US2018327802A1PendingUtilityA1
Separatome-based protein expression and purification platform
Est. expiryMar 13, 2032(~5.6 yrs left)· nominal 20-yr term from priority
C12N 15/70C12N 15/74C12P 21/02B01D 15/203C12P 21/00B01D 15/363B01D 15/362C12N 1/20C12N 9/00B01D 15/424B01D 15/3804C12N 15/79
64
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Claims
Abstract
Provided is a separatome-based recombinant peptide, polypeptide, and protein expression and purification platform based on the juxtaposition of the binding properties of host cell genomic peptides, polypeptides, and proteins with the characteristics and location of the corresponding genes on the host cell chromosome, such as that of E. coli , yeast, Bacillus subtilis or other prokaryotes, insect cells, mammalian cells, etc. The separatome-based protein expression and purification platform quantitatively describes and identifies priority deletions, modifications, or inhibitions of certain gene products to increase chromatographic separation efficiency, defined as an increase in column capacity, column selectivity, or both, with emphasis on the former. Moreover, the separatome-based protein expression and purification platform provides a computerized knowledge tool that, given separatome data and a target recombinant peptide, polypeptide, or protein, intuitively suggests strategies leading to efficient product purification. The separatome-based protein expression and purification platform is an efficient bioseparation system that intertwines host cell expression systems and chromatography.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . An isolated host cell for expression of a target recombinant peptide, polypeptide, or protein,
wherein the chromatographic separation efficiency of said target recombinant peptide, polypeptide, or protein expressed in said isolated host cell is improved in an amount in the range of from about 3% to about 50%, wherein the genome of said isolated host cell:
i) is a reduced genome,
ii) a modified genome, or
iii) a genome in which expression of genes is reduced or completely inhibited;
wherein genes that are deleted, modified, or the expression of which is reduced or completely inhibited in said isolated host cell code for isolated host cell proteome peptides, polypeptides, or proteins that impair the chromatographic separation efficiency of said target recombinant peptide, polypeptide, or protein expressed in said isolated hast cell; wherein said genes are identified, quantified, scored, and ranked according to chromatographic importance in adversely affecting the chromatographic separation efficiency of said target recombinant peptide, polypeptide, or protein; and wherein deletion, modification, reduction of expression, or complete inhibition of expression of said genes in said isolated host cell improves the chromatographic separation efficiency of said target recombinant peptide, polypeptide, or protein expressed in said isolated host cell in an amount in the range of from about 3% to about 50% compared to the chromatographic separation efficiency of said target recombinant peptide, polypeptide, or protein expressed in said isolated host cell when said genes are not deleted, not modified, or the expression of which is not reduced or completely inhibited in said isolated host cell, respectively.
2 . The isolated host cell of claim 1 , wherein said genes are identified and quantified by:
1) fractionating (a) a lysate of said isolated host cell in the case where said target recombinant peptide, polypeptide, or protein is not secreted from said isolated host cell, or (b) the culture medium in which said isolated host cell is grown in the case where said target recombinant peptide, polypeptide, or protein is secreted from said isolated host cell, on an affinity chromatography column comprising an affinity ligand bound to a solid phase, or on an adsorption-based, non-affinity chromatography column comprising an adsorption-based, non-affinity chromatography medium, wherein each of said columns is equilibrated using a mobile loading or eluting phase or an operational variable, and then washed with an elution gradient to elute isolated host cell proteome peptide-, polypeptide-, and protein-containing fractions from each of said columns; and 2) determining and quantifying the isolated host cell proteome peptides, polypeptides, and proteins present in each of said eluted fractions.
3 . The isolated host cell of claim 2 , wherein determining the isolated host cell proteome peptides, polypeptides, and proteins present in each of said eluted fractions in 2) is performed by liquid chromatography-tandem mass spectroscopy, and quantifying the isolated host cell proteome peptides, polypeptides, and proteins present in each of said eluted fractions in 2) is performed by either (a) spectral counting or (b) a combination of Bradford protein assay, 2-dimensional electrophoresis, and densitometry.
