Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-08-21
2002-04-16
Ponnaluri, Padmashri (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S235100, C435S320100, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500, C530S387300
Reexamination Certificate
active
06372425
ABSTRACT:
SUMMARY OF THE INVENTION
This invention relates to a process for optimizing ligand-target molecule system selection and using a selected system for preparing large scale quantities of macromolecules in particularly pure form.
BACKGROUND OF THE INVENTION
The production of an array of ligands to select a ligand or a set of ligands for use in a macromolecule purification scheme has traditionally been by organic synthesis methods. Such methods include solid-phase and liquid-phase synthesis. Solid-phase methods are capable of producing longer ligands than liquid-phase methods thus making solid-phase methods preferable. However, ligands produced using either of these methods are difficult to identify and amplify.
Ligand synthesis in a biological system is advantageous over organic synthesis because of the ability to identify and amplify large quantities of ligands. Until recently, biological system synthesis of an array of ligands, such as an epitope library, was limited. However, random peptide libraries that take advantage of the ability of filimentous phage coat protein gene pIII to accept and express foreign DNA on its surface have been described as being useful to identify millions of potential ligands quickly. See, e.g., Scott, J. K. and Smith, G. P.,
Science
, 449, 386-390 (1990), Devlin, J. J., et al.,
Science
, 449, 404-406 (1990), Cwirla, S. E. et al.,
Proc. Nat. Acad. Sci. USA
, 87, 6378-6382 (1990) and U.S. Pat. No. 5,223,409.
Historically, ligand-target molecule system selection for macromolecules have relied upon secondary indication methods to determine appropriate ligands. Such methods include RIA, ELISA, and biotin-avidin complex formation assays. Although these methods identify ligand-target molecule systems that have an unusually high association constants, it is only with subsequent rounds of screening that one skilled in the art can identify ligand-target molecule systems with sufficient binding characteristics for use in subsequent macromolecule purification. These techniques have other deficiencies including the ability to produce false positives, being time consuming and lacking the ability to differentiate between active and nonactive macromolecules during purification.
Surface plasmon resonance (SPR) has been known for quite some time, Kreetschmann, E., & Raether, H., Z. Naturforsch. A23, 2135 (1968). However, it was not until recently that the use of SPR to study ligand-target molecule interactions was described, Karlsonn, R., et al.,
J. Immunol. Methods
, 145, 249 (1991).
It is well known that affinity chromatography is an effective purification approach that exploits a macromolecule's biological function. Most macromolecules possess active sites that perform unique functions. These active sites are involved in the recognition and the catalysis of selected small molecules or restricted regions of other macromolecules. It is the property of recognition upon which the principles of affinity chromatography have been developed. The fundamental requirement of affinity chromatography is that the comparative rate constants reflect reasonable affinity, and that the qualitative nature of the ligand and target molecule reflect reasonable stereochemical specificity.
It is desirable that an interacting ligand-target molecule system be chosen such that the ligand-target molecule complex is not chemically altered as a result of the interaction. Many nonenzymatic interacting systems do not exhibit such chemical alteration and are, therefore, ideally suited for affinity chromatography purification. Such interacting systems theoretically include antigens, antibodies, vitamin and drug binding proteins, biological receptors, and transport proteins.
It is also desirable that the interacting ligand-target molecule system be chosen such that the target molecule binds sufficiently fast to the ligand and that the ligand-target molecule system exhibit a sufficiently slow dissociation, thereby allowing large quantities of the target molecule to couple with the ligand without significant loss of target molecules before elution. Following these parameters it is possible to increase the purity and amount of target molecule ultimately recovered.
The aforementioned techniques are themselves individually known. However, the combination of these techniques to identify ligand-target molecule systems with specific association and dissociation constants for subsequent purification of target molecules and the subsequent purification of target molecules using identified ligand-target molecule systems is not known.
ABBREVIATIONS AND DEFINITIONS
The following terms are used herein according to the definitions.
TERM
DEFINITION
AIDS
Acquired Immune Deficiency
Syndrome
HIV
The generic tenn for the
presumed etiological agent of
AIDS, ARC or both; so referred
to as strains HTLV-III, ARV and
LAV.
Library
A collection of DNA or
oligopeptide sequences, of defined
length, with or without limited
sequence restrictions.
Ligand
An oligopeptide that binds target
molecules. Ligands may differ
one from another in their binding
affinities for the target molecule.
Macromolecule
Any biologically active compound,
including but not limited to
antibodies, antigens, proteins, or
enzymes.
PCR
Polymerase Chain Reaction
Recombinant fusion polypeptide
Polypeptide or oligopeptide
(RFP)
expressed as a contiguous
translation product from a spliced
foreign DNA in a recombinant
eukaryotic or prokaryotic
expression system, wherein the
spliced foreign DNA is derived
from two or more coding
sequences of different origin, and
joined together by ligation or
PCR.
Recombinant protein
A polypeptide or oligopeptide
expressed by foreign DNA in a
recombinant eukaryotic or
prokaryotic expression system.
Recombinant expression system
A cell containing a foreign DNA
expressing a foreign protein or a
foreign oligopeptide.
SPNE
Selected Principle Neutralization
Epitope, which is the principle
neutralization determinant bound
by one or more broadly
neutralizing antibodies. SPNE is
defined to include consensus
sequences.
SPR
Surface Plasmon Resonance.
Target molecule
Any compound of interest for
which a ligand is desired. A
target molecule can be any
macromolecule.
The terms “protein,” “peptide,” “oligopeptide,” and “polypeptide” and their plurals will be used interchangeably to refer to chemical compounds having amino acid sequences of five or more amino acids. “Amino acid” refers to any of the 20 common amino acids for which codons are naturally available and are listed in the table of amino acids.
As used herein, all amino acid three letter and single letter designations conform to those designations which are standard in the art, and are listed as follows:
Alanine
Ala
A
Arginine
Arg
R
Asparagine
Asn
N
Aspartic acid
Asp
D
Cysteine
Cys
C
Glutamic acid
Glu
E
Glutamine
Gln
Q
Glycine
Gly
G
Histidine
His
H
Isoleucine
Ile
I
Leucine
Leu
L
Lysine
Lys
K
Methionine
Met
M
Phenylalanine
Phe
F
Proline
Pro
P
Serine
Ser
S
Threonine
Thr
T
Tryptophan
Trp
W
Tyrosine
Tyr
Y
Valine
Val
V
When any variable, e.g., SPNE, occurs more than one time in any constituent, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents, variables, or both are permissible only if such combinations result in stable compounds.
DETAILED DESCRIPTION OF THE INVENTION
The present invention overcomes the problems of previous methods for identifying specific ligands for the purification of macromolecules. One embodiment of the present invention is to prepare RFPs selected as SPNEs of phage libraries that bind to a specific target molecule. Another embodiment of the present invention is to determine, in real time, the association and dissociation constants for the RFPs and the target molecule of choice and then select RFPs that meet predetermined binding characteristics in order to optimally purify the target molecule. A further embodiment of the present invention is to provide sufficient qualities of identified RFPs for target molecule purification. Yet a further embodiment of the present invention is to
Arnold Beth
Conley Anthony J.
Keller Paul M.
Shaw Alan R.
Tung Jwu-Sheng
Cocuzzo Anna L.
Merck & Co. , Inc.
Ponnaluri Padmashri
Tribble Jack L.
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