WO2012078936A2

WO2012078936A2 - Methods to produce a purified peptide chain fraction

Info

Publication number: WO2012078936A2
Application number: PCT/US2011/064052
Authority: WO
Inventors: Andrea S. Diener; David C. Greene
Original assignee: Glaxosmithkline Llc
Priority date: 2010-12-10
Filing date: 2011-12-09
Publication date: 2012-06-14
Also published as: WO2012078936A3

Abstract

Methods to produce a purified peptide chain fraction are disclosed. The methods comprise the step of adding a hexadecyltrimethylammonium salt to a first liquid portion containing a peptide chain of interest and the step of adjusting the pH of the second liquid portion.

Description

METHODS TO PRODUCE A PURIFIED PEPTIDE CHAIN FRACTION

FIELD OF THE INVENTION

Methods to produce a purified peptide chain fraction are disclosed. BACKGROUND OF THE INVENTION

The use of therapeutic molecules comprising peptide chains, such as recombinant proteins, is an effective approach to treating various diseases. Such molecules are typically produced by recombinant expression from living cells. The purification of therapeutic cytokine, antibody, and fusion molecules (e.g., domain antibody:: albumin fusions) is necessary to prepare these peptide chain molecules for therapeutic use.

A common problem in the purification of recombinant proteins is the need to separate undesired contaminants, such as cellular components, from a peptide chain of interest. In particular, it is necessary to substantially remove cellular debris from a first liquid portion containing a peptide chain of interest, such as a cell lysate or cellular growth medium, and obtain a purified peptide chain fraction containing a peptide of interest at a desirable purity and yield.

Limiting the contaminant load in such a purified peptide chain fraction is especially important if subsequent filtration or chromatographic steps are required to further purify a protein of interest.

Thus, a need exists for methods to produce a purified peptide chain fraction.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 Percentage of hIL-18 recovery (i.e., % yield) in supernatants prepared from cell lysates as a function of the % CTAB ([g* 100%]/ml) at pH 7.5.

Fig. 2 Percentage of hIL-18 recovery (i.e., % yield) in supernatants prepared from cell lysates as a function of the % CTAB ([g* 100%]/ml) at pH 4.5.

Fig. 3 Percentage of hIL-18 recovery (i.e., % yield) in supernatants containing different % CTAB ([g* 100%]/ml) prepared from cell lysates as a function of pH.

Fig. 4 Percentage of hIL-18 recovery (i.e., % yield) in supernatants containing different % CTAB ([g* 100%]/ml), in the absence of ethanol, prepared from cell lysates as a function of pH.

Fig. 5 Percentage of hIL-18 recovery (i.e., % yield) in supernatants containing different % CTAB ([g* 100%]/ml), in the presence of ethanol, prepared from cell lysates as a function of pH. Fig. 6 Percentage of hIL- 18 purity (i.e., % purity) in supernatants containing different % CTAB ([g* 100%]/ml), in the presence of ethanol, prepared from cell lysates as a function of pH.

SUMMARY OF THE INVENTION

One aspect of the disclosure is a method of producing a purified peptide chain fraction comprising the steps of: a) providing a first liquid portion containing a peptide chain of interest;

b) adding a hexadecyltrimethylammonium salt to the first liquid portion to obtain a second liquid portion containing about 9.6 mM to about 13.72 mM of the

hexadecyltrimethylammonium salt; c) adjusting the pH of the second liquid portion to a pH of about 4.3 to about 7.5; and

d) separating the second liquid portion into an insoluble fraction and a purified peptide chain fraction containing the peptide chain of interest; whereby a purified peptide chain fraction is produced.

Another aspect of the disclosure is a method of producing a purified peptide chain fraction comprising the steps of: a) providing a first liquid portion comprising a cell lysate that contains a peptide chain of interest; b) adding hexadecyltrimethylammonium bromide to the first liquid portion to obtain a second liquid portion containing about 13.72 mM

hexadecyltrimethylammonium bromide (CTAB); c) adjusting the pH of the second liquid portion to a pH of about 4.4; d) holding the second liquid portion at about 18 °C to about 22 °C for about 15 minutes to about 24 hours; and e) separating the second liquid portion into an insoluble fraction and a purified peptide chain fraction containing the peptide chain of interest by centrifugation, wherein the peptide chain of interest is about 40% to about 70% of a total peptide chain population in the purified peptide chain fraction; whereby a purified peptide chain fraction is produced.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

