared by the method of the invention are characterized by a sharp peak on the above-mentioned HPLC columns as well as a single narrow band in polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) in the presence of 2-mercaptoethanol. Gel extraction gave a single sharp peak of antiviral activity that was consistent with the protein band. The specific activities of pure interferon species range from n.

2.6-4.0 x 10 units / mg with MDBK cells (with bovine epigelic kidney cells) and between 1.5-4 x 10 units / mg with the human cell line Agl732. Molecular weights ranged from about 16,000 to 21,000 (cf. Table 4, page 22). The results of the amino acid analysis are summarized in Table 5 (page 24).

Interferons have antiviral, anti-tumor, anti-growth and anti-rejection effects. These effects could be observed even on a clinical scale with the administration of 1-10 x 10θ units / day of relatively impure formulations containing less than 1% human interferon. Homogeneous species of interferon purified by the method of this invention can be used in the same manner as previously known interferon preparations by adjusting the dosage according to the degree of purity achieved. Individual species of interferon may be administered alone or in admixture with each other. Such mixtures can be obtained either by mixing the isolated species or by interrupting the purification process at a point where more interferon species are present but no interferon-inactive proteins at all.

Induction of interferon production, primary enrichment of interferon and fractionation, including gel filtration, can be performed by methods known per se. These process steps, which provide an aqueous solution of impure interferons, are not an object of the present invention.

The invention is illustrated by the following examples.

Example 1

Homogeneous human leukocyte interferon from normal blood donors

A. Preparation of interferon

Interferon was prepared by incubating for 16 hours human leukocytes obtained from the blood of normal blood donors with γ (10 cells / ml) Newcastle disease viruses (15 haemagglutinin units / ml) in serum-free minimal medium containing 10 mg / ml casein. An average interferon titer of 5,000 units / ml was obtained. The methods used corresponded to those used by Mogensen, KE et al., Pharmacology and Therapeutics A, 1977, 369-381; Wheelock, EF, J. Bacteriol. 92, 1415-1421 (1966) and Cantell, K. et al., Appl. Microbiol. 22, 625-628 (1971), with a few minor modifications. The interferon titer was determined by an assay method based on inhibition of cytopathic effect, which could be performed within 16 hours. All interferon titers are given in units / ml and calibrated against the National Institut of Health (USA) reference standard for human leukocyte interferon.

B. Interferon enrichment and first fractionation

Unless otherwise stated, work at a temperature ranging from 0 to 4 ° C. At the end of the incubation, cells and cell debris were removed by centrifugation (15 minutes, 500 xg). Casein was precipitated by acidification with HCl to pH 4.0. After 2 hours, the mixture was centrifuged (10 minutes, 12,000 xg) and the precipitate was discarded. The supernatant (10 L) was adjusted to 1.5% (w / v) trichloroacetic acid. 1 After 1 hour, the precipitate was centrifuged apart (10 minutes, 12,000 xg) and redissolved in 50 ml of 0.1 M

NaHCO 3 - After adding 0.5 g of Triton X-100 and 1.5 ml of acetic acid (dropwise and stirring), the mixture was allowed to stand for 1 hour at 0 ° C and then for 16 hours at -20 ° C. After thawing, centrifuged for 10 minutes at 17,000 xg. The residue was discarded and the supernatant was adjusted to 4% trichloroacetic acid. After one hour, the mixture was centrifuged for 10 minutes at 12,000 xg and the precipitate was dissolved in 5 ml of 0.5 M NaHCO 3.

C. Gel filtration

The resulting concentrated interferon solution was mixed with 1.5 g of urea and applied to a column (2.6 x 90 cm) of fine Sephadex G-100 pre-equilibrated with 4M urea / 0.1 M sodium acetate buffer. The column was eluted at room temperature and 0.5 ml / min. at a flow rate of 4M urea / 0.1 M sodium acetate, pH 7.5. 12.5 ml fractions were collected. Interferon activity eluted in fractions 19-23.

D. HPLC

The combined fractions 19-23 were transferred from the Sephadex G-100 column directly via pump to a Lichrosorb RP-8 column (10 μl, 4.6 x 250 mm).

The column was pre-equilibrated with 1M sodium acetate buffer (pH 7.5).

containing 0.01% (v / v) thiodiglycol and eluted with a linear gradient of n-propanol in the same buffer / 1 hour, 0-20%, 3 hours, 20-40% v / v / v at a flow rate of 0.25 ml / min. 0.75 ml fractions were collected. Interferon eluted in fractions of 23-40 / 25-30% (v / v) n-propanol /.

Fractions 27-33, which contained most of the interferon activity, were pooled, mixed with n-propanol to a final concentration of% (v / v) and transferred directly to a Lichrosorb-Diol column via pump (10 μl, 4.6 x 250 mm) pre-equilibrated with 0.1 M sodium acetate solution with 80% n-propanol (v / v). The column was then eluted for one hour with a linear gradient of 72-50% (v / v) n-propanol in 0.1Μ sodium acetate at a flow rate of

0.25 ml / min. 0.75 ml fractions were collected. Interferon activity was eluted in the form of three distinct major peaks that varied quantitatively from sample to sample. These peaks were named according to their elution order.-k, p and / or. the fraction eluted at% n-propanol, the p-fraction at 66.5% n-propanol and the '/' fraction at 65.5% n-propanol. The yield of total inter9 ferron activity was greater than 80%.

Fractions from each peak were combined and further purified in several steps. Since the peak β was incomparably the largest and appeared to be the most distinct from the other components, it was chosen for further purification. Fractions 54-56 from diol columns containing peak I * were combined and the n-propanol was removed by two extractions with equal amounts of hexane. The remaining hexane was removed under a stream of nitrogen. Pyridine and formic acid were added to a final concentration of 1M and 2M, respectively, and the solution was applied to a Lichrosorb RP-8 column (10 μ; 4.6 x 250 mm) pre-equilibrated with 1M pyridine and 2M formic acid (pH 4.0 with formic acid). The column was eluted for 3 hours with a linear gradient of n-propanol (20-40%) in 1 M pyridine / formate buffer at a flow rate of 0.2 ml / min. 0.6 ml fractions were collected. The main peak of activity was consistent with the peak of protein. Fractions 45 and 46 (32% v / v) propanol) corresponding to this peak were combined and rechromatographed under the same conditions. Interferon eluted in fraction 31 (32% v / v).

O propanol). The specific activity of this fraction was estimated to be 4 x 10 units / mg compared to bovine serum albumin. This material was further used for characterization. The fluorescence profiles of the HPLC were so well reproducible that they gave a permanent characteristic to the whole method.

The purification results are summarized in Table 1. The total purification, from the incubation medium to the second RP-8 column, was 60,000-80,000-fold. The cumulative yield from step 1 to the diol step was 30-50%. After this step, each of these interferon peaks was purified.

4J tn 'ä

-g

•B

I 44 • h -S

4-> 0)

cr>

• OJ |

II

E. Polyacrylamide gel electrophoresis

Interferon samples (1.5 x 10 units) were incubated

With SDS-PAGE and 2-mercaptoethanol and applied to a plate gel.

After electrophoresis, a single sharp band was obtained after staining with Coomassie blue. The apparent molecular weight was determined to be 17,500 (compared to standard proteins). The gel was cut into 1 mm strips, each strip being homogenized in 0.4 ml of 0.5 M NaHCO 3 / O 2. % SDS-PAGE and examined for interferon activity. A single peak with antiviral activity was found that was consistent with a single protein band.

F. Amino acid analysis

Amino acid analysis of homogeneous human leukocyte interferon (peak γ) was performed on a Fluorescamine amino acid analyzer

natural interferon and then hydrolyzed in 6M HCl under reducing conditions (0.1% thioglycolic acid). Under these conditions, cysteine is measured as S-carboxymethylcysteine and cysteine as free cysteine. The results of the amino acid analysis are summarized in Table 2. The specific activity, calculated from the amino acid content, was determined in g

2-4 x 10 units / mg.

Table 2

Amino acid composition of human leukocyte interferon

<td>Amino-</td><td>Scraps</td>

<td>Ask (total)</td><td> 3,3+0,7</td>

<td>1/2 cystine+</td><td> 1,8+0,2</td>

<td>Cysteine +</td><td> 1,5+0,5</td>

* Fixed time 0.

