CA2137237C

CA2137237C - Purified forms of dnase

Info

Publication number: CA2137237C
Application number: CA002137237A
Authority: CA
Inventors: John Frenz; Steven J. Shire; Mary B. Sliwkowski
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 1992-06-08
Filing date: 1993-05-28
Publication date: 2004-10-26
Anticipated expiration: 2013-05-28
Also published as: EP1013284B1; BR9306670A; US5279823A; GR3034718T3; DE69329200D1; TJ396B; ATE455557T1; US6932965B2; WO1993025670A1; HU9403512D0; ES2150447T3; NO944752L; SK282957B6; BG99234A; DE4392749T1; GB2282140B; KR100323357B1; AU682822B2; CZ293105B6; SK149594A3

Abstract

The present invention provides the identification and characterization of two components of a recombinant preparation of DNase. These components are the purified deamidated and non-deamidated human DNases. Taught herein are the separation of these components and the use of the non-deamidated species as a pharmaceutical per se, and in particular in compositions wherein the species is disclosed within a plastic vial, for use in administering to patients suffering from pulmonary distress.

Description

PUt2IFIED FORIdS OF DN71SE
Related Patent ADplications The present application is related in subject matter to the disclosure contained in International Patent Application Publication No. WO 90/07572.
Field of the Invention The present invention is related to results obtained from research on deoxyribonuclease (DNase), a phosphodiesterase that is capable of hydrolyzing polydeoxyribonucleic acid. It relates generally to the separation of several forms of said DNase, to these forms per se, to pharmaceutical compositions by which their utility can be exploited clinically, and to methods of using these DNases and compositions thereof.
Background of the Invention DNase is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid. DNase has been purified from various species to various degrees. The complete amino acid sequence for a mammalian DNase was first made available in 1973. See e-g., Liao, et al., J. Biol. Chem. 248:1489 (1973).
DNase has a number of known utilities and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscoelasticity of pulmonary secretions in such diseases as pneumonia and cystic fibrosis, thereby aiding in the clearing of respiratory airways. See e-g., Lourenco, et al., Arch. Intern. Med.
142:2299 (1982); Shak, et al., Proc. Nat. Acad. Sci. 87:9188 (1990);
Hubbard, et al., New Engl. J. Med. 326:812 (1992).
DNA encoding human DNase I has been isolated and sequenced and that DNA has been expressed in recombinant host cells, thereby enabling the production of human DNase in commercially useful quantities. See e-Q., Shak, et al., Proc. Nat. Acad. Sci. 87:9188-9192 11990). Recombinant human DNase (rhDNase) has been found:to be useful clinically, especially in purified form such that the DNase is free from proteases and other proteins With which it is ordinarily associated in nature. See e-cr., Hubbard, et al., New Engl. J. Med.
x:812 (1992).
The means and methods by Which human DNase can be obtained in pharmaceutically effective form is described in the patent applications cited above. Various specific methods for the purification of DNase are known in the art. See e-a., Khouw, et al., U.S. Patent No. 4,065,355 (issued 27 Decembqr 1977); Mazkey, FEBS