As used herein and in the claims, the singular forms "a," "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to

"a peptide chain" is a reference to one or more peptide chains and includes equivalents thereof known to those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the methods of the disclosure, exemplary compositions and methods are described herein. Any of the aspects and embodiments of the disclosure described herein may also be combined. For example, the subject matter of any dependent or independent claim disclosed herein may be multiply combined (e.g. , one or more recitations from each dependent claim may be combined into a single claim based on the independent claim on which they depend).

Ranges provided herein include all values within a particular range described and values about an endpoint for a particular range. The figures and tables of the disclosure also describe ranges, and discrete values, which may constitute an element of any of the methods disclosed herein.

Concentrations described herein are determined at ambient temperature and pressure. This may be, for example, the temperature and pressure at room temperature or in within a particular portion of a process stream. Preferably, concentrations are determined at a standard state of 25 °C and 1 bar of pressure.

The term "about" means a value within two standard deviations of the mean for any particular measured value.

The term "peptide chain" means a molecule that comprises at least two amino acid residues linked by a peptide bond to form a chain. Large peptide chains of more than 50 amino acids may be referred to as "polypeptides" or "proteins." Small peptide chains of less than 50 amino acids may be referred to as "peptides."

The term total "percent solids" or "% solids" means the value obtained by dividing the mass of wet solids present in a sample volume by the total mass of the sample volume and multiplying by 100. The unit of mass used in the calculation should be the same (e.g., grams) and the unit of volume (e.g., milliliters) used in the calculation should be the same.

Mathematically this can be expressed as: percent solids =

[mass of wet solids in a sample volume / total mass of the same sample volume] * 100%

For a cell suspension, the percent solids value may be determined, for example, as follows. First, a 25 ml volume of cell suspension is collected and its total mass in grams is determined. Second, the 25 ml volume of cell suspension is centrifuged in a JA- 17 rotor (Beckman Coulter Inc., Brea, CA) at 30,000 x g for 20 min. The liquid supernatant portion is then decanted and the wet mass in grams of the wet cell pellet is then determined. The value obtained for the wet mass of the wet cell pellet in the 25 ml volume of cell suspension is then divided by the value obtained for the total mass of the 25 ml volume of cell suspension. The value produced by this mathematical operation is then multiplied by 100 to produce the percent solids value for the cell suspension.

The term "population" means at least two items such as two molecules, including peptide chains.

One aspect of the disclosure is a method of producing a purified peptide chain fraction comprising the steps of a) providing a first liquid portion containing a peptide chain of interest;

hexadecyltrimethylammonium salt; c) adjusting the pH of the second liquid portion to a pH of about 4.3 to about 7.5; and d) separating the second liquid portion into an insoluble fraction and a purified peptide chain fraction containing the peptide chain of interest; whereby a purified peptide chain fraction is produced.

In the methods of the disclosure, the first liquid portion may be any part of a fluid containing a peptide chain of interest and may be obtained from a variety of sources. For example, the first liquid portion may be a medium, such as cell culture media, comprising the peptide chain of interest that has been secreted by cells into the media. The first liquid portion may also be a cell lysate prepared by disrupting prokaryotic, eukaryotic, or archeal cells capable of expressing the peptide chain of interest. Preferably the cell lysate is prepared by disruption of the prokaryote Escherichia coli having a partial genotype of cybcB-IRIA, gpEA, 6pgl⁺ which is deficient in cytochrome b562 expression due to the cybcB-IRIA allele, has a phage interrupt allele that is gpEA, expresses active 6-phosphogluconolactonase from a functional 6pgl allele and is capable of maintaining a pET28 based plasmid vector as well as expressing a peptide chain of interest via the T7 promoter in this vector.

It is preferred that the cell lysate be prepared from an Escherichia coli cell strain having a genotype that is:

F^", ompT, hsdS_B, (¾^", m_B ^~), dcm, gal, λ(ΟΕ3), zae-502::TN10,/¾wA21, cyb c::Zeo^r, gpE::Cm^T and which contain a λ phage (DE3) which has been made replication defective by inactivation of the integrase gene int and transformation with a red recombinase vector. It is also preferred that this Escherichia coli cell strain be transformed with a plasmid vector having the features shown in Table 6. This preferred plasmid vector is capable of driving expression, after isopropyl β-D- 1 -thiogalactopyranoside (IPTG) induction, of a single peptide chain with the amino acid sequence shown in SEQ ID NO: 1 that contains four cysteine residues (38C, 68C, 76C and 127C) with free sulfhydryl groups and which lacks glycosylation.

Table 6: Plasmid vector features.

In the methods of the disclosure, the first liquid portion containing the peptide chain of interest may also be a portion of fluid stream subjected to the method of the disclosure. For example, the first liquid portion may be a portion of a fluid stream containing a peptide chain of interest that has previously been subjected to other operations such as chromatography, salting out, dialysis, isoelectric focusing, filtration, centrifugation and the like which are used in the purification of peptide chains.

In the methods of the disclosure, the step of providing a first liquid portion containing a peptide chain of interest can be performed by any technique which results in the provision of such a first liquid portion. For example, this can be accomplished in a continuous process in which a portion of liquid from a process stream is provided so that the disclosed methods can be performed. Alternatively, this can be accomplished by providing a discrete volume of a liquid.

In the methods of the disclosure, the peptide chain of interest may be any peptide chain. It is preferred the peptide chain have a molecular weight of about 18,216.8+/-0.1 Da as measured by electrospray ionization time of flight mass spectroscopy and a pi of about 5.4 as measured by vertical gel iso-electric focusing. For example, the peptide chain of interest may comprise a human IL- 18 (hIL- 18) peptide chain having the amino acid sequence shown in SEQ ID NO: 1. This IL-18 protein may consist of, or comprise, a single peptide chain with the amino acid sequence shown in SEQ ID NO: 1 that contains four cysteine residues (38C, 68C, 76C and 127C) with free sulfhydryl groups and which lacks glycosylation.

In the methods of the disclosure, the step of adding a hexadecyltrimethylammonium salt to the first liquid portion to obtain a second liquid portion containing about 9.6 mM to about 13.72 mM of the hexadecyltrimethylammonium salt can be performed by adding a quantity of the hexadecyltrimethylammonium salt in an aqueous solution or as a solid to the first liquid portion. The hexadecyltrimethylammonium salt may be at least one selected from the group consisting of hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium tosylate, hexadecyltrimethylammonium tosilate, hexadecyltrimethylammonium sacchrinate and hexadecyltrimethylammonium hydroxide.

In the methods of the disclosure, hexadecyltrimethylammonium salt is added to the first liquid portion to obtain a second liquid portion containing about 10.98 mM to about 13.72 mM of the hexadecyltrimethylammonium salt.

In the methods of the disclosure, the step of adjusting the pH of the second liquid portion to a pH of about 4.3 to about 7.5 can be performed using any appropriate technique which adds acid ([H⁺]) or base ([OH ]) equivalents to a liquid to obtain a desired pH. For example, such adjustment may be performed by the addition of a liquid containing such equivalents, a gas (e.g. , CO₂) to produce such equivalents, the salt of a compound to provide such equivalents or via ion exchange processes as desired. It is preferred that acetic acid be used to adjust the pH of the second liquid portion in the methods of the disclosure.

In the methods of the disclosure, the step of adjusting the pH of the second liquid portion is an adjustment of the pH is to a pH of about 4.4 to about 4.6.

In the methods of the disclosure, the step of adjusting the pH of the second liquid portion is to a pH of about 4.4.

In the methods of the disclosure, the step of separating the second liquid portion into an insoluble fraction and a purified peptide chain fraction containing the peptide chain of interest can be performed using any appropriate technique that results in the separation of insoluble materials from a liquid. Such techniques include centrifugation, filtration (e.g., alternating tangential flow filtration, tangential flow filtration, direct filtration etc.), settling, magnetism based separations, electrostatic based separations and laminar flow based separations.

In one embodiment of the methods of the disclosure the

hexadecyltrimethylammonium salt is hexadecyltrimethylammonium bromide. In one embodiment of the methods of the disclosure the first liquid portion comprises a cell lysate. Such cell lysates may be prepared by a variety of techniques suitable for disrupting prokaryotic, eukaryotic or archeal cells. Such techniques include shear (e.g., sonication, French press, Dounce homogenizer, Gaulin homogenizer, AVESTIN™

homogenizer, blender and/or bead mill), enzymatic treatment (e.g., proteases and/or lysozyme), viral lysis, osmolysis and detergent treatment or any combination of these.

In another embodiment of the methods of the disclosure, the cell lysate comprises 50 mM Tris-HCl, 5 mM EDTA and 20 mM DTT at a pH of 7.5. Tris-HCl is an abbreviation for tris hydrochloride. EDTA is an abbreviation for ethylenediaminetetraacetic acid. DTT is an abbreviation for dithiothreitol.

Alternatively, in the methods of the disclosure, the cell lysate comprises 50 mM tromethamine or a salt thereof, 5 mM EDTA and 20 mM DTT at a pH of 7.5.

In another embodiment of the methods of the disclosure, the second liquid portion of step c) is held at about 18 °C to about 22 °C for about 15 minutes to about 24 hours. The hold step may be performed in any appropriate vessel. Alternatively, in the methods of the disclosure, the second liquid portion of step c) is held at about 18 °C to about 22 °C for about 12 hours to about 24 hours.

In another embodiment of the methods of the disclosure, a centrifuge is used in step d) to separate the second liquid portion. Such a centrifuge may be, for example, a disc stack centrifuge.

In another embodiment of the methods of the disclosure, the cell lysate is prepared from a cell suspension containing about 15% solids to about 30% solids. Cell suspensions containing about 15% solids to about 30% solids can be prepared using dilution or concentration techniques as necessary. Dilution can be accomplished with any liquid compatible with the methods of the disclosure such as cell culture media.

In another embodiment of the methods of the disclosure, the peptide chain of interest is about 40% to about 70% of a total peptide chain population in the purified peptide chain fraction. The percentage value that a peptide chain of interest is of a total peptide chain population in a purified peptide chain fraction can be determined by SDS-PAGE separation of a purified peptide chain fraction, visualization of the peptide chain bands in the fraction and quantification of the total amount of peptide chains in the sample (e.g. , by densitometry or similar analyses) as well as the total amount of the peptide chain of interest in the sample followed by dividing the amount of the peptide chain of interest by the amount of the total peptide chains in the sample and multiplying by 100%. This value may be alternatively referred to herein as the "purity" or "percent purity" of a peptide chain (e.g. , hIL- 18). It is preferred that SDS-PAGE separations used for these determinations performed using a NUPAGE™ NOVEX™ Bis-Tris 4- 12% polyacrylamide gel (Invitrogen Corp., Carlsbad, CA) run with a volume of the purified peptide chain fraction containing about 1 μg to about 5 μg of total protein, as determined using the PIERCE™ BCA (bicinchoninic acid) assay (Thermo Fisher Scientific Inc., Rockford, IL), that has been reduced at 95 °C for 5 minutes and is then loaded on the SDS-PAGE gel for electrophoresis at 200 V until the peptide chain of interest is resolved, after which the resolved peptide chains in the gel may be visualized by silver staining or Coomassie blue staining. Preferred percent purity ranges include about 40% to about 70%, about 20% to about 90%, about 20% to about 70%, about 25% to about 40% and about 20% to about 65%.

"Percent recovery" values can be similarly determined by SDS-PAGE. This is done as follows. A SDS-PAGE separation of a sample volume from a purified peptide chain fraction is performed, followed by visualization of the peptide chain band of interest in the sample from the purified peptide chain fraction and quantification (e.g., by densitometry or similar analyses), followed by calculation of the total amount of the peptide chain of interest in the total volume of the purified peptide chain fraction obtained by performing the disclosed methods. A SDS-PAGE separation of a sample volume from a first liquid portion (e.g., a cell lysate) is also performed, followed by visualization of the peptide chain band of interest in the sample from the first liquid portion and quantification (e.g. , by densitometry or similar analyses), followed by calculation of the total amount of the peptide chain of interest in the total volume of the first liquid portion. The total amount of the peptide chain of interest in the purified peptide chain fraction volume obtained by performing the disclosed methods is then divided by the total amount of the peptide chain of interest in the volume of the first liquid portion and multiplied by 100%. This value may be alternatively referred to herein as the "% recovery," "percent yield" and/or "% yield" of a peptide chain (e.g., hIL- 18). It is preferred that SDS-PAGE separations for these determinations be performed using a NUPAGE™ NOVEX™ Bis-Tris 4-12% polyacrylamide gel (Invitrogen Corp., Carlsbad, CA) run with a volume of the purified peptide chain fraction, or the first liquid portion, containing about 2 μg to about 5 μg of total protein as determined using the PIERCE™ BCA (bicinchoninic acid) assay (Thermo Fisher Scientific Inc., Rockford, IL), that has been reduced at 95 °C for 5 minutes and is then loaded on the SDS-PAGE gel for electrophoresis at 200 V until the peptide chain of interest is resolved, after which the resolved peptide chains in the gel may be visualized by silver staining or Coomassie blue staining. Preferred percent yield ranges include about 64% to about 95%, about 20% to about 100%, about 50% to about 100%, about 55% to about 100%, about 55% to about 90% and about 50% to about 75%. In another embodiment of the methods of the disclosure, the peptide chain of interest is a cytokine. For example, the peptide chain of interest may comprise a human IL- 18 (hlL- 18) peptide chain having the amino acid sequence shown in SEQ ID NO: 1. The IL-18 protein may consist of, or comprise, a single peptide chain with the amino acid sequence shown in SEQ ID NO: 1 that contains four cysteine residues (38C, 68C, 76C and 127C) with free sulfhydryl groups and which lacks glycosylation.

Another aspect of the disclosure is a method of producing a purified peptide chain fraction comprising the steps of: a) providing a first liquid portion comprising a cell lysate that contains a peptide chain of interest; b) adding hexadecyltrimethylammonium bromide to the first liquid portion to obtain a second liquid portion containing about 13.72 mM hexadecyltrimethylammonium bromide; c) adjusting the pH of the second liquid portion to a pH of about 4.4; d) holding the second liquid portion at about 18 °C to about 22 °C for about 15 minutes to about 24 hours; and e) separating the second liquid portion into an insoluble fraction and a purified peptide chain fraction containing the peptide chain of interest by centrifugation, wherein the peptide chain of interest is about 40% to about 70% of a total peptide chain population in the purified peptide chain fraction; whereby a purified peptide chain fraction is produced.

In another embodiment of the methods of the disclosure, the cytokine comprises the amino acid sequence shown in SEQ ID NO: 1.

The present invention will now be described with reference to the following specific, non-limiting examples.

Example 1

Recovery of a Purified hIL-18 Peptide Chain Fraction from Cell Lysate

as a Function of CTAB Concentration at pH 7.1 to 7.5

A purified hIL- 18 peptide chain fraction was recovered from cell lysates after addition of about 2.74 mM to about 137.19 mM CTAB at pH 7.1 to 7.5. See Fig. 1, Table 1 and Table 2. For convenience, Table 3 shows CTAB concentrations expressed in % ([g* 100%]/ml), g/L and mM. Table 1: Recovery of a purified hIL- 18 peptide chain in fractions from cell lysate after addition of about 2.74 mM to about 137.19 mM CTAB at pH 7.5.

Table 3: CTAB concentrations.

Escherichia coli cells having a partial genotype of cybc -IRIA, gpEA, 6pgl which are deficient in cytochrome b562 expression due to the cybclS-IRIA allele, have a phage interrupt allele that is gpEA, express active 6-phosphogluconolactonase from a functional 6pgl allele and are capable of maintaining a pET28 based plasmid vector as well as expressing an hIL- 18 peptide chain of interest (SEQ ID NO : 1 ) via the T7 promoter from this vector were cultured under standard conditions. Cells were then pelleted by centrifugation and diluted to 10% solids with 50 mM Tris-HCl, 5 mM EDTA and 20 mM DTT at a pH of 7.5 to form a cell suspension. A cell lysate was then prepared by a double pass of the cell suspension through a glycol cooled homogenizer. The cell lysate was then treated with the CTAB concentrations indicated in Table 1, Table 2 and Fig. 1 at a pH of either 7.1 (Table 2) or 7.5 (Table 1) to obtain a second liquid portion follow by centrifugation to separate the resulting second liquid portion into an insoluble fraction and a purified peptide chain fraction containing hIL-18. The hlL- 18 peptide chain was about 25% to about 40% of the total peptide chain population in the purified peptide chain fraction (i.e., had a purity of about 25% to about 40%) and was obtained at a percent yield (i.e., % recovery) of from about 3.36% to about 89.31% as indicated in Fig. 1, Table 1 and Table 2.