+ Measured after carboxymethylation of natural interferon ++ Measured after hydrolysis in 6M Hcl / 4% thioglycolic acid

II

Example 2

Homogeneous human leukocyte interferon from leukocytes in leukemia patients

Interferon was obtained by incubating human leukocytes isolated from the blood of leukemia patients with chronic myelogenous leukemia (CML) by leukophoresis with Newcastle disease viruses in serum-free, proteinaceous medium with interferon titers ranging from 5,000 to 40,000 units / ml.

The purification procedure was the same as described for Example 1 for interferon from the blood of normal blood donors and involved selective precipitation with 0.5 M acetic acid in the presence of Triton X-100, gel filtration with Sephadex G-100 in 4M urea, HPLC pH 7.5 Lichrosorb With RP-8, HPLC with Lichrosorb-Diol and HPLC with Lichrosorb-RP-8 at pH 4.0.

The fractions corresponding to the A, * and / peaks of the Lichrosorb-Diol column were collected and then purified separately in further steps.

From the combined fractions 43-46 (peak A), n-propanol was removed by two extractions with equal amounts of n-hexane. Traces of hexane were removed with a stream of nitrogen gas. Pyridine and formic acid were added to a final concentration of 1M and 2M, respectively, and the solution was applied to a Lichrosorb RP-8 column (10 μl, 4.6 x 250 mm) pre-equilibrated with 1M pyridine / 2M formic acid (pH 4.0). The column was eluted for three hours with a straight gradient of n-propanol (20-40%), v / v in 1M pyridine formate buffer at a flow rate of 0.2 ml / min. 0.6 ml fractions were collected. Interferon activity eluted as broad peaks at n-propanol concentrations ranging from 31-35% (v / v). These fractions were combined and rechromatographed under the same conditions. Interferon activity eluted to two major peaks (<^ and <k2) In 31 and 32% (v / v) n-propanol. The secondary components eluted at 35% (v / v) n-propanol.

Combined fractions 47-50 (peak β) from a Lichrosorb-Diol column were treated in the same manner and chromatographed on Lichrosorb RP-8 as described for peak, a. Interferon activity eluted at two major peaks: β2 In 32% (v / v) n-propanol and P1 2 in 34% (v / v) n-propanol. Chromatography was not necessary in this case. A peak in M31% (v / v) n-propanol could be observed in some samples.

Combined fractions 52-54 of the Lichrosorb-diol column (peak treated in the same manner and chromatographed on Lichrosorb RP-8 as described for the peak. Interferon activity eluted with five major peaks: (31% n-propanol, v / v)), T ~ 2 (32% n-propanol, v / v), (34% n-propanol, v / v), f (35% n-propanol, v / v) ) and (35.5% n-propanol, v / v). Re-chromatography was not necessary in this case.

The purification results for the preparation of individual interferon species are summarized in Table 3.

Species Λ 2 3a^ 2 v° to the east further separates Lichrosorb-Diol: 11a: -k on the basis of its elution properties 2 elutes with 68% (v / v) n-propanol and 66.5%: se11a (v / v) with n-propanol.

Samples of interferon species (1.5 x 10 units) were incubated with SDS-PAGE and 2-mercaptoethanol and plated on a gel. After electrophoresis, the peaks yielded Ά, λ2, P2· · ^2 3a ^ 4 each gave a single ribbon, while peaks 7 ^, and ^^ each yielded two ribbons. The apparent molecular weights ranged from 16,000 to 18,000, except for 16,500 which gave one band and another

At 21000 jap / j, which gave a band at 21000 (see Table 4).

oo mm

<td>Ή</td><td> *—1</td><td>r-4</td><td> 1—4</td><td></td><td>Ή</td><td>r — 4</td><td>Ή f—

Total 4 3 6

Purification of leukocyte interferon from CML cells

I, * '

I o

I 04

x • H nJ>

O 'T cL co

I Q. tx

X} P 0 ω 0 li

X u • H kJ * *

B

·*

C Γ — I · aj .Η ω

H • H ^ 4> · Mh en

H r- n H nj

O) -m

P-: 5 5 CO <rH

+ + + + + + + + +

O rH <0 EH

UP and CM + 1 • m

Amino acid analysis of purified human leukocyte interferon fragments was performed on a Fluorescamine amino acid analyzer with 0.1 μg samples. Hydrolysis was performed in 6N HCl under reducing conditions (0.1% thioglycolic acid). The results of the analyzes are summarized in Table 5.

Amino acid analysis of human leukocyte interferon

Accuracy + 1.5 residues H Mixture of both tapes

Trypsin degradation and HPLC of the particles

Purified human leukocyte interferon species were dissolved in each 300 p-mole of aqueous sodium bicarbonate (50 mmol pH 8, 50 μL). After adding 0.1 μg trypsin 2 μl HCl (pH 3) to each incubated for 14 hours at 37 ° C, 5 μl acetic acid was added and the mixture was applied to a Lichrosorb RP-8 column (particle size 10 / U; 4, 6 x 250 mm). The column was eluted for one hour with a linear gradient of 0-40% (v / v) n-propanol in 0.1 M formic acid / 0.03 M pyridine buffer (pH 3) at a flow rate of 0.5 ml / min.

Particles were detected by the Fluorescamin method. The results are summarized in Table 6, where the position of the peaks is expressed in% n-propanol and the relative particle sizes are given (S = small; M = medium; L = large).

Table 6

<td colspan="4">Human white blood cell interferon</td><td colspan="2">tryptic</td><td>peptides</td><td rowspan="2"></td>

<td>Species</td><td></td><td colspan="3">Elution%:</td><td>in n</td><td>-propanol</td>

14.5S, γ2

14.5M, 16S, 17.5M, 18M, 29M

3L, 4L, 4.2M,

16S, 18L, 29M

3M, 4M, 4.2M,

16S, 18L, 20S,

3L, 4L, 4.2M, 4.5M, 7S, 7.5S, 10S,

12.5S, 14S, 14.5M, 16S, 18L, 24.5S,

4.5S, 5S, 11.5S, 12.5S,

11.5M, 12.5S, 13.5S,

32M

14.5M, γ3 γ5

11.5L,

25.5S, 32S

Purified human leukocyte interferon species were subjected to an amino sugar assay to detect amino sugar between 50 and 100 pmol. In all cases, less than one residue per glucose amine, galactosamine, or mannose amine was found. In most cases, it was interfered with by a small number of peptides that eluted in the vicinity of the amino sugar by this assay. It is therefore possible that the peaks considered to belong to amino sugars belong in part or in whole to the peptides.

Finally, no attempt is made to sequence 1 nmol of pure γ 2~ interferon by manual Edman digestion, which included backhydrolysis and lactic acid analysis using Fluorescamine, yielded amino acids in the first two cycles. 100 pmoles of pure human white blood cells \2~ treatment of interferon with leucine aminopeptidase and aminopeptidase M20 for 1 hour at 37 ° C does not affect biological activity; no amino acids could be detected in the incubation medium. By treating the supernatant of the induction medium (leukocytes and Newcastle disease viruses in minimal medium) with aminopeptididase M, no loss of interferon activity could be detected. This suggests that the interferon molecule already contains a protected amino acid before the purification procedure. For comparison, crude interferon, pure interferon, and insulin N-chain were incubated with aminopeptidase M. While insulin was partially degraded (amino acids detectable), there was no loss of interferon activity.

76 members in 39 offices

This recordMember, by publication datePriority claim29 more offices are in the list below

Priority claims12

Priority claims
Document	Office	Kind	Date
96325778	United States of America	A	1978-11-24
96325778	United States of America	A	1978-11-24
6237479	United States of America	A	1979-07-31
6237479	United States of America	A	1979-07-31
7771079	United States of America	A	1979-09-21
7771079	United States of America	A	1979-09-21
62374	–	–	–
77710	–	–	–
963257	–	–	–
US19780963257	–	–	–
US19790062374	–	–	–
US19790077710	–	–	–