y WO 93/25670 ,~ ~N ~ '' ~ w PCT/US93/05136 Letters 167:155 (1984); Nefsky, et al., Eur: J. Biochem. 179:215 (1989) .
Although it was not appreciated at the time the above-referenced patent applications were filed, the DNase product obtained from cultures of recombinant host cells typically comprises a mixture of deamidated and non-deamidated forms of DNase. The existence of deamidated forms of DNase remained~unappreciated notwithstanding that the phenomenon of deamidation of asparagine and glutamine residues in some proteins is known. See e-a., Eipper et al., Ann. Rev. Physiol.
50:333 (1988); Kossiakoff, Science 240:191 (1988); Bradbuxy et al., Trends in Biochem. Sci. 16:112 (1991); and Wright, Protein Engineering 4:283 (1991).
The present invention is predicated upon the previously unappreciated fact that recombinant human DNase may exist as a mixture of deamidated and non-deamidated forms. Using the methods of the present invention, it has been found that deamidated human DNase is less active enzymatically than non-deamidated human DNase. Thus, the presence of the deamidated DNase and non-deamidated,DNase together in a mixture, and the potential for further deamidation occurring, such as has been found to occur upon in vitro storage of preparations of human DNase, may complicate efforts to provide consistent uniformity in a DNase product being administered clinically. Therefore, as the existence and characteristics of deamidated DNase were not known prior to the present invention, the methods for identifying'deamidated DNase and separating it from preparations of DNase in which 'it may be found were unobvious at the time this invention was made.
a~, r~ of the Invention The present invention is directed to processes for separating the deamidated and non-deamidated human DNase forms from a mixture thereof. This process in preferred embodiments comprises subjecting the mixture to chromatography using a resin, or other', support medium, having bound hereto a cationic polymer such as heparin or a non-hydrolyzable deoxyribonucleic acid tDNA).analog, or chromatography using a'so-called tentacle cation exchange resin. The present invention also is directed to the use of those chromatographic methods with non-human DNases; such as bovine DNase.
The present, invention also is directed; to deamidated human DNase as a purified product, substantially free of non-deamidated human DNase.
The present invention also is directed to non-deamidated human DNase as a purified product, substantially free of deamidated human DNase: It has been.found herein that purified non-desmidated human DNase is fully enzymatically active as compared with deamidated human DNase'.
-z-WO 93/25670 Z ~ ~ ~ ~'~ PCT/US93/05136 The present invention also is directed to pharmaceutical, compositions consisting of eithAr purified deamidated human DNase or purified non-deamidated human DNase as the active principle, optionally together with a pharmaceutically acceptable excipient.
The present invention also is directed to a method comprising administering a therapeutically effective amount of purified deamidated human DNase or purified non-deamidated human DNase for the treatment of a patient, for example those having an accumulation of viscous, DNA-containing material. The administration of~'such purified DNases preferably is effected by direct inhalation into the lungs.
The present invention is particularly directed to a,method of treating a patient having a pulmonary disease such as chronic bronchitis, cystic fibrosis, or emphysema; that comprises administering a herapeutically effective: amount of purified non-15' deamidated human'DNase, preferably directly into the air~ray passages.
The present-invention also is directed to pharmaceutical compositions comprising non-deamidaGed~human DNase t: are disposed within a plastic vial, optionally in the.presence of a pharmaceutically acceptable excipient.
Brief Description of the Drawinas Figure:l depicts; the amino, acid (SEQ:ID:NO. 1) and DNA sequences ($EQ.ID.NO. 2) of human DNase I. The native signal sequence is uaderli,ned, the potential initiation codons are Circled,'and the 25 mature sequence is bracketed.
Figure:2 depicts the'correlation between enzymatic activity and extent of deamidati,on of samples of human DNase. Specific activity W aswdetera~i~,ed by normalizing the DNase activity as determined by a -methjrh green (1HG) assay (in concentration units relative' to a standard .30 cuxve) to the DNase concentration measured by an enzyme-'linked immu~oabsorbent assay (ELISA).. Percent deamidation was',determined-by Cryptic mapping. "Day of'Harveat" samples. of human DNase were purified fr~n a .culture of recombinant Chinese hamster civary (CHO) cells expressing DNA-encoding human DNase I, :'Such samples were taken 35 at 3, 5, 7f, .9, 11, 13, and 20 days after he culture Brae started.
"FIigh pH" .samples .were day 1,3 samples of purified DNase that were 'incubated in vitro for two days: at pH 8 at 37°. "Stability" sales v~ere day 13 saatples of purified DNase that were stored in v' r at 5°, 25°, or 37° C for various periods of time.
4Q Figrure 3 is a~a example of a~ txyptic map of DNase employed for WO 93/25670 . ;1'~ '~~ ~~'~ ~c PCT/US93/05136 N l"
DNase. Deamidation converts the Asn-74 to either an aspartic acid (Asp) or an iso-aspartate (iso-Asp) residue. Each of the three forms of DNase yields, on digestion with trypsin, a gair of peptides that indicates the identity of the particular form of DNase.
S Figure 5 is a chromatogram of a human DNase sample fractionated on a tentacle cation exchange (TCX) column. The sample shown is 67%
M
deamidated DNase.
Figure 6 shows tzyptic maps of the two peak fractions from the TCX .separation shown in Figure 5. The absence of tryptic peptide T6-7 from the map of the Peak 2 digest indicates the absence of deamidated DNase.
Figure 7 shows chromatograms of several human DNase samples fractionated on a TCx column. The sample designated "M1-28 STD." is a preparation of human DNase obtained from a culture of Chinese hamster ovary'(CHO) cells transformed with DNA encoding native human DNase I.
The sample designated "DNase ASP Mutant" is DNase having an aspartic acid residue (rather than an asparagine residue) at amino acid position 74, and Which thus has the same amino acid sequence as the Asp form of deamidated DNase shown in Figure 4. The DNase ASP Mutant was obtained from a culture of cells transformed with DNA encoding that mutant form of human DNase. The DNA encoding the DNase ASP
Mutant was prepared by site-directed mutagenesis of DNA encoding native human Dl~ase. Comparison of the chromatograms shows that one of the fozms of human DNase in the M1-28 STD. elutes from the TCX column at the same position as the DNase Asp Mutant.
Figure 8 shows chromatograms of several human DNase samples fractionated on a TSK-Heparin column (Toso Haas, Montgomeryville, Pennsylvania). The sample designated "12K #8" is a preparation of human DNase obtained from a culture of Chinese hamster ovary (CHO) cells transformed with DNA encoding native human DNase I. The sample designated "Deamidated Standard" is purified deamidated human DNase.
The sample designated "Non-deamidated standard" refers to purified non-deamidated human DNase. Purified deamidated human DNase and purified non-deamidated human DNase were prepared by TCX
chromatography. ' Figure 9 shows chromatograms of several human DNase samples fractionated on an immobilized DNA analog column. The sample designated "M1-28" is a preparation of human.DNase obtained from a culture of Chinese hamster ovary (CHO) cells transformed with DNA
encoding native human DNase I. The sample designated "Deamidated Standard" is purified deamidated human DNase. The sample designated "Non-deamidated standard" refers to purified non-deamidated human DNase. Purified deamidated human DNase and purified non-deamidated humaiz DNase was prepared by~TCX chromatography. The sample. designated WO 93/25670 ~ ~ ~ ~ Z ~ ~ PCT/US93/05136 "DNase ASP Mutant" is DNase having an aspartic acid residue (rather than an asparagine residue) at amino acid position 74.
Detailed Description A. Definitions By the term "human DNase" herein is meant a polypeptide having the amino acid sequence of human mature DNase I set forth in Figure 1 as well as amino acid sequence variants thereof (including allelic variants) that are enzymatically active in hydrolyzing DNA. Thus, the term "human DNase" herein denotes a broad definition of those materials disclosed and prepared in the patent applications described above.
The term "human DNase" necessarily embraces native mature human DNase having an asparagine (Asn) residue at amino acid positrt~n 74 of the polypeptide. That asparagine has been found herein to b susceptible to deamidation, which deamidation may produce a n-._..cure of deamidated and non-deamidated forms of human DNase. Instead of the Asn residue at amino acid position 74, deamidated DNase has aspartic acid (Asp) or an iso-aspartate (iso-Asp) residue (~:,-. Figure 4) .
The term "deamidated human :.°ase" as used herein thus mear- human DNase that is deamidated at the asparagine residue that occurs position 74 in the amino acid sequence of native mature human . 3se.
It has been found that deamidated human DNase may arise during the production of human DNase by recombinant means, and may be found in preparations of human DNase obtained from recombinant host cells.
Additionally, deamidated human DNase may arise upon in vitro storage of non-deamidated human DNase.
Although the asparagine residue at amino acid position 7 in the amino acid sequence of native mature human DNase also may be deamidated (in addition to the asparagine residue at amino acid position 74), such doubly deamidated DNase has been found to be enzymatically inactive. .
The term "mixture" as used herein in reference to preparations of human DNase means the presence of both deamidated and non-deamidated foxins of human DNase. It has been found, for example, that in preparations of human DNase obtained from recombinant expression, as much as about 50% to 80% or more of the human DNase is deamidated.
. The term "purified deamidated human DNase" as used herein means deamidated human DNase that is substantially free of non-deamidated human DNase. In other words, non-deamidated human DNase will comprise less than about 10%, preferably less than about 5%, and most preferably less than about 1% by weight of the total DNase in the . ,purified de~amidated human'DNase composition.