4 . The isolated host cell of claim 2 , wherein scoring and ranking the chromatographic importance of said isolated host cell proteome peptides, polypeptides, and proteins in adversely affecting the chromatographic separation efficiency of said target recombinant peptide, polypeptide, or protein comprise:
(1) determining for each of said isolated host cell proteome peptides, polypeptides, and proteins present in each of said eluted fractions:
(a) its adsorption strength;
(b) its adsorption specificity;
(c) its adsorption abundance;
(d) its steric effect; and
(e) its metabolic necessity;
(2) multiplying the values of (a) through (e) in 1) for each of said isolated host cell proteome peptides, polypeptides, and proteins to produce an importance score for each of said isolated host cell proteome peptides, polypeptides, and proteins in each of said eluted fractions; and (3) summing each of said importance scores for each of said isolated host cell proteome peptides, polypeptides, and proteins in each of said eluted fractions to determine its overall importance score, and comparing each of said overall importance scores for each of said isolated host cell proteome peptides, polypeptides, and proteins to the overall importance scores of each of the other isolated host cell proteome peptides, polypeptides, and proteins eluted from said affinity chromatography column or said adsorption-based, non-affinity chromatography column, respectively, wherein a higher overall importance score identifies an isolated host cell proteome peptide, polypeptide, or protein having a greater adverse effect on the chromatographic separation efficiency of said target recombinant peptide, polypeptide, or protein than a proteome peptide, polypeptide, or protein having a lower overall importance score.
5 . The isolated host cell of claim 4 , wherein:
adsorption strength in (1)(a) is manifested as the ratio of the concentration of mobile phase eluent in each of said eluted fractions compared to the maximum mobile phase eluent concentration with which said column is washed; adsorption specificity in (1)(b) is manifested as the ratio of the amount of each individual isolated host cell proteome peptide, polypeptide, or protein present in an eluted fraction compared to the total amount thereof bound on said column; adsorption abundance in (1)(c) is manifested as the ratio of the amount of each individual isolated host cell proteome peptide, polypeptide, or protein present in an eluted fraction compared to the total amount of all isolated host cell proteome peptides, polypeptides, and proteins present in said eluted fraction; steric effect in (1)(d) is manifested as the ratio of the molecular weight of each of said isolated host cell proteome peptides, polypeptides, and proteins compared to the molecular weight of a reference protein within said isolated host cell proteome; and metabolic necessity in (1)(e)) is assessed by bioinformatics or as disclosed in published literature.
6 . The isolated host cell of claim 4 , wherein scoring and ranking of isolated host cell proteome peptides, polypeptides, and proteins in adversely affecting the chromatographic separation efficiency of said target recombinant peptide, polypeptide, or protein are performed employing the following equation:
importance
i
=
∑
j
[
b
1
(
y
c
j
y
max
)
(
h
i
,
j
h
i
,
total
)
(
h
i
,
j
h
j
,
total
)
(
M
W
i
M
W
ref
)
α
]
i
wherein b 1 =scaling parameter; y cj and y max =concentration of mobile phase eluent in fraction (j) and maximum value, respectively; h i,j and h i,total =the amount of protein (i) in fraction (j) and total bound protein (i), respectively; h j,total =amount of protein in fraction (j); MW i =molecular weight of protein (i); MW ref =molecular weight of a reference protein within said isolated host cell proteome; a=steric factor; and i=protein,
wherein:
ratios of y's and h's adopt values between 0 and 1;
a protein that remains bound and requires stringent conditions for elution exhibits a y ratio
(
y
c
j
y
max
)
close to, or equal to, unity;
a protein that emerges as a tight peak has an h ratio
(
h
i
,
j
h
i
,
total
)
close to unity and a j ratio
(
h
i
,
j
h
j
,
total
)
close to unity if it constitutes the majority of fraction (j);
a non-zero α between 0 and 1 indicates steric effects; and
wherein the quantitative ranking for a protein is calculated by multiplying
b
1
×
(
y
c
j
y
max
)
×
(
h
i
,
j
h
i
,
total
)
×
(
h
i
,
j
h
j
,
total
)
×
(
M
W
i
M
W
ref
)
α
,
and summing the product for each fraction (j) where (i) is present.
7 . The isolated host cell of claim 1 , which is selected from a bacterium, a fungus, a mammalian cell, an insect cell, a plant cell, or a protozoal cell.
