Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2003-05-21
2004-12-07
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
Reexamination Certificate
active
06828118
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is directed to an assay for determining the amount of Chinese Hamster Ovary (CHO)-produced tPA present in samples of recombinant human tPA with native sequence or its variants produced in CHO cells.
2. Description of Related Art
Tissue-type plasminogen activators (tPA) are endogenous serine proteases involved in a cascade of events leading to the dissolution of a blood clot (Astrup and Permin,
Nature
, 159, 681-682 (1947); Camiolo et al.,
Proc. Soc. Exp. Biol. Med
., 138, 277-280 (1971); Collen,
J. Cell. Biochem
., 33, 77-86 (1987); Hoylaerts et al.,
J. Biol. Chem
., 257, 2912-2919 (1982)). ACTIVASE® is the recombinant form of human tPA (r-tPA), used in the management of acute myocardial infarction and pulmonary embolism (Grossbard,
Pharm. Res
., 4, 375-378 (1987)). ACTIVASE® is also now approved for treating ischemic stroke (Smith et al.,
Acad. Emergency Medicine
, 6(6), 618-25 (1999); Kwiatkowski et al.,
New Eng. J. Med
., 340(23), 1781-1787 (1999)). It is a glycoprotein produced by expressing the complementary DNA (cDNA) for natural human tPA in CHO cells. TNK-tPA is a genetically engineered variant of human tPA cloned and expressed in CHO cells (Keyt et al.,
Proc. Natl. Acad. Sci USA
., 91, 3670-3674 (1994)). Site-directed mutations were introduced at three specific sites of human tPA to create the TNK-tPA variant. They are Thr103 to Asn (T103N), Asn 117 to Gln (N117Q), and Lys-His-Arg-Arg (SEQ ID NO:1) 296-299 to Ala-Ala-Ala-Ala (SEQ ID NO:2) (KHRR296-299AAAA). When compared to tPA, TNK-tPA exhibits similar in vitro biological activity, an increased resistance to plasminogen activator inhibitor and an enhanced fibrin specificity, and is cleared more slowly from plasma (Keyt et al.,
Proc. Natl. Acad. Sci USA
., 91, 3670-3674 (1994); Thomas et al.,
Stroke
, 25:10, 2072-2079 (1994); Benedict et al.,
Circulation
, 92:10, 3032-3040 (1995); Modi et al.,
Thromb Haemost
, 79, 134-139 (1998)). It is currently awaiting regulatory approval as a single bolus administered form of r-tPA. CHO cells biosynthesize endogenous hamster tPA called CHO-PA. CHO-PA has a similar fibrinolytic activity to human tPA as determined by the clot lysis assay. The amino acid sequence of CHO-PA is 80% identical to that of human tPA. Many of the substitutions are semi-conservative such as: Arg<
>Lys, Glu<
>Asp, Phe<
>Tyr, Val<
>Ala, Ile<
>Leu, or Thr<
>Ser. Using a model of the human tPA protease domain based upon the bovine chymotrypsin structure, it is observed that virtually all of the substitutions in CHO-PA are localized at or near the protein surface.
r-tPA, TNK-tPA, and CHO-PA are all single polypeptide chains composed of 527 amino acids with 17 disulfide bonds (Nguyen and Ward, “Stability Characterization and Formulation Development of Altepase, a Recombinant Tissue Plasminogen Activator,” in
Stability and Characterization of Protein and Peptide Drugs: Case Histories
, Y. J. Wang, R. Pearlman, eds., (Plenum Press: New York, 1993), pp. 91-135). For all three proteins, the peptide bond between Arg
275
and Ile
276
is particularly susceptible to protease cleavage. The cleavage results in two fragments: one consisting of the N-terminal 275 amino acids and the other consisting of the C-terminal 252 amino acids. The N-terminal chain contains regions which are homologous to the kringle regions found in plasminogen and prothrombin and, therefore, is often referred to as the “kringle fragment” (Nguyen and Ward, supra; de Vos et al.,
Biochem
., 31, 270-279 (1992)).