Example 2

Recovery of a Purified hIL-18 Peptide Chain Fraction from Cell Lysate as a Function of CTAB Concentration at pH 4.5

A purified hlL- 18 peptide chain fraction was recovered from cell lysates after addition of about 5.49 mM to about 13.72 mM CTAB at pH 4.5. See Fig. 2 and Table 4.

Table 4: SDS-PAGE analysis of hIL-18 peptide chain purity and percent recovery in purified hIL-18 peptide chain fractions from cell lysates after addition of about 5.49 mM to about 13.72 mM CTAB at pH 4.5.

Escherichia coli cells, as described in Example 1 , were cultured under standard conditions. Cells were then pelleted by centrifugation and diluted to 20% solids with 50 mM Tris-HCl, 5 mM EDTA and 20 mM DTT at a pH of 7.5 to form a cell suspension. A cell lysate was then prepared by a double pass of the cell suspension through a glycol cooled homogenizer. The cell lysate was then treated with the CTAB concentrations indicated in Fig. 2 and Table 4 for 15 min at 18 °C to obtain a second liquid portion. The pH of the resulting second liquid portion was then adjusted to pH 4.5 by titration. The second liquid portion was then held for 15 min at 18 °C followed by centrifugation in a JA-17 rotor (Beckman Coulter Inc., Brea, CA) at 15,200 x g for 20 min at 4 °C to separate the resulting second liquid portion into an insoluble fraction and a purified peptide chain fraction containing hIL-18.

The hIL- 18 peptide chain was about 65% to about 69% of the total peptide chain population in the purified peptide chain fraction (i.e., had a % purity of about 65% to about 69%) and was obtained at a percent yield (i.e., % recovery) of from about 53% to about 74% as indicated in Fig. 2 and Table 4. Additionally, it is apparent from Table 4 that maximal product recovery (i.e., percent yield) with good purity was obtained from about 9.60 mM (0.35% CTAB) to about 13.72 mM (0.5% CTAB) at pH 4.5.

Example 3

Recovery and Purity of a Purified hIL-18 Peptide Chain Fraction from Cell Lysate as a Function of CTAB Concentration and pH

The recovery and purity of a purified hIL- 18 peptide chain fraction from cell lysates a function of CTAB concentration and pH was analyzed to identify optimal CTAB concentrations and pH values. See Fig. 3 and Table 5. Table 5: SDS-PAGE analysis of hIL-18 peptide chain percent recovery and percent purity in purified hIL-18 peptide chain fractions from cell lysates after addition of about 10.98 mM to about 13.72 mM CTAB at pH values of about 4.4 to about 4.6.

Escherichia coli cells, as described in Example 1 , were cultured under standard conditions. Cells were then pelleted, diluted to by centrifugation and diluted to 20% solids with 50 mM Tris-HCl, 5 mM EDTA and 20 mM DTT at a pH of 7.5 to form a cell suspension. A cell lysate was then prepared as described in Example 2. The cell lysate was then treated with the CTAB concentrations (0.2% CTAB to 0.7% CTAB) indicated in Fig. 3 and Table 5 for 15 min to 30 min at 18 °C to obtain a second liquid portion. The pH of the resulting second liquid portion was then adjusted to the pH values indicated in Fig. 3 and Table 5 (pH 4.0 to pH 4.8) by titration. The second liquid portion was then held for 30 min at 4 °C followed by centrifugation as described in Example 2 to separate the resulting second liquid portion into an insoluble fraction and a purified peptide chain fraction containing hIL-18.

Maximal hIL- 18 peptide chain percent recovery (percent yield) and percent purity occurred at about 0.4% CTAB to about 0.5% CTAB at pH values of about 4.4 to about 4.6. See Fig. 3 and Table 4. Thus, these values represent optimal CTAB concentrations and pH values for producing a purified peptide chain fraction containing a peptide chain of interest. Example 4

Recovery and Purity of a Purified hIL-18 Peptide Chain Fraction from Cell Lysate as a Function of CTAB Concentration, pH and the presence of Ethanol

The recovery and purity of a purified hIL- 18 peptide chain fraction from cell lysates as a function of CTAB concentration, pH and the presence of ethanol was analyzed. See Fig. 4, Fig. 5 and Fig. 6.

Escherichia coli cells, as described in Example 1 , were cultured under standard conditions. Cells were then pelleted by centrifugation and diluted to 18% solids with 50 mM Tris-HCl, 5 mM EDTA and 20 mM DTT at a pH of 7.5 to form a cell suspension. A cell lysate was then prepared by a double pass of the cell suspension through a glycol cooled homogenizer. The cell lysate was then treated with the CTAB concentrations indicated in Fig. 4, Fig. 5 and Fig. 6 to obtain second liquid portions containing 5.49 mM, 10.98 mM and 21.95 mM CTAB. Stock CTAB solutions contained 10% (v/v) ethanol in water and the final concentration of ethanol in CTAB treated second liquid portions was approximately from about 0.4% (v/v) to about 0.8% (v/v). The pH of the resulting second liquid portion was then adjusted by titration to have pH values from about 3.0 to about 7.5, as indicated in Fig. 4, Fig.

5 and Fig 6. The second liquid portion was then held for about 15 min to about 30 min at about 4 °C to about 8 °C followed by centrifugation to separate the resulting second liquid portion into an insoluble fraction and a purified peptide chain fraction containing hIL-18.

The hIL- 18 peptide chain was about 40% to about 65% of the total peptide chain population in the purified peptide chain fraction (i.e., had a % purity of about 40% to about 65%) and was obtained at a percent yield (i.e., % recovery) of from about 40% to about 95% as indicated in Fig. 4, Fig. 5 and Fig. 6. Additionally, it is apparent from Fig. 4, Fig. 5 and Fig.

6 that maximal product recovery (i.e., percent yield) with good purity was obtained at about 10.98 mM (0.4% CTAB) both in the presence or absence of ethanol.

Example 5

Large Scale Production of a Purified hIL-18 Peptide Chain Fraction from Cell Lysates at a CTAB Concentration of 12.35 mM and pH of about 4.4 to about 4.5

Escherichia coli cells, as described in Example 1 , were cultured under standard conditions in a small-scale model, fermentor containing 2 L of growth media for modeling large scale hIL- 18 production by a fermentor containing about 1700 L of growth media.

The following large scale process was applied based on the modeled large scale hlL- 18 production process. First, about 1700 L of growth media in a large-scale fermentor was inoculated with an Escherichia coli cell strain having a genotype that is:

F^", ompT, hsdS_B, (¾^~, m_B ^~), dcm, gal, λ(ΟΕ3), zae-502::TN10,/¾wA21, cyb c::Zeo^r, gpEy.Cm¹ and which contain a λ phage (DE3) which has been made replication defective by inactivation of the integrase gene int and transformation with a red recombinase vector. This Escherichia coli cell strain was also transformed with a plasmid vector having the features shown in Table 6 above. This plasmid vector is capable of driving expression, after isopropyl β-D- 1 - thiogalactopyranoside (IPTG) induction, of a single peptide chain with the amino acid sequence shown in SEQ ID NO: 1 that contains four cysteine residues (38C, 68C, 76C and 127C) with free sulfhydryl groups and which lacks glycosylation. Growth of the cells in the fermentor was then monitored and IPTG was added to induce expression of the amino acid sequence shown in SEQ ID NO: 1. Growth media from the fermentation was then fed into either an Alfa Laval Separator (Alfa Laval Inc., Richmond, VA), a CARR® POWERFUGE® P12 separator (Pneumatic Scale Corp., Cuyahoga Falls, OH) or a Westfalia CSC6 separator and centrifuged at a maximum temperature of 18 °C to obtain a cell suspension containing about 60% solids. This cell suspension was then be diluted with a lysis buffer containing 50 mM Tris-HCl, 5 mM EDTA and 20 mM DTT at a pH of 7.5 to form a second cell suspension containing about 15% to about 30% solids that was maintained at a temperature of 2 °C to 8