t ~ ~ ~' 1 pCT/US93/05136 WO 93/25670 ~~ ?~ ~ ~ N
N
The'lterm "purified non-deamidated human DNase" as used herein means non-deamidated human DNase that is substantially free of deamidatec7 human DNase. In other words, deamidated human DNase will comprise less than about 25%, preferably less than about 5%, and most preferably less than about 1% by weight of the total DNase in the purified non-deamidated human DNase composition. M
By the term "excipient" herein is meant a pharmaceutically acceptable material that is employed together with DNase for the proper and successful administration of the DNase to a patient.
Suitable excipients axe well known in the art, and are described, for example, in the Physicians Desk Reference, the Merck Index, and Remington's Pharmaceutical Sciences.
A preferred formulation for human DNase is a buffered or unbuffered aqueous solution, and preferably is an isotonic salt solution such as 15O mM sodium chloride containing 1.0, mM calcium chloride at pH 7. These solutions are particularly adaptable for use in commercially-available nebulizers including jet nebulizers and ultrasonic nebulizers useful for administration, for example directly into the airways or lungs of an affected patient. Reference is made to the above-identified patent applications for further detail concerning how human DNase can be formulated and administered for effective use.
By the term "therapeutically effective amount " herein, is meant dosages of from about l Ng to about 100 mg of human DNase per kilogram .
of body v~reight of the patient, administered within pharmaceutical compositions, as described herein. The therapeutically effective amount of human DNase will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. In view of the differences in enzymatic activity between deamidated and non-deamidated DNases described herein, it may be. that the amount .of purified non-deamidated DNase required to achieve a therapeutic effect will be less than the amount of purified deamidated human DNase or a mixture of the two forms necessary to achieve the same effect under the same conditions.
The purified DNases hereof, particularly the non-deamidated form, are employed for,enzymatic alteration of the viscoelasticity of mucous. Such purified human DNases are particularly useful for the 4Q treatment of patients with pulmonary disease who have abnormal viscous, purulent secretions and conditions such as acute or chronic bronchial pulmonary disease, including infectious pneumonia, bronchitis or tracheobronchitis, bronchiectasis, cystic fibrosis, ,. . ,asthma, tuberculosis, and'fungal infections. For such therapies, a solution or finely divided dry preparation of purified deamidated WO 93/25670 ~' ~ ~ ~ PCT/US93/05136 human DNase or purified non-deamidated human DNase is instilled in conventional fashion into the~bronchi, for example by aerosolization.
B. Preferred Embodiments After the successful cloning and expression of human DNase in recombinant host cells, it was discovered after substantial research that the DNase product obtained from such recombinant expression typically existed as a mixture of as then yet undefined components.
In particular, isoelectric focusing (IEF) analysis of human DNase purified from cultures of recombinant Chinese hamster ovary (CHO) cells revealed a complex pattern of DNase species. The various DNase species were determined to result from several post-translational modifications of the DNase, including deamidation.
Two assays were used to detezmine the presence and extent of deamidated DNase in such preparations. Dne method involved txyptic digestion of the starting preparation of DNase and analysis of the resulting geptides by reverse phase HPLC. In this method, the amount of deamidated DNase in the starting preparation was determined by measuring the quantities of six deamidation-indicating tryptic peptides.
The other method involved chromatography of the starting preparation of DNase on a tentacle cation exchange (TCX) column. It Was discovered that the TCX column is capable of resolvin_ deamidated human DNase and non-deamidated human DNase, such that each form of DNase could be effectively separated from the other, and obtained in purified form. In this method, the amount of deamidated and non-deamidated DNase in the starting preparation was determined by measuring on chromatograms the peak areas corresponding to the separated forms of DNase.
Although these two methods are about equally effective in determining and quantitating deamidated DNase, the TCX method is espec'sally efficient, requiring far less time and labor than ti. other method. Moreover, TCX chromatography provides a means for separating deamidated and non-deamidated fozms of DNase, whereas conventional.
canon exchange resins and various other chromatography resins that were analyzed were not capable of such separation.
The general principles of TCX chromatography have been described, for example, by Miller, J. Chromatography ,~Q:133 (1990); Janzen et al., J.~Chromatography ?2:77 (1990); and Hearn et al., J.
Chromatography X4$:117 (1991). Without limiting the invention to any particular mechanism or theory of operation, it is believed that the Asn-74 residue in human DNase that is susceptible to deamidation is located within the DNA-binding groove of the enzyme, by analogy to the . . . known crystal structure of bovine DNase. The DNA-binding groove .
contains basic amino acid residues (in .order_to bind DNA) and this WO 93/25670 ~~ ~~ '~ ~ ~ PCT/US93/05136 ~1'~~ ..
groove apparently is accessible to the ligands of the tentacle cation exchange resin but not to the much shorter ligands of conventional cation exchange resins. Presumably the ligands of the tentacle cation exchange resin mimic natural nucleic acid substrates. Therefore, it is expected that tentacle action exchange chromatography will be useful for the purification of other'nucleases, such as ribonuclease M
(RNase) or restriction endonucleases, as well as DNA binding proteins.
Alternatively, the separation of deamidated and non-deamidated fortes of DNase may be accomplished by chromatography using a resin or other support matrix containing covalently bound cationic polymers such as heparin or a synthetic non-hydrolyzable DNA analog.
Immobilized heparin chromatography columns are commercially available (for example, from Toso Haas Co., Montgomeryville, Pennsylvania).
Non-hydrolyzable DNA analogs have been described, for example, by Spitze,r et al., Nuc. Acid. Res. 16:11691 (1988). An immobilized non-hydrolyzable DNA analog column is conveniently prepared by synthesizing such a DNA analog with an amino acid group at the 3'-end of one or both of its complementary strands. The amino group is then available for coupling to an epoxy-activated column, as described, for example, in literature published by Rainin Biochemical LC Products (woburn, Massachusetts).
Following the successful separation of deamidated and non-deamidated human DNases according to the methods of the present invention, it was found that deamidated human DNase has diminished enzymatic activity as compared to non-deamidated human DNase, as detezmined by a methyl green (MG) assay. Kurnick, Arch. Biochem.
29:41 (1950). It was found that deamidated human DNase exhibits just over half of the enzymatic activity of non-deamidated human DNase.
Thus, by combining the purified deamidated DNases and the purified non-deamidated DNase of the present invention in various proportions, it is possible to prepare pharmaceutical compositions~of human DNase having any desired specific activity in the range between the specific activities of the individual components, as may be optimal for treating particular disorders: ~ , The following examples are offered by way of illustration only and are not intended to limit the invention in any manner.
C. Examples 1. Trwtic Mappinct.
, The procedure used for tryptic mapping of human DNase is summarized as follows:
Step 1. Bring concentration of 1 mg sample of DNase to 4 mg/ml by'concentration on Amicon Centricon-10 device or by , ~45 ~ dilution with excipient. Final, volume: ,250 ~1~., _g_ Step 2. Add 250 ~1 of pretreatment buffer (40 mM BisTris, 10 mM
EGTA, pH 6.0) to sample. Incubate 1 hour at 37.

Step 3. Buffer exchange sample into digest buffer (100 mM Tris, pH 8) using Pharmacia NAP-5* column. Final volume:

ml.

Step 4. Add 10 ~1 trypsin solution (1 mg/ml trypsin, 1 mM HC1) to sample and incubate 2 hours at 37.

_ Step 5. Add second 10 ~1 aliquot of trypsin solution to sample and incubate additional 2 hours at 37.