The C-terminal chain contains the catalytically active site and, therefore, is commonly referred to as the “protease fragment” (Pennica et al.,
Nature
, 301, 214-221 (1983)). The cleaved two chains are linked by a single disulfide bond formed between Cys
264
and Cys
395
. The cleaved molecule is commonly referred to as “two-chain tPA” as opposed to “single-chain tPA” or the intact form.
r-tPA contains four potential sites for N-linked glycosylation identified by the sequence Asn-X-Ser/Thr (Nguyen and Ward, supra). These are Asn
117
, Asn
184
, Asn
218
, and Asn
448
. r-tPA exists as two glycosylation isozymes designated type I and type II. Type I r-tPA is glycosylated at Asn
117
, Asn
184
, and Asn
448
; whereas type II r-tPA is glycosylated only at Asn
117
and Asn
448
. Asn
218
is not glycosylated in either isoform. TNK-tPA has the same glycosylation pattern as r-tPA, except that the Thr103 to Asn and Asn117 to Gln mutations effectively moved the glycosylation site from position 117 to 103 (Keyt et al., supra). The glycosylation pattern for CHO-PA is not fully characterized (Rijken and Collen,
J. Biol. Chem
., 256: 7035-7041 (1981)).
ACTIVASE® is a trademark for the recombinant form of human tissue-type plasminogen activator (r-tPA), used in the management of acute myocardial infarction and pulmonary embolism. ACTIVASE® brand tPA is also now approved for treating ischemic stroke. It is produced by expressing the complementary DNA (cDNA) for natural human tPA in CHO cells (U.S. Pat. No. 5,753,486). TNK-tPA is a genetically engineered variant of r-tPA with enhanced efficacy and lower incidence of bleeding compared with ACTIVASE® r-tPA. It was created by three site-directed mutations (T103N, N117Q and KHRR (SEQ ID NO:1) 296-299AAAA (SEQ ID NO:2)), and is also cloned and expressed in CHO cells (U.S. Pat. No. 5,612,029). CHO cells biosynthesize endogenous hamster tPA called CHO-PA. The amino acid sequence of CHO-PA is highly homologous (80% identical) to that of r-tPA. All three thrombolytic proteins exist as heterogeneous isoforms, mainly due to proteolysis/hydrolysis and differential glycosylation.
A method for purifying human tPA from CHO-PA is described in U.S. Pat. No. 5,411,864. This method-comprises contacting a fluid containing the human tPA with antibodies specifically binding the corresponding endogenous CHO-PA and recovering the human tPA. Preferably the contacting step involves passing the fluid through a chromatographic bed having the antibodies immobilized thereon.
The development of recombinant DNA-derived protein pharmaceuticals has been facilitated by the introduction of new analytical methods that can be used to characterize protein and/or to demonstrate consistency of manufacture of a protein. Peptide mapping is a key method for monitoring the amino acid sequence and is able to detect small changes in small-to moderate-size proteins, for example, insulin and human growth hormone. The analysis of a much larger protein, e.g., fibrinogen (molecular mass of 350,000), or the heterogeneous glycoproteins, such as antibodies (molecular mass of 150,000), is hindered by the complexity of the range of peptides generated by an enzymatic digestion. Such complexity makes a single reversed-phase high-performance liquid chromatography (RP-HPLC) separation combined with on-line ultraviolet detection of limited utility.
The advent of commercially available combined HPLC and electrospray ionization mass spectromety (LC-ES-MS) systems compatible with convention HPLC has increased the power of peptide mapping considerably (Ling et al.,
Anal. Chem
., 63: 2909-2915 (1991); Guzzetta et al.,
Anal. Chem
., 65: 2953-2962 (1993)). LC-EM-MS in combination with in-source collisionally induced dissociation (CID) has been used effectively to identify sites of N-and O-linked glycosylation (Carr et al.,
Protein Sci
., 2: 183-196 (1993); Huddleston et al.,
Anal. Chem
., 65: 877-884 (1993); Conboy and Henion,
J. Am. Soc. Mass Spectrom
., 3: 804-814 (1992)).
However, even this technique is limited by insufficient resolution resulting from the large number of very similar peptides caused by variable protein glycosylation and enzymatic digests of moderately sized glycoproteins. It is therefore necessary to employ a range of techniques with orthogonal selectivity to characterize such samples.
The use of combinations of high-performance capillary electrophoresis, HPLC, LC-ES-MS, and matrix-assisted laser desorption ionization-time of flight mas
Carlson Karen Cochrane
Genentech Inc.
Hasak Janet E.
Snedden Sheridan
LandOfFree
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