°C. A cell lysate was then be prepared from the cell suspension containing about 15% to about 30% solids by homogenization with two passes through a Gaulin homogenizer at 10,000 psi to produce a cell lysate. The cell lysate was maintained at a temperature of 2 °C to 8 °C between passes through the Gaulin homogenizer. After the second pass through the homogenizer, the preferred temperature for the cell lysate was from 18 °C to 25 °C. A quantity of 5.0% CTAB (([g*100%]/ml); 137.19 mM); in 10% ethanol (10 ml volume of ethanol/100 ml total volume of ethanol and water mixture) was then added to the cell lysate over a period of 10 min to 20 min with mixing to produce a second liquid portion containing about 0.45% CTAB (12.35 mM). The pH of this second liquid portion was then adjusted to a pH of from 4.4. to 4.5 with 3 M acetic acid over 15 minutes with mixing at a temperature of from 18 °C to 25 °C.

Optionally, after mixing, this second liquid portion may be held without further mixing at about 18 °C to about 22 °C for about 15 minutes to about 24 hours, or about 12 hours to about 24 hours. The second liquid portion was then separated into an insoluble fraction and a purified peptide chain fraction containing the hIL- 18 peptide chain by continuous

centrifugation; whereby a purified peptide chain fraction was obtained. Table 7 shows yield data obtained after performing the large scale process described here on seven different batches to produce a purified hIL- 18 peptide chain fraction with the amino acid sequence shown in SEQ ID NO: 1.

Table 7: hIL- 18 peptide fraction production from seven large scale process batches produced using good manufacturing practices (GMP).

The claimed methods now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

What is claimed is: 1. A method of producing a purified peptide chain fraction comprising the steps of: a) providing a first liquid portion containing a peptide chain of interest;

hexadecyltrimethylammonium salt;

c) adjusting the pH of the second liquid portion to a pH of about 4.3 to about 7.5; and d) separating the second liquid portion into an insoluble fraction and a purified peptide chain fraction containing the peptide chain of interest; whereby a purified peptide chain fraction is produced.

2. The method of Claim 1, wherein the hexadecyltrimethylammonium salt is hexadecyltrimethylammonium bromide .

3. The method of Claim 1, wherein the first liquid portion comprises a cell lysate.

4. The method of Claim 3, wherein the cell lysate comprises 50 mM Tris-HCl, 5 mM EDTA and 20 mM DTT at a pH of 7.5.

5. The method of Claim 1, wherein the second liquid portion of step c) is held at about 18 °C to about 22 °C for about 15 minutes to about 24 hours.

6. The method of Claim 1, wherein a centrifuge is used in step d) to separate the second liquid portion.

7. The method of Claim 3, wherein the cell lysate is prepared from a cell suspension containing about 15% solids to about 30% solids.

8. The method of Claim 7, wherein the peptide chain of interest is about 40% to about 70% of a total peptide chain population in the purified peptide chain fraction.

9. The method of Claim 1 , wherein the peptide chain of interest is a cytokine.

10. A method of producing a purified peptide chain fraction comprising the steps of: a) providing a first liquid portion comprising a cell lysate that contains a peptide chain of interest;

b) adding hexadecyltrimethylammonium bromide to the first liquid portion to obtain a second liquid portion containing about 13.72 mM hexadecyltrimethylammonium bromide;

c) adjusting the pH of the second liquid portion to a pH of about 4.4;

d) holding the second liquid portion at about 18 °C to about 22 °C for about 15 minutes to about 24 hours; and

e) separating the second liquid portion into an insoluble fraction and a purified peptide chain fraction containing the peptide chain of interest by centrifugation, wherein the peptide chain of interest is about 40% to about 70% of a total peptide chain population in the purified peptide chain fraction; whereby a purified peptide chain fraction is produced.

1 1. The method of Claim 10, wherein the cell lysate is prepared from a cell suspension containing about 15% solids to about 30% solids.

12. The method of Claim 1 1, wherein the cell lysate comprises 50 mM Tris-HCl, 5 mM EDTA and 20 mM DTT at a pH of 7.5.

13. The method of Claim 1 1, wherein the peptide chain of interest is a cytokine.

14. The method of Claim 13, wherein the cytokine comprises the amino acid sequence shown in SEQ ID NO: 1.