Step 6. Stop digestion by addition of 6 ~l trifluoroacetic acid (TFA). Store samples at or below 5 until chromatographed.

Step 7. Separate the peptide mixt,.re by HPLC under the following conditions:

Column: Nucleosil-~C18*, 5 Vim, 100 ~, 2.0 x 150 mm (Alltech, Co., Deerfield, Illinois).

Column temperature: 40.

Eluent A: 0.12% TFA in water.

Eluent B: 0.10% TFA in acetonitrile.

Gradient profile:

Time (min) %A %B

Flow rate: 0.25 ml/min. Sample injection volume:
250u1.
Post-run column reequilibration time at 100% A: 20 min.
Autosampler compartment temperature: 5°.
Detection: Absorbance at 214 and 280 nm.
Step 8. Identify T7, (D)T7, T7-8, (D)T7-8, T6-7-8, and T6-7 tryptic peptides by retention time comparison with standard.
Step 9. Integrate chromatogram obtained at 280 nm. Check quality of integration by inspection of baseline and separation of closely eluting peaks. Special attention must be paid to the early-eluting T7 and (D)T7 peptides that may not be well-resolved.
Step 10. Normalize peak areas of the six reporter peptides to tyrosine content. Peptides T7, (D)T7, T7-8, and (D)T7-8 each contain a single Tyr residue, while T6-7-8 and T6-7 contain three Tyr residues. Calculate the proportion of deamidated species based on the normalized peak areas of (D)T7, (D)T7-8, T6-7-8,~and T6-7 relative to the total normalized peak areas of the six peptides.
One milligram of DNase in a volume of 250 ul is required in order to accurately carzy out the tryptic mapping method for determination of deamidated DNase according to the procedure outlined above. Hence, the initial sample preparation for this method requires either *-trademark concentration or dilution of the sample to achieve that result. DNase in the presence of calcium is highly resistant to proteases, including trypsin. Therefore the next step in the procedure is to partially remove calcium ions by treatment with [ethylene bis(oxyethylenenitrilo)) tetraacetic acid (EGTA). Over-treatment with EGTA can denature and aggregate DNase, so this step must be performed with care. The EGTA-treated sample in a volume of 0.5 ml is then exchanged into 1 ml of the digest buffer, trypsin added, and the sample incubated at 37~ for two hours. A second aliquot of trypsin is then added and the sample incubated an additional two hours.
Digestion is stopped by acidification, and the sample is either stored for later analysis or loaded on the HPLC column directly.
250 ul (250 fig) of the peptide mixture resulting from the tryptic digestion is separated on a reversed phase HPLC column according to the conditions outlined above. A typical tryptic map of human DNase is shown in Figure 3. HPLC was performed with a Hewlett-Packard model 1090M HPLC. The column effluent was monitored simultaneously at 214 and 280 nm by the diode array detector that is a feature of this instrument. Since the early portion of the peptide map is critical to the quantitation of deamidated DNase, as described below, other instruments with larger gradient delay and other extra-column volumes may not be suited to this analysis. Each analysis by this procedure requires 70 minutes for the gradient separation and 20 minutes to re-equilibrate the column for a total HPLC turnaround time of 90 minutes.
The rationale and approach to peak integration for determination of deamidated DNase in a sample are described below.
Deamidation of human DNase occurs at least at the asparagine residue that is present at amino acid position 74 (Asn-74) in native mature human DNase. Asn-74 is on the C-terminal side of a tryptic cleavage site at the arginine residue at amino acid position 73 (Arg-73), as seen in the list of expected tryptic peptides of human DNase shown in Table I.
*Ttrademark WO 93!25670 TABLE I. PEPTIDES EXPECTED TO BE PRODUCED UPON DIGESTION OF NATIVE
MATURE HUMAN DNASE
WITH TRYPSTN.
10 Residues Amino Acid Sequence of Peptide T2 3-15 IAAFNIQ.TFGETK (SEQ.ID.NO. 3) M

T3 16-31 MSNATLVSYIVOILSR (SEQ.ID.NO. 41 T4 32-41 YDIALVQEVR (SEQ.ID.NO. 51 T5 42-50 DSHLTAVGK (SEQ.ID.NO. 6) T6 51-73 LLDNLNQDAPDTYHYVVSEPLGR (SEQ.ID.NO. 7) T7 74-77 NSYK (SEQ.iD.NO. 8) T9 80-111 YLFVYRPDaVSAVDSYYYDDGCEPCGNDTFNR (SEQ.ID.NO.

T10 112-117 EPAIVR (SEQ.ID.NO. 10) T1 1 1 18-121 FFSR (SEQ.ID.NO. 11 ) T12 122-126 FTEVR (SEQ.ID.NO. 121 T13 127-157 EFAIVPLHAAPGDAVAEIDALYDVYLDVQEK (SEQ.ID.N0.13) T14 158-185 WGLEDVMLMGDFNAGCSYVRPSQWSSIR (SEn.ID.NO.
t4) T15 186-213 LWTSPTFnWLIPDSADTTATPTHCAYDR (SEQ.ID.NO.
15) T16 214-222 IVVAGMLLR (SEQ.ID.NO. 161 (SEQ.ID.NO.

17) ~ Instead of the Asn (single letter designation ~~N~~) residue at residue 74 in native, non-deamidated human DNase, deamidated human DNase has either an Asp or iso-Asp residue, as shown in Figure 4. Iso-Asp is an isomeric, beta-amino acid form of aspartic acid. The peptide bond between Arg-73 and iso-Asp is resistant to cleavage by trypsin, so deamidated human DNase yields a characteristic tryptic pegtide containing residues.51-77 and called T6-7 since it is the conjoined peptides T6 and T7. Under conditions employed for tryptic mapping, the Arg-73-Asn-74 peptide bond in non-deamidated~human DNase and the Arg-73-Asp-74 peptide bond in the Asp fornl of deamidated human DNase are cleaved by trypsin.
Hence, non-deamidated DNase is indicated in the tryptic map by the presence of T7 peptide shown ~in Table I, while the' Asp-74 form of deamidated human DNase is indicated in the tryptic map by the presence of the deamidated T7 peptide, called (D)T7. These three reporter peptides are labelled in Figure 3: Unfortunately, trypsin only partially cleaves the peptide bond at the C-terminal side of T7,'between residues 77 and 78, so that each of the reporter peptides T7, (D)T7 and T6-7 has a T8-conjugate, T7-8, (D).T7:.8 and T6-7-8, respectively. These six reporter peptides must therefore be accounted for in order to quantitate deamidated htunan DNase by the txyptic mapping method.
In principle; the (D)T7, '(D)T7-8, T6-7 and T6-7-8 peptides represent deamidated human DNase and the T7 and T7-8 peptides represent non-deamidated human DNase and knowledge of the relative proportions of these peptides permits a straightforward calculation of the extent of deamidation in a preparation of DNase. In order to calculate the .fraction of the sample that is deamidated DNase, knowledge of the molar ratios .of deaniidated,and non-deamidated species is required, but the WO 93/25670 1 ~~r'~ ' ,a v ~ PCT/US93105136 t,,.
'~,, l"
There are two additional problems in the tryptic mapping procedure that must be overcome: one chromatographic problem and one detection problem.
The chromatographic problem is that the T2 peptide coelutes with T6-7, and so impedes the integration of an accurate peak area of this deamidation-indicating peptide. This problem can be overcome by integration of the chromatogram obtained at 280 nm, since all six of the relevant peptides have at least one tyrosine (Tyr) residue, and so absorb strongly at 280 nm, while T2 contains no Tyr or tryptophan (Trp) residues and thus absorbs negligibly at this wavelength. The detection problem is that the T6-7 and T6-7-8 peptides each contain three Tyr residues while the other four peptides each contain only one. Thus the T6-containing peptides have a higher molar absorptivity than do the peptides that contain only T7, and a simple comparison of peak areas would tend to overestimate the content of deamidated species in a sample. This problem is overcome by normalizing the peak areas of the six peptides to the number of Tyr residues in the peptide. Normalizing the peak areas in this manner implies that all tyrosine residues in each of the peptides is in an equivalent chemical environment, which is probably a good assumption for relatively small peptides such as considered here. Upon normalization, the corrected peak areas for deamidated and non-deamidated peptides can be compared to arrive at an estimate of the content of deamidated DNase in a sample.

2. Tentacle Cation Exchancre Chromatoaraphv~.
Tentacle cation exchange (TCX) resins, unlike conventional cation exchange resins, have polyionic ligands bound to a silica surface. The ligands of the LiChrospher° 1000 S03 column (EM Separations, Gibbstown;
New Jersey) used in this example are advertised as containing between 25 and 50 sulfopropyl groups along a polyethylene backbone that is joined at one end to the silica surface.
The TCX chromatogram of a sample of recombinant human DNase run on.
a LiChrospher° 1000 S03 column is shown in Figure 5., Recombinant human DNase was gurified from cultures of Chinese hamster ovary (CHO) cells transformed .with DNA encoding human DNase . Shak, et al . , Proc . Nat .
Acad. Sci: 87:9188-9192 (1990); Shak, et al., International Patent Application Publication No. W0 90/07572 (published l2 July 1990).
The two peaks obtained were collected and subjected to several analyses in order to identify them as the dorms of DNase differing only at the residue at amino acid position 74. Figure 6 shows tryptic maps of the two peaks collected from the TCX column, confirming that they are, respectively, the deamidated and non-deamidated forms of human DNase.
The tryptic map also reveals that both forms of deamidated DNase (having Asp and iso-Asp at amino acid position 74) are present in the first peak from the TCX ,separation. Table II shows the specific activities measured ~45~ ~ for the two peaks, confirming therelationship between deamidation and WO 93/25670 '~ ~ ~ ? '~ ~ PGT/US93/05136 specific activity inferred from the correlation shown in Figure 2, and further supporting the identification of the TCX fractions. Activity of the DNase fraction was determined by a methyl green (MG) assay.
TABLE II. ACTIVITIES OF FRACTIONS COLLECTED FROM
TCX COLUNIN.
MG and ELTSA concentrations are the averages of determinations on two samples.
MG ELISA Specific Sample (~Cg/ml) (~g/ml) ' Activity Starting preparation of 8315 7828 1.06 recombinant human Dnase (load) TCX Peak 1 (deamidated) 85.3 119.7 0.71 TCX Peak 2 (non-deamidated) 149.2 99.4 1.50 A mutant form of human DNase, having an Asp residue at amino acid position 74, was produced by site-directed mutagenesis of the DNA
encoding native mature human DNase. This mutant coelutes with the first peak obtained in the above chromatography, as shown in Figure 7.
The: following is the procedure used to pack the LiChrospherm 1000 SO; tentacle cation exchange resin: Another tentacle cation exchange resin similarly :useful for separation of deami.dated and non-deamidated forms of human DNase is Fractogel° tentacle cation exchange resin (EM
Separations, Gibbstown, New Jersey). LiChrospher and Fractogel are registered trademarks of EM Industries, Inca,, Hawthorne, N.Y., or E. Merck, Darmstadt, West Germany. The "strong~~ forms of the tentacle cation exchange resins (whether LiChraspher or Fractogel), having a SO;
functional group, appear at this time to give the best results.

3. HPLC Column 'PackincL Prgcedure for LiChrosvher~ 1000 SO,' Resin:
a . Mxrcri gl_s= an_d Eg~uibment 1. Superfonnance glass,cartridge 1.0 cm x 5.0 cm bed.
2. Packing Buffer: lOmM sodium' acetate, 1mM CaCl=, pH to 4 .5 with acetic acid. Filter through a 0.:2~ filter. ' 45 ,.
3. Column packing reservoir with a capacity of 20 ml.
(Alltech part # 9501 or equivalent).

4. Empty 4.6mm x 50mm stainless steel column with 0:5 ~ cut-50 ~ off frita 5. HPLC pump capable of maintaining a back~pressuie of 2000 psi (Waters Model 510 or equivalent). .
_13_ b. Packing Procedure.
1. De-fine resin:
a) Unpack 1.0 cm x 5.0 cm Superformanc2~ glass column (Bed volume = 3.93 ml resin). Resuspend resin to 20 mls in a clear glass, capped vessel with column packing buffer. Slurry into a uniform suspension and divide into 2 x 10 ml aliquots. Add 10 mls of column packing buffer to each aliquot to achieve suspensions of approx. 1.95 mls resin in 20 mls packing buffer.
b) Slurry resin to achieve a uniform suspension. Allow to settle until particles form a solid bed on the bottom of the vessel (2-4 hours) . Carefully pour off the supernatant containing fine particles.
c) Add 20 mls. packing buffer to resin and repeat step b). This procedure should be repeated at least four times to assure removal of all fine resin particles.
2. Column Packing:
a) Connect 4.6 mm x 50 mm empty HPLC column to packing reservoir. Slurry resin in 20 mls of packing buffer.
b) Add slurried resin to reservoir and quickly cap.
Pump packing buffer at a pressure that does not exceed 2000 psi. Adjust flow rate so that packing pressure remains constant at about 2000 psi and flow for 15 minutes after pressure stabilizes. Remove column and attach top end. Column may be used directly or stored in 0.02% sodium azide.
For most samples, including DNase formulated in 150 mM NaCl, no sample preparation is required prior to injection of the sample onto the column. The column is equilibrated with a ph 4.5 acetate buffer containing calcium ions, the sample is injected, and the column then is eluted with a salt gradient. The following procedure is useful for small-scale separations of deamidated and non-deamidated forms of human DNase. The proportions of the peak areas on the resulting chromatogram are equal to the proportions of deamidated and non-deamidated DNase in the sample.
Step 1. Load sample, containing up to 150 mM NaCl and at a pH up to 9 into autosampler vial. Harvested cell culture fluid samples require adjustment of pH to 4.5 and centrifugation to remove proteins that are insoluble in the buffers used in this procedure.
Step 2. Separate the two forms of DNase by HPLC under the following conditions:
Column: TCX LiChrospher~ 1000 S03 repacked into a steel column. Column dimensions of 4.6 x 50 mm and 4.6 x 150 mm have been packed and employed.
Column temperature: ambient.
Eluent A: 10 mM sodium acetate, 1 mM CaCl2, pH 4.5.
Eluent B: 1 M NaCl in buffer~A.
Gradient profile:
*-trademark -14-Time (min) %J~ %B

30.1 5 95 Flow rate: 0.8 ml/min (50 mm column), 0.5 mI/min (150 mm column).
Sample injection volume: up to 250 ul.
Post-run column reequilibration time at 100% A: 20 min.
Autosampler compartment temperature: 5°.
Detection: Absorbance at 280 nm.
Step 3. Integrate chromatogram. Calculate the proportion of deamidated species based on the peak area of the earlier eluting deamidated DNase relative to the total peak area of both forms .
Tentacle cation exchange chromatography also provides a means for separating, at large scale, the deamidated and non-deamidated forms of human DNase. Large scale separations are more conveniently carried out using simplified elution operating conditions than are described above for small-scale analytical separations of the two forms of DNase. Hence, larger scale separations have been carried out on the Fractogel-supported tentacle cation exchanger according to the following pH-elution procedure:
Step 1. Pack 31.6 column (1.6 cm i.d. x 15.7 cm high) with Fractogel EMD SO,-650M tentacle cation exchange resin (EM
Separations, Gibbstown, New Jersey).
Step 2. Diafilter'~DNase load with equilibration buffer (30 mM
sodium acetate (NaAc), 1 mM calcium chloride (CaCl,), 50 mM sodium chloride (NaCl), pH 5). Concentrate by ultrafiltration to volume of 355 mls and concentration of 2.5 mg/ml.
Step 3. Wash column with 2.5 column volumes (CV) of 2% sodium hydroxide INaOH).
Step 4. Wash column with 2.5 CV of pre-equilibration buffer (300 mM NaAc, 1 M NaCl, pH 5).
Step 5. wash column with 2.5 CV of equilibration buffer.
Step 6. Load column with 1-1.3 g of diafiltered / ultrafiltered DNase (from Step 2) . Begin collecting fractions of column effluent upon commencement of DNase load.
Step 7. wash column with 5 CV of equilibration buffer.
Step 8. Wash column with 5 CV of pH 5.3 wash buffer (25 mM
succinate, 1 mM CaCl=, pH 5.3).
Step 9. Wash column with 10 CV of pH 5.4 wash buffer (25 mM
succinate, 1 mM CaCl=, pH 5..4) .
Step 10. Wash column with 10 CV of pH 6 wash buffer (25 mM MES, 1 mM CaCl;, pH 6 . 0 ) .

*-trademark Step 11. Combine fractions collected during Steps 6-8 to make a pool consisting predominantly of deamidated DNase.
Combine fractions collected during Step 10 to make a non-deamidated DNase pool. Fractions collected during Step 9 contain a mixture of the two forms of DNase and may be recycled.
The protocol described above is one example of the use of a tentacle cation exchange resin for a preparative purification of the two fozms of recombinant human DNase in a manner that is scaleable to large-scale recovery of purified deamidated and purified non-deamidated DNase.
4. Heparin and Immobilized DNA Analog Chromatoqraphv. ' In Figure 8 chromatograms are aligned of analyses on a TSK-Heparin column (Toso Haas, Montgomeryville, Pennsylvania) of samples containing either a mixture of deamidated and non-deamidated forms of human DNase, purified deamidated human DNase, or purified non-deamidated human DNase.
The TSK-Heparin column*was run under the same conditions as described above for running the analytical TCX column. The aligned chromatograms demonstrate that the column of immobilized heparin resolves deamidated and non-deamidated forms of DNase.
As described above, another means of separating the deamidated and non-deamidated forms of DNase is to employ a column containing an immobilized analog of DNA that is resistant to hydrolysis by DNase. One example of this approach to an immobilized DNA analog column involved the synthesis of the phosphorothioate oligonucleotide 5'-GCGCGCGCGCGCGCGCGCGCGC-NH,-3'. This self-complementary sequence can be annealed into a double-stranded form, and coupled to a Rainin Hydropore-EP column*(Rainin Co., Woburn, Massachusetts). Figure 9 shows aligned chromatograms of the analyses on this column of samples containing either a mixture of deamidated and non-deami dated forms of human DNase, purified deamidated human DNase, purified non-deamidated human DNase, or purified mutant human DNase having an aspartic acid residue (rather than an asparagine residue) at amino acid position 74. The column was run for these analyses in a buffer containing 1 mM calcium chloride,'~5 mM MES at a pH of 6, and eluted with a linear gradient in salt concentration to 1 M sodium chloride over 20 minutes at a flow rate of 1 ml/min. As shown in Figure 9, under these conditions deamidated and non-deamidated DNase forms are partially separated from each other. In addition, the two isomeric forms of deamidated DNase, that differ at amino acid position 74 of the DNase sequence by having either aspartic acid or iso-aspartic acid at this position, are also resolved by this column. Thus an additional benefit of this chromatographic method is that it allows the isolation of the two isomers that arise on deamidation of human DNase.
*-trademark -16- ' -5. Enzymatic Activity of Deamidated Human DNase and Non-deamidated human DNase.
Several analytical methods have been used to examine the effect of deamidation on the enzymatic activity of human DNase. Purified deamidated human DNase and purified non-deamidated human DNase for use in these studies were prepared by TCX chromatography, as described above.
In one method for determination of DNase enzymatic activity, synthetic double stranded DNA, 25 base pairs in length, was labeled with dinitrophenol (DNP) on one end and with biotin on the other end.
Hydrolysis of the substrate by DNase was detected by capture of the reaction products on microtiter plate wells coated with antibody to DNP
and by quantitation of the intact probe with streptavidin-horseradish peroxidase. The specific activity of stability samples was correlated (r==0.613;n=5) with the extent of DNase deamidation (range 27% - 93%).
Extrapolation of the least squares linear equation provided an estimate that the specific activity of desmidated human DNase was approximately 77% lower than that of non-deamidated human DNase.
Another method for determination of DNase enzymatic activity involved hydrolysis of the chromogenic substrate p-nitrophenyl phenylphosphonate (PNPP) as described by Liao, et al., Biochem. J. 55:
781-787 (1988). The kinetics of PNPP hydrolysis by human DNase are sigmoidal and were fit to the Hill equation by nonlinear regression. By this method the V~" of fully deamidated human DNase was determined to be 77% lower than that of non-deamidated human DNase. The substrate concentration for half maximal activity (S°.~) did not differ significantly for the deamidated and non-deamidated human DNase samples.
Another method for determination of DNase enzymatic activity is the assay described by Kunitz, J. Gen. Physiol. X3_:349 (1950), preferably modified such that the enzymatic reaction is carried out at about pH 7.0 - 7.5. By this method, the enzymatic activity of deamidated human DNase also was determined to be lower than that of non-deamidated human DNase.

6. In Vitro Storacre of Human DNase.
Human DNase purified from recombinant CHO cells was dissolved at a concentration of 4 mg/ ml in an unbuffered aqueous solution,bf 150 mM
NaCl and 1 mM CaCl... Samples of the resulting DNase solution were then placed into glass and plastic vials. Two different types of plastic vials were used, one being made of Dupont 20 plastic resin*(manufactured by E.I. du Pont de Nemours & Co., Inc., Wilmington, Delaware 03A), and the other being made of Escorene plastic resin*(manufactured by Exxon Corp.). Both of those plastics are low density polyethylene, but containers formulated with other plastics, such as polypropylene, polystyrene, or other polyolefins also may be used. The vials containing the DNase solution were stored at either '-70° C, 2-8° C, or 25° C.
Initially, about 60ic - 65ic of the DNase in the solutions was deamidated.
*-trademark -17-~ v ~ PCT/US93/05136 WO 93/25670 ~ ~ ~ ~ .
f'~
The DNase solutions in the vials were assayed at several times after initial storage to determine the extent of deamidation of the DNase. The results of those assays are shown in Table III.
TABLE III. % DEAMIDATION OF RECOMBINANT HUMAN
DNASE STORED IN GLASS AND PLASTIC VIALS.
Sample Day -70°C 2-8°C 25°C
Glass 83 66 66 78 174 63 66 81 , .
Dupont 20 83 65 66: 71 Escorene 83 65 66 71 After 83 and 174 days storage at -70° C or 2-8°C; no difference Was found in the amount of deamidated DNase in he plastic vials and the amount of deamidated DNase in the glass vials. In each such case, approximately 64% (+/- 2%) of the DNase in the vial s was deamidated DNase.
Unexpectedly, however, after 83 or 174 days storage at 25° C, there was a difference in the amQUnt of deamidated DNase in the plastic vials and the amount of deamidated DNase in the glass vials. Significantly less deamidated DNase was present in the plastic vials. In particular;
of er 83 days storage at 25° C, 78% of the DNase in the glass vials was deamidated DNase, whereas only about 70% of the DNase in he plastic vials was deamidated DNase. After 174 days storage at 25° C, 81% of the DNase in the'glass vials was deamidated DNase, whereas only about 71%: of the DNase in the plastic vials was deamidated DNase;
Without: limiting the invention to any particular mechanism or theory of operation, it maybe that the differences in deamidation of DNase in plastic and glass vials may kie a consequence of differences in the pH of the solutions in he vials. Initially, the pH of the DNase solution in the glass vials was slightly higher than that in the plastic vials (approximately pH,6.7 and approximately pH 6.5, respeqti.vely). The pH
of the DNase solution in the glass vials continued to increase slightly over time.(to approximately pH 6.9 after 83 days storage at 25° C, and approximately pH 7.0 after 174 days storage at 25°C), perhaps as consequence of silicates or ions from the glass surface dissolving in the solution, At higher pH, the rate of deamidation of human DNase is increased. Since it. was not appreciated that deamidation of human DNase occurs at elevated pH; it is an embodiment of this invention to formulate WO 93/25670 "~' ~ ~ PCT/US93/05136 and/or store human DNase in solutions having acidic pH, typically at about pH 4.5 - 6.8 and most preferably at about ph 0 - 6.8.
Thus, a significant improvement in the stabi~... 1~ of human DNase in solution is obtained by placing such DNase solution in plastic vials rather than glass vials, with apparently less deamidation of the DNase occurring over time in the plastic vials than in t ~ glass vials. This M
finding may be especially relevant to the choice ~. packaging c' ~tunan DNase for therapeutic use, where it is especially desirable - the human DNase be capable of storage for extended periods of time ~. ..:.izout significant loss of enzymatic activity. Of course, glass vials with non-glass coatings, for example, plastic linings, would be equally useful.
What is important is to avoid storing DNase it contG :pith glass, especially for storage exceeding about l5 - 30 General Remarks The foregoing description details specific methods which can be employed to practice the present invention. Having'detailed specific methods used to identify, characterize, separate and use the pure deamidated and non-deamidated human DNase hexeof, and ther disclosure as to specific model systems pertaining thereto, those _led in the art will well enough know how to devise alternative reliable methods for arriving at the same information in using the fruits of the present invention, Thus, however detailed the forgoing may appear in text, it should not be construed as limiting the overall scope hereof ; ra- ', the ambit of the present invention is to be determined only by tr .awful construction of the appended claims.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Genentech, Inc.
(ii) TITLE OF INVENTION: PURIFIED FORMS OF DNase (iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE ADDRESS:.
(A) ADDRESSEE: Genentech, Inc.
(B) STREET: 460 Point San Bruno Blvd (C) CITY: South San Francisco (D) STATE: California (E) COUNTRY: USA
(F) 2IP: 94080 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 5.25 inch, 360 Kb floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: patin (Genentech) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAN~: Johnston, Sean A.
(B) REGISTRATION NUMBER: 35,910 (C) REFERENCE/DOCKET NUMBER: 747 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415/225-3562 (B) TELEFAX: 415/952-9881 (Cl TELEX: 910/371-7168 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 346 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: S~Q ID NO: l:
Ser Cys Thr Gly Ser Ala Leu Lys Cys Phe Phe Arg Asp Leu Ser Ser Xaa Thr Thr Phe Phe Ser Leu Ser Ser Lys Arg Arg Lys Leu Ser Ser Lys Asp Ile Pro Asp Ser Xaa Gln His Ser Arg His Leu Xaa Gly His His His His Leu Arg Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu Leu Gln Gly Ala Val Ser 65 70 ~ 75 Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys *-trademark ~,i;
WO 93/25670 "t ~- ~ ~ "'' '' ~ PCF/US93/05136 Met Ser Asn Thr Leu Val Ser Ile GlnIle LeuSer Ala Tyr Val g5 100 105 Arg Tyr Asp Ala Leu Val Gln Val AspSer HisLeu Ile Glu Arg Thr Ala Val Lys Leu Leu Asp Leu AsnGlnAsp AlaPrv Gly Asn Asp Thr Tyr Tyr Val Val Ser Pro LeuGlyArg AsnSer His Glu Tyr Lys G1u Tyr Leu Phe Val Arg ProAspGln ValSer Arg Tyr Ala Val Asp Tyr Tyr Tyr Asp Gly CysGluPro CysGly Ser Asp Asn Asp Thr Asn Arg Glu Pro Ile ValArgPhe PheSer Phe Ala Arg Phe Thr Val Arg Glu Phe Ile ValProLeu HisAla Glu Ala Ala Pro Gly Ala Val Rla Glu Asp AlaLeuTyr AspVal Asp Ile Tyr Leu Asp Gln Glu Lys Trp Leu GluAspVal MetLeu Val Gly Met Gly Asp Asn Ala GIy Cys Tyr ValArgPro SerGln Phe Ser ' Trp Ser Ser Arg Leu Trp Thr Pro ThrPheGln TrpLeu Ile Ser Ile Pro Asp Ala Asp Thr Thr Thr ProThrHis CysAla Ser Ala Tyr Asp Arg Val Val Ala Gly Leu LeuArgGly AlaVal IIe Met Val Pro Asp Ala Leu Pro Phe Phe GIn Ala TyrGly Ser Asn Ala Leu Ser Asp Ser Asp Tyr Pro Gln Leu Ala His Val Gln Ala Ile Glu Va1 Met His Xaa Leu Lys Xaa Thr Thr Ala Ala Pro Ser Pro 335 340 . 345 Ala (2) INFORMATIONFOR SEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:
1039 bases (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY:
linear (xi) SEQUENCE DESCRIPTION: SEQ N0:2: -ID

,, ,1.
WO 93/25670 '~ ~~~~ r ~ r' P~I'/US93/05136 ~.

CACCAGACAC CTATCACTAC GTGGTCAGTG AGCCACTGGG ACGG.AACAGC 450 AGGGAGTTTG CC~TTGTTCC CCTGCATGCG GCCCCGGGGG ACGCAGTAGC 650 (2).INFORMATION FOR SEQ ID N0:3:
(i) SEQiJENCE CHARACTERISTICS:
(A) LENGTH: 13 amino'acids . .
(B)'TYPE: amino acid'.
(D) TOPOLOGY: linear WO 93/25670 =I ' '~'~ ,'~ '.~ ''~ PCT/US93/05136 ;,~ 1 ~ ~.~ ..~
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids ..
(B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ~ , (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Tyr.Asp Ile Ala Leu Val Gln Glu Val Arg (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Asp Ser His Leu Thr Ala Val Gly Lys (2) INFORMATION FOR SEQ ID NO:7:
(z) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids (B) TYPE: amino acid S O (D ) TOPOLOGS~ : l inear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Leu Leu Asp ASn Leu Asn Gln Asp Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg (2) INFORMATION FOR SEQ ID N0:8: ' (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear v (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
_23_ :.
~ ~a Asn '~~~'yr Lys (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: amino aci d (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser 1 5 10 , 15 Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Asn Arg (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid , (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Glu Pro Ala Ile Val Arg (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Phe Phe Ser Arg (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE : amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Phe Thr Glu Val Arg (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CFiAR,ACTERISTICS:
(A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear . (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
' - Glu Phe Ala.Ile Val Pro Leu His Ala Ala Pro Gly Asp~Ala Val WO 93/25670 ~ ~, ~'~ ~ 2 ~ ~ PCT/US93/0~136 Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu 20 ~ 25 30 Lys (2} INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTTCS: ", (A) LENGTH: 28 amino acids (B) TYPE: amino acid (D} TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg (2) INFORMATION FOR SEQ ID N0:15:

(i) SEQUENCE CHARACTERISTICS:

~ (A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ
ID N0:15:

Leu Trp Thr Ser Pro Thr Phe Gln Leu Pro Asp Ser Ala Trp Ile Asp Thr Thr Ala Thr Pro Thr His Ala Asp Arg Cys Tyr (2) INFORMATION FOR SEQ ID NO:16:

(i)'SEQUENC& CHARACTERISTICS:

40' (A) LENGTH: 9 amino acids (g) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ
ID N0:16:

21e Val Val Ala Gly Met Leu Leu Arg (2) INFORMATION FOR SEQ ID N0:17:

(i) SEQUENCE CHARACTERISTICS: ' (A) LENGTH: 38 amino acids f8)-TYPE: amino acid ' (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ
ID N0:17:

Gly Ala Val Val Pro Asp Ser Ala Pro Asn Phe Gln Ala Leu Phe Ala Tyr Gly Leu Ser Asp Gln Leu Gln Ile Ser Asp His Ala Ala Tyr Pro Val Glu Val Met Leu Lys

Claims

1. A process for preparing a purified human DNase I that is non-deamidated at the amino acid residue corresponding to Asn 74 of native human DNase I, comprising separating a mixture of deamidated and non-deamidated human DNase I with a tentacle cation exchange resin.

2. A process for preparing a purified human DNase I that is non-deamidated at the amino acid residue corresponding to Asn 74 of native human DNase I, comprising separating a mixture of deamidated and non-deamidated human DNase I with an immobilized heparin resin.

3. A process for preparing a purified human DNase I that is non-deamidated at the amino acid residue corresponding to Asn 74 of native human DNase I, comprising separating a mixture of deamidated and non-deamidated human DNase I with an immobilized non-hydrolyzable DNA analog resin.

4. A purified human DNase I that is non-deamidated at the amino acid residue corresponding to Asn 74 of native human DNase.

5. A DNase I composition comprising a human DNase I non-deamidated at the amino acid residue corresponding to Asn 74 of native human DNase I and a human DNase I
deamidated at said Asn 74 residue, wherein said deamidated human DNase I is present in the composition in an amount less than25% by weight of the total human DNase I.

6. The DNase I composition according to claim 5, wherein said deamidated human DNase I is present in the composition in an amount less than 5% by weight of the total human DNase I.

7. The DNase I composition according to claim 5, wherein said deamidated human DNase I is present in the composition in an amount less than 1% by weight of the total human DNase I.

8. The DNase I composition according to claim 5 in a plastic vial.

9. The DNase I composition according to claim 6 in a plastic vial.

10. The DNase I composition according to claim 7 in a plastic vial.