8 . The isolated host cell of claim 7 , wherein said bacterium is selected from E. coli, L. lactis, B. subtilis, B. licheniformis, B. amyloliquefaciens, P. fluorescens , or C. glutamicum ; said fungus is a yeast selected from S. cerevisiae, K. pastoris , or P. methanolica ; said mammalian cell is selected from a CHO cell, a HEK cell, a mouse myeloma cell, a BHK cell, or a human retinal cell; said insect cell is selected from an S. frugiperda cell, a T. ni cell, or a D. melanogaster cell; said plant cell is selected from a tobacco cell, an alfalfa cell, a rice cell, a tomato cell, a soybean cell, or an algal cell; and said protozoal cell is a L. tarentolae cell.
9 . A method of preparing a pharmaceutical or veterinary composition comprising a recombinant therapeutic peptide, polypeptide, or protein, comprising the steps of:
a) expressing said recombinant therapeutic peptide, polypeptide, or protein in said isolated host cell of claim 1 ; b) in the case where said recombinant therapeutic peptide, polypeptide, or protein is not secreted from said isolated host cell, preparing a lysate of said isolated host cell containing said recombinant therapeutic peptide, polypeptide, or protein, producing an initial recombinant therapeutic peptide-, polypeptide-, or protein-containing mixture; or c) in the case where said recombinant therapeutic peptide, polypeptide, or protein is secreted from said isolated host cell, harvesting culture medium in which said isolated host cell is grown, containing said recombinant therapeutic peptide, polypeptide, or protein, thereby obtaining an initial recombinant therapeutic peptide-, polypeptide-, or protein-containing mixture; d) chromatographing said initial recombinant therapeutic peptide-, polypeptide-, or protein-containing mixture of step b) or step c) on an affinity chromatography column comprising an affinity ligand bound to a solid phase or on an adsorption-based, non-affinity chromatography column comprising an adsorption-based, non-affinity chromatography medium, and collecting elution fractions, thereby obtaining one or more fractions containing an enriched amount of said recombinant therapeutic peptide, polypeptide, or protein relative to other peptides, polypeptides, or proteins in said one or more fractions compared to the amount of said recombinant therapeutic peptide, polypeptide, or protein relative to other peptides, polypeptides, or proteins in said initial recombinant therapeutic peptide-, polypeptide-, or protein-containing mixture; e) further chromatographing an enriched fraction of step d) to obtain said recombinant therapeutic peptide, polypeptide, or protein in a desired degree of purity; f) recovering purified recombinant therapeutic peptide, polypeptide, or protein of step e); and g) formulating said purified recombinant therapeutic peptide, polypeptide, or protein of step f) with a pharmaceutically or veterinarily acceptable carrier, diluent, or excipient to produce a pharmaceutical or veterinary composition, respectively.
10 . The method of claim 9 , wherein said genes that are deleted, modified, or the expression of which is reduced or completely inhibited in said isolated host cell are identified and quantified by:
1) fractionating (a) a lysate of said isolated host cell in the case where said recombinant therapeutic peptide, polypeptide, or protein is not secreted from said isolated host cell, or (b) the culture medium in which said isolated host cell is grown in the case where said recombinant therapeutic peptide, polypeptide, or protein is secreted from said isolated host cell, on an affinity chromatography column comprising an affinity ligand bound to a solid phase or on an adsorption-based, non-affinity chromatography column comprising an adsorption-based, non-affinity chromatography medium, wherein each of said columns is equilibrated using a mobile loading or eluting phase or an operational variable, and then washed with an elution gradient to elute isolated host cell proteome peptide-, polypeptide-, and protein-containing fractions from said affinity chromatography column or said adsorption-based, non-affinity chromatography column, respectively; and 2) determining and quantifying the isolated host cell proteome peptides, polypeptides, and proteins present in each of said eluted fractions.
11 . The method of claim 10 , wherein scoring and ranking the chromatographic importance of said isolated host cell proteome peptides, polypeptides, and proteins in adversely affecting the chromatographic separation efficiency of said recombinant therapeutic peptide, polypeptide, or protein comprise:
(1) determining for each of said isolated host cell proteome peptides, polypeptides, and proteins present in each of said eluted fractions:
(a) its adsorption strength, manifested as the ratio of the concentration of mobile phase eluent in each of said eluted fractions compared to the maximum mobile phase eluent concentration with which said affinity chromatography column or said adsorption-based, non-affinity chromatography column, respectively, is washed;
(b) its adsorption specificity, manifested as the ratio of the amount of each individual isolated host cell proteome peptide, polypeptide, or protein present in an eluted fraction compared to the total amount thereof bound on said affinity chromatography column or on said adsorption-based, non-affinity chromatography column, respectively;
(c) its adsorption abundance, manifested as the ratio of the amount of each individual isolated host cell proteome peptide, polypeptide, or protein present in an eluted fraction compared to the total amount of all isolated host cell proteome peptides, polypeptides, and proteins present in said eluted fraction;
(d) its steric effect, manifested as the ratio of the molecular weight of each of said isolated host cell proteome peptides, polypeptides, and proteins compared to the molecular weight of a reference protein within said isolated host cell proteome; and
(e) its metabolic necessity, assessed by bioinformatics or as disclosed in published literature;
(2) multiplying the values of (a) through (e) in 1) for each of said isolated host cell proteome peptides, polypeptides, and proteins to produce an importance score for each of said isolated host cell proteome peptides, polypeptides, and proteins in each of said eluted fractions; and (3) summing each of said importance scores for each of said isolated host cell proteome peptides, polypeptides, and proteins in each of said eluted fractions to determine its overall importance score, and comparing each of said overall importance scores for each of said isolated host cell proteome peptides, polypeptides, and proteins to the overall importance scores of each of the other isolated host cell proteome peptides, polypeptides, and proteins eluted from said affinity chromatography column or said adsorption-based, non-affinity chromatography column, respectively, wherein a higher overall importance score identifies an isolated host cell proteome peptide, polypeptide, or protein having a greater adverse effect on the chromatographic separation efficiency of said recombinant therapeutic peptide, polypeptide, or protein than an isolated host cell proteome peptide, polypeptide, or protein having a lower overall importance score.
12 . The method of claim 9 , wherein said isolated host cell is selected from a bacterium, a fungus, a mammalian cell, an insect cell, a plant cell, or a protozoal cell.
13 . The method of claim 12 , wherein said bacterium is selected from E. coli, L. lactis, B. subtilis, B. licheniformis, B. amyloliquefaciens, P. fluorescens , or C. glutamicum ; said fungus is a yeast selected from S. cerevisiae, K. pastoris, or P. methanolica ; said mammalian cell is selected from a CHO cell, a HEK cell, a mouse myeloma cell, a BHK cell, or a human retinal cell; said insect cell is selected from an S. frugiperda cell, a T. ni cell, or a D. melanogaster cell; said plant cell is selected from a tobacco cell, an alfalfa cell, a rice cell, a tomato cell, a soybean cell, or an algal cell; and said protozoal cell is a L. tarentolae cell.
14 . The method of claim 9 , wherein said recombinant therapeutic peptide, polypeptide, or protein is an antibody, an antibody fragment, a vaccine, α 1 -Antitrypsin, an enzyme, a growth factor, a blood clotting factor, a hormone, a nerve factor, an interferon, an interleukin, tumor necrosis factor, lung surfactant protein, or serum albumin.
15 . The method of claim 9 , wherein said adsorption-based, non-affinity chromatography column is an ion exchange chromatography column.
16 . A method of purifying a recombinant enzyme, comprising the steps of:
a) expressing said recombinant enzyme in said isolated host cell of claim 1 ; b) in the case where said recombinant enzyme is not secreted from said isolated host cell, preparing a lysate of said isolated host cell containing said recombinant enzyme, producing an initial recombinant enzyme-containing mixture; or c) in the case where said recombinant enzyme is secreted from said isolated host cell, harvesting culture medium in which said isolated host cell is grown, containing said recombinant enzyme, thereby obtaining an initial recombinant enzyme-containing mixture; d) chromatographing said initial recombinant enzyme-containing mixture of step b) or step c) on an affinity chromatography column comprising an affinity ligand bound to a solid phase, or on an adsorption-based, non-affinity chromatography column comprising an adsorption-based, non-affinity chromatography medium, and collecting elution fractions, thereby obtaining one or more fraction, containing an enriched amount of said recombinant enzyme relative to other peptides, polypeptides, or proteins in said one or more fractions compared to the amount of said recombinant enzyme relative to other peptides, polypeptides, or proteins in said initial recombinant enzyme-containing mixture; e) optionally, further chromatographing an enriched fraction of step d) to obtain said recombinant enzyme in a desired degree of purity; and f) recovering purified recombinant enzyme.
17 . The method of claim 16 , wherein said genes that are deleted, modified, or the expression of which is reduced or completely inhibited in said isolated host cell are identified and quantified by:
1) fractionating (a) a lysate of said isolated host cell in the case where said recombinant enzyme is not secreted from said isolated host cell, or (b) the culture medium in which said isolated host cell is grown in the case where said recombinant enzyme is secreted from said isolated host cell, on an affinity chromatography column comprising an affinity ligand bound to a solid phase, or on an adsorption-based, non-affinity chromatography column comprising an adsorption-based, non-affinity chromatography medium, wherein each of said columns is equilibrated using a mobile loading or eluting phase or an operational variable, and then washed with an elution gradient to elute isolated host cell proteome peptide-, polypeptide-, and protein-containing fractions from said affinity chromatography column or said adsorption-based, non-affinity chromatography column, respectively; and 2) determining and quantifying the isolated host cell proteome peptides, polypeptides, and proteins present in each of said eluted fractions.
18 . The method of claim 17 , wherein scoring and ranking the chromatographic importance of said isolated host cell proteome peptides, polypeptides, and proteins in adversely affecting the chromatographic separation efficiency of said recombinant enzyme comprise:
(1) determining for each of said isolated host cell proteome peptides, polypeptides, and proteins present in each of said eluted fractions:
(a) its adsorption strength, manifested as the ratio of the concentration of mobile phase eluent in each of said eluted fractions compared to the maximum mobile phase eluent concentration with which said column is washed;
(b) its adsorption specificity, manifested as the ratio of the amount of each individual isolated host cell proteome peptide, polypeptide, or protein present in an eluted fraction compared to the total amount thereof bound on said affinity chromatography column or on said adsorption-based, non-affinity chromatography column, respectively;
(c) its adsorption abundance, manifested as the ratio of the amount of each individual isolated host cell proteome peptide, polypeptide, or protein present in an eluted fraction compared to the total amount of all isolated host cell proteome peptides, polypeptides, and proteins present in said eluted fraction;
(d) its steric effect, manifested as the ratio of the molecular weight of each of said isolated host cell proteome peptides, polypeptides, and proteins compared to the molecular weight of a reference protein within said isolated host cell proteome; and
(e) its metabolic necessity, assessed by bioinformatics or as disclosed in published literature;
(2) multiplying the values of (a) through (e) in (1) for each of said isolated host cell peptides, polypeptides, and proteins to produce an importance score for each of said isolated host cell proteome peptides, polypeptides, and proteins in each of said eluted fractions; and (3) summing each of said importance scores for each of said isolated host cell proteome peptides, polypeptides, and proteins in each of said eluted fractions to determine its overall importance score, and comparing each of said overall importance scores for each of said isolated host cell proteome peptides, polypeptides, and proteins to the overall importance scores of each of the other isolated host cell proteome peptides, polypeptides, and proteins eluted from said affinity chromatography column or said adsorption-based, non-affinity chromatography column, respectively; wherein a higher overall importance score identifies an isolated host cell proteome peptide, polypeptide, or protein having a greater adverse effect on the chromatographic separation efficiency of said recombinant enzyme than an isolated host cell proteome peptide, polypeptide, or protein having a lower overall importance score.
19 . The method of claim 16 , wherein said isolated host cell is selected from a bacterium, a fungus, a mammalian cell, an insect cell, a plant cell, or a protozoal cell.
20 . The method of claim 19 , wherein said bacterium is selected from E. coli, L. lactis, B. subtilis, B. licheniformis, B. amyloliquefaciens, P. fluorescens , or C. glutamicum ; said fungus is a yeast selected from S. cerevisiae, K. pastoris, or P. methanolica ; said mammalian cell is selected from a CHO cell, a HEK cell, a mouse myeloma cell, a BHK cell, or a human retinal cell; said insect cell is selected from an S. frugiperda cell, a T. ni cell, or a D. melanogaster cell; said plant cell is selected from a tobacco cell, an alfalfa cell, a rice cell, a tomato cell, a soybean cell, or an algal cell; and said protozoal cell is a L. tarentolae cell.
21 . The method of claim 16 , wherein said adsorption-based, non-affinity chromatography column is an ion exchange chromatography column.Join the waitlist — get patent alerts
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