Members76

Family members, oldest first
Document	Office	Kind
IT7927524D0	Italy	D0	1979-11-23
PT70495A	Portugal	A	1979-12-01
IL58759A0	Israel	A0	1980-02-29
IL58759D0	Israel	D0	1980-02-29
BE880201A	Belgium	A	1980-05-22
IE792247L	Ireland	L	1980-05-24
DK496779A	Denmark	A	1980-05-25
FI793649A	Finland	A	1980-05-25
FI793649A7	Finland	A7	1980-05-25
NL7908516A	Netherlands (Kingdom of the)	A	1980-05-28
NO793819L	Norway	L	1980-05-28
AU5313679A	Australia	A	1980-05-29
DE2947134A1	Germany	A1	1980-06-12
SE7909721L	Sweden	L	1980-06-16
FR2442054A1	France	A1	1980-06-20
GB2037296A	United Kingdom	A	1980-07-09
JPS5594320A	Japan	A	1980-07-17
MC1300A1	Monaco	A1	1980-10-03
ES486263A0	Spain	A0	1980-11-01
ES8100502A1	Spain	A1	1980-11-01
ZA796175B	South Africa	B	1980-11-26
MTP857B	Malta	B	1981-04-29
AR222359A1	Argentina	A1	1981-05-15
LU81918A1	Luxembourg	A1	1981-06-04
MW3379A1	Malawi	A1	1981-08-12
US4289690A	United States of America	A	1981-09-15
ATA745379A	Austria	A	1981-12-15
RO79132A	Romania	A	1982-07-06
AT367769B	Austria	B	1982-07-26
CA1129409A	Canada	A	1982-08-10
IN150740B	India	B	1982-12-04
IL58759A	Israel	A	1983-02-23
KR830000910A	Republic of Korea	A	1983-04-28
RO79132B	Romania	B	1983-05-30
GB2037296B	United Kingdom	B	1983-07-27
FR2442054B1	France	B1	1983-09-09
YU286179A	Yugoslavia, later Serbia and Montenegro (until 2006)	A	1983-10-31
JPS58192896A	Japan	A	1983-11-10
GR72828B	Greece	B	1983-12-06
KR830002616B1	Republic of Korea	B1	1983-12-06
AU1586683A	Australia	A	1983-12-08
KE3347A	Kenya	A	1983-12-16
HU182500B	Hungary	B	1984-01-30
HK26684A	Hong Kong, China	A	1984-03-30
AU535936B2	Australia	B2	1984-04-12
SG71183G	Singapore	G	1984-08-03
NO151157B	Norway	B	1984-11-12
NO151157C	Norway	C	1985-02-20
US4503035A	United States of America	A	1985-03-05
IE48882B1	Ireland	B1	1985-06-12
NZ192201A	New Zealand	A	1985-07-31
FI69476BThis record	Finland	B	1985-10-31
CH653347A5	Switzerland	A5	1985-12-31
MY8500222A	Malaysia	A	1985-12-31
FI69476C	Finland	C	1986-02-10
CH654843A5	Switzerland	A5	1986-03-14
IT1127253B	Italy	B	1986-05-21
IT7927524A0	Italy	A0	1986-05-21
CH658459A5	Switzerland	A5	1986-11-14
PH20763A	Philippines	A	1987-04-10
AU561672B2	Australia	B2	1987-05-14
JPS6261040B2	Japan	B2	1987-12-18
SE454276B	Sweden	B	1988-04-18
DE2947134C2	Germany	C2	1988-04-21
JPS63164897A	Japan	A	1988-07-08
JPS6338330B2	Japan	B2	1988-07-29
JPH031320B2	Japan	B2	1991-01-10
DK161048B	Denmark	B	1991-05-27
DK161048C	Denmark	C	1991-11-04
DOP1979002953A	Dominican Republic	A	1992-11-10
LU88314I2	Luxembourg	I2	1994-05-04
DE2954574C2	Germany	C2	1995-03-09
JPH07224098A	Japan	A	1995-08-22
US4503035B1	United States of America	B1	1996-03-19
NL193085B	Netherlands (Kingdom of the)	B	1998-06-02
NL193085C	Netherlands (Kingdom of the)	C	1998-10-05

2 legal events, as the office reported them to INPADOC

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Events

Legal events, newest first
	Event	Code
2004-08-31	Patent expiredExpiredMA	MA
1998-04-10	Application lapsedLapsedFD	FD