Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/62—Detectors specially adapted therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/575—Hormones
  • G01N2333/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography

Definitions

• BACKGROUND Purification of a protein from a cell culture mixture is affected by several factors including, but not limited to, the amount of material loaded onto a chromatographic column as well as the types and levels of contaminating materials present in the protein mixture .
• Immobilized protein A affinity chromatography is an important technique for industrial purification of pharmaceutical antibodies because it provides a method to extract antibodies from complex solutions (Hjelm, H et al , FEBS Letters ( 1972) 28( 1 ), Leser, E W et al , Journal of Chromatography ( 1992) 584 43) Protein
• A binds to IgG of different subclasses, primarily to the F c region but also in some cases to the F ab (Surolia,
• Dynamic capacity is the amount of antibody that can be loaded before antibody breakthrough occurs, and it is affected by several factors including column media, column length, and flow rate (Horstmann, B J et al , Chem Eng Res Des (1989) 62 243, van Sommeren, A P G et al , Preparative
• Rapid assay technology allows product analysis to be run on-line in real-time to find product breakthrough
• On-line means that the preparative column effluent is directly sampled by the assay, and real time means that a quantitative measurement of antibody breakthrough (such as assay peak area) is available as soon as the assay is run
• Assay technology such as high performance liquid chromatography, flow injection immunoassay, biosensors, and perfusion chromatography can produce protein analysis m 20 seconds to ten minutes (Chen, H et al , Journal of Chromatography A (1995) 705 3, Nilsson, M , et al ,
• a method is provided herein to analyze effluent composition by using the on-line assay to quickly measure analyte breakthrough or elution of impurities.
• the information gained from analyzing the effluent is used to control loading of a cell culture mixture onto a first chromatographic column (such as a preparative column) and/or to control collection of effluent.
• a method of separating a protein of interest from a contaminant comprises a) loading a protein mixture onto a first chromatography column, wherein the mixture comprises an analyte including, but not limited to, a protein of interest and at least one contaminant, and b) monitoring the amount of analyte in effluent from the first chromatography column as the analyte elutes from the first column by deflecting column effluent to a separate on-line assay apparatus for a period of time sufficient to provide enough analyte to be monitored in the separate assay apparatus, wherein the monitoring requires less than twenty percent of the total elution time of the analyte on the chromatography column and wherein the monitoring measures the amount of analyte in the effluent from the chromatography column.
• the first chromatography column is a preparative chromatography column.
• the separate on-line assay apparatus comprises a second
• the method further includes controlling a chromatographic device when the amount of analyte in the effluent reaches a predetermined amount.
• the chromatographic device to be controlled is a loading device capable of directing a protein mixture onto the column or away from the column, wherein the loading device is controlled based on information from the monitoring step.
• the controlling may be by manual, mechanical or electronic means, or a combination of means.
• the chromatographic device to be controlled is an effluent collection device capable of directing column effluent into a collection reservoir or away from the collection reservoir, wherein the device is controlled based on information from the monitoring step.
• the controlling may be by manual, mechanical or electronic means, or a combination of means.
• the chromatographic device to be controlled comprises a loading device and an effluent collection device.
• the monitoring requires less than ten percent of the total elution time of analyte on the chromatography column, preferably less than five percent of the total elution time of the protein of interest from the chromatography column.
• the protein of interest is a fusion protein wherein a protein is fused covalently to a portion of an IgG molecule.
• the protein of interest includes, but is not limited to, C2B8, rhuMAb-HER2 (recombinant human monoclonal antibody to HER2), an anti-IgE antibody (for example, E25 and E26), a TNF receptor-IgG fusion (for example, TRY), human DNase (hDNase), and IGF-1.
• the contaminant is a protein including, but not limited to, an aggregate of IGF proteins, and host cell proteins (HCPs) such as CHOP (Chinese hamster ovary cell proteins), ECP (E. coli proteins), and insect cell proteins.
• HCPs host cell proteins
• CHOP Choinese hamster ovary cell proteins
• ECP E. coli proteins
• insect cell proteins such as CHOP (Chinese hamster ovary cell proteins), ECP (E. coli proteins), and insect cell proteins.
• an isolated protein of interest is provided by a method comprising: a) loading a protein mixture onto a first chromatography column, wherein the mixture comprises an analyte including, but not limited to, the protein of interest and at least one contaminant, and b) monitoring the amount of analyte in effluent from the first chromatography column as the analyte elutes from the first column by deflecting column effluent to a separate on-line assay apparatus for a period of time sufficient to provide enough analyte to be monitored in the separate assay apparatus, wherein the monitoring requires less than twenty percent of the total elution time of the analyte on the chromatography column and wherein the monitoring measures the amount of analyte in the effluent from the chromatography column.
• the separate on-line assay apparatus comprises a second chromatography column.
• the purified protein of interest is C2B8, rhuMAb-HER2, IGF-1, or hDNase.
• the isolated protein of interest is present in the final product at a level of at least 80%, more preferably at least 90%, and most preferably at least 95% of the total protein in the product at the level of detection used for standard assay of the product.
• the purified protein of interest is provided by further including controlling a chromatographic device when the amount of analyte in the effluent reaches a predetermined amount.
• the first chromatography column is a protein A affinity column when the protein of interest is an IgG-containing protein, such as an IgG fusion protein.
• the assay used in the separate on-line assay apparatus is a chromatographic assay comprising a second chromatographic column.
• the second chromatographic column may contain the same material as the first (preparative) chromatographic column or it may be different according to what analyte is being monitored by the on-line assay.
• the first and second chromatography columns and the detection are chosen to be appropriate for the purification of the protein of interest.
• the separate on-line assay comprises the second chromatography column, which column comprises anti-HCP antibodies immobilized onto a solid support matrix.
• the HCP is CHOP and the second chromatography column comprises anti-CHOP antibodies immobilized onto a solid support matrix.
• detection of analyte is by UV absorbence. Other preferred detection methods include, but are not limited to, absorption in the visible or infrared spectrum, NMR, biosensor, and the like.
• breakthrough refers to a rapid increase in analyte concentration in the column effluent indicating that the analyte is no longer being retarded by the column bed material. Where the analyte is the protein of interest, breakthrough is monitored for the purpose of determining loading parameters for the first chromatographic column and/or to control the amount of protein of interest loaded onto the column.
• monitoring refers to periodic analysis of chromatographic column effluent such that the concentration of a chosen an analyte is detected by any means appropriate for such detection. Such detection may include, but is not limited to, UV absorption, antibody interaction, and visible light detection.
• loading refers to the amount of a cell culture mixture comprising a protein of interest applied to a chromatography column. Preferably loading is ceased as breakthrough of a chosen analyte (such as the protein of interest) begins.
• protein of interest refers to a protein that may be an analyte and is a protein that the user wishes to isolate from contaminants in a cell culture mixture.
• contaminant refers to a component in the cell culture mixture comprising the protein of interest, or to a component introduced during the purification of the protein of interest, which component is undesirable in the final product-
• the contaminant or impurity may include, but is not limited to, a protein, nucleic acid, host cell, host cell debri, serum, peptone, degraded protein of interest, detergent, antifoam material, pluronic polyol, buffer component, retrovirus and the like.
• analyte refers to either a protein of interest or a contaminant or impurity, the detection of which is monitored in the column effluent.
• column effluent refers to the solution and proteins contained therein flowing from the chromatography column.
• Elution time refers to the time from loading of a protein on the column to the time that the protein emerges from the column.
• control refers generally to the manipulation of a chromatographic device, which manipulation is performed when the amount of an analyte reaches a predetermined level in the first chromatographic column effluent.
• Manipulation includes, but is not limited to manual, mechanical or electronic means.
• a chromatographic device includes, but is not limited to, a loading device such as a loading loop, a collection device such as a fraction collector or product pool collector, a detection device such as a UV detector, a computer for the calculation of data from the monitoring step or for subsequent control of other devices.
• Fig. 1A is a chromatogram of an on-line assay for monitoring antibody breakthrough. The eluted peak is antibody.
• Figs. 2A - 2D are graphs generated using the on-line assay to monitor complete breakthrough curves for (Fig. 2A) Sepharose A, (Fig. 2B) Poros 50 A, (Fig. 2C) Prosep A at five different flow rates, and (Fig. 2D) Prosep A at six column lengths.
• Figs. 3A and 3B are graphs showing breakthrough capacity determined from the breakthrough curves of Fig. 2.
• Fig. 3 A is a graph showing the effect of flow rate on breakthrough capacity for three column media.
• Fig. 3B is a graph showing the effect of column length on capacity.
• Figs. 4A and 4B Figs. 4A and 4B.
• Fig. 4A is a chromatogram of an on-line assay for monitoring IGF-I purity.
• Fig. 4B is a chromatogram resulting from monitoring the preparative column.
• Fig. 5 is a bar graph showing the results from 5 runs pooling IGF-I from 85% to 85% purity. Percent for IGF and aggregate is percent of total peak area by the off-line HPLC analysis for both the load an pool. Yield is percent yield by the off-line HPLC analysis. Error bars are one standard deviation for five runs.
• Figs. 6A and 6B are a chromatogram of the on-line assay for monitoring CHOP.
• Fig. 6B is a chromatogram of preemptive purification of C2B8, with results from the on-line CHOP assay overlaid.
• HCCF HCCF, where cells and cell debris are removed by tangential flow filtration
• load material was obtained from Genentech, Inc. (South San Francisco, CA).
• the HCCF contained a humanized monoclonal antibody (with a murine F v and a human F c , produced in Chinese hamster ovary cells) at a concentration of about 0.5 g/L.
• the humanized monoclonal antibodies analyzed herein are rhuMAb-HER2 and C2B8 produced in separate cell cultures.
• on-line assay parameters were used for monitoring the breakthrough of rhuMAb-HER2 or C2B8 during protein A affinity chromatographic purification of these proteins of interest.
• On-line analysis was performed at room temperature by a perfusion affinity assay.
• the column was a 30 mm length X 2.1 mm diameter Poros A/M with a 20 ⁇ m particle size.
• Buffer A was 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2
• buffer B was 10 mM sodium phosphate, 150 mM sodium chloride, pH 2.2. Two flow rates were used: 20 ml/min for purge, 3.5 ml/min for run.
• Injection volume was 20 ⁇ l from the process stream using an in-line sampling T to directly sample the preparative column effluent. Detection was at 280 nm.
• the method was: purge 3.5 ml A, run 15 column volumes (CV) A, inject, run 12 CV A, purge 3.5 ml B, run 20 CV B.
• Chromatography was run on a BioCAD/RPM. On the BioCAD instrument, purging takes the column out of the flow path, allowing increased flow rate to flow path to rapidly fill with buffer; the flow rate is decreased and the column is then placed back in line. This purge/run method decreases assay time. The BioCAD/RPM software automatically integrates the antibody peak and displays the result immediately after the assay is finished.
• Example 3 Preparative Chromatography for Protein A Affinity Chromatography
• Preparative protein A affinity chromatography used 0.66 cm diameter columns. Columns were equilibrated with at least 5 CV of 25 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.1 and eluted with at least 3 CV of 0.1 M acetic acid, pH 3.5. All buffers and load material were filtered through a 0.22 ⁇ m filter prior to use. Chromatography was run on a BioCAD.
• Example 4 Purification of a Humanized Monoclonal Antibody by On-line Protein Breakthrough Assay
• the following purification method was applied to purification of rhuMAb-HER2 or C2B8.
• the effluent from the preparative protein A affinity column was monitored for C2B8 product breakthrough using an immobilized protein A perfusion chromatography assay, shown in Fig. 1A, where convective mass transport allows the assay to be run at 6000 cm/hr, resulting in a 2.3 minute assay.
• the assay provides a fast, accurate, and reliable method for real-time analysis of antibody breakthrough.
• the on-line assay measures protein breakthrough in real-time during loading, where antibody breakthrough is obscured at 280 nm by flow-through of contaminant proteins.
• the preparative assay for the protein of interest (C2B8 in this example) is shown in Fig. IB.
• the assay must be fast enough to run about ten assays during product breakthrough for accurate breakthrough measurement. For this study, a 2.3 minute assay is sufficient, but the assay time could be reduced by increasing the flow rate on the assay column or by decreasing the volume of equilibration.
• the shape of the breakthrough curve for affinity absorption results from the ability of the antibody to transfer onto the absorbate surface, which is determined by three mass transfer mechanisms (Cowan, G.H. et al.. Journal of Chromatography (1986) 363:37): film diffusion (mass transfer from the mobile phase to the surface of the particle), pore diffusion (diffusion into the particle), and the kinetics of the interaction between the antibody and protein A. With no mass transfer effects (ideal behavior), C j /C 0 would increase to one the instant that saturation capacity was reached, resulting in a very sharp breakthrough curve. Diffusion changes the shape of the breakthrough curve because with diffusion antibody starts breaking through before the column is saturated (Mao, Q.M. et al, Journal of Chromatography (1995) 691 :273).
• Breakthrough curves were plotted as C j /C 0 vs. antibody loaded, where the antibody loaded is F-C 0 t V c
• the column capacity in grams of antibody per liter of column volume
• the column capacity can be directly read from the plot, and it corrects for differences in flow rate or column length used to generate the breakthrough curves.
• Poros 50 A has symmetric breakthrough curves, indicating short diffusion path lengths.
• the breakthrough curves for Prosep A have a good shape when C j /C 0 ⁇ 0.5, but degenerate when C j /C 0 > 0.5 at higher flow rates, indicating that some binding sites are deep in the media.
• a column loading device is manipulated such that loading is stopped when C./C 0 is approximately 0 05, or when the amount of protein of interest unretained by the first chromatography column (such as a preparative column) reaches an unacceptable level with regard to reduced purification efficiency
• the on-line assay measures antibody breakthrough during loading of preparative protein A chromatography, leading to a fast evaluation of breakthrough capacity
• the assay can be applied to other antibody processes where real-time measurement of antibody concentration is needed By immobilizing different affinity hgands on the assay media, this assay could be modified to measure breakthrough of other proteins in real-time It is useful for monitoring processes where breakthrough is obscured by flow through of contaminant proteins
• the on-line assay described herein may be applied using any affinity column specific for the protein of interest, where the protein of interest may be a whole protein, a fragment thereof, or a fusion protein and includes, for example, antibodies or fusion proteins containing antibody fragments
• the protein of interest includes without limitation such proteins as IgG fusion proteins, C2B8, rhuMAb-HER2, E25, E26, TRY, hDNase, and IGF-l
• the contaminant includes without limitation an aggregate of IGF proteins
• the protein of interest is produced CHO cell culture, the contaminant
• the detection method may be any method suitable for the protein of interest and suitable for on-line, real-time analysis
• Table II shows the results from loading to breakthrough (determined by the on-line assay), compared to loading by volume for rhuMAb-HER2 and C2B8 produced CHO cell culture In each case, yield and purity (determined by CHOP ELISA) was equivalent TABLE II
• IGF-I is separated from aggregated IGF-I.
• Purified IGF-I is difficult to reliably collect because the preparative chromatogram does not indicate the elution positions of IGF-I and aggregated IGF-I.
• An on-iine reversed-phase assay can assay for IGF-I and aggregated IGF-I every 4 minutes and enable real-time control of product pooling.
• Preparative IGF-I purification was performed on a 1 X 25 cm (20 ml) Bakerbond C4 40 ⁇ m 275 A column (Fig. 4B).
• Buffer A was 50 mM acetic acid 50 mM citric acid pH 3.0
• Buffer B was 50 mM acetic acid 20 mM citric acid pH 3.0 with 50% hexylene glycol.
• the load was 12.5 mg IGF/ml CV of folded pool.
• the column was run at a temperature of 30 C.
• a flow rate of 20 column volumes (CV)/hr was used for the Equilibrate/Load/Wash, and a flow rate of 9 CV/hr was used for the Gradient/Regenerate.
• the method was: Equilibrate 3 CV 100% Buffer A, Load, Wash 2 CV 100% Buffer A, Gradient 0-50% Buffer B/ 15 CV, Regenerate 2 CV 100% Buffer B. Detection was at 280 nm. Off-line analysis was performed by an HPLC assay.
• the column was a 4.6 X 25 mm Vydac C18 5 ⁇ m
• Buffer A was 0.12% trifluoroacetic acid (TFA) in water and Buffer B was 0.1% TFA in acetonitrile.
• the flow rate was 2 ml/min.
• the temperature was 50° C.
• a volume of 100 ⁇ l was injected, containing about 10 ⁇ g of IGF-I.
• the method was: 27.5-28.5% B / 9 min, 28.5-40% B / 4 min. 40-90% B / 2 min, hold 90% B for 1 min, 27.5% B for 4 min.
• Detection was at 214 nm.
• On-line analysis was performed by a rapid reversed-phase assay (Fig. 4A).
• the column was a 2.1 X
• IGF-I pool can be reliably collected (Fig. 5).
• Example 6 Improved Purification of a Protein of Interest by Monitoring Contaminant in the Column Effluent
• the method of the invention disclosed herein may be adapted to monitor the concentration of the contaminant(s) such that the purity of the protein of interest in the collected material is monitored and/or enhanced. Collection of the protein of interest as it elutes from the chromatography is controlled such that the amount of contaminating protein is kept to a relatively low acceptable level in the collected material.
• the methods of the invention may be applied together to the purification of a protein of interest such that both loading and collection are monitored and controlled, thereby limiting the amount of contaminating proteins significantly.
• ELISA ELISA of host cell proteins as trace contaminants in a preparation of a protein of interest (such as recombinant protein) produced in CHO cell cultures (CHO proteins or CHOPs).
• Process validation involves documenting the removal of host cell impurities by the purification process (Chen, A.B.
• the separation, monitoring and control method disclosed herein is applied to, but not limited to, C2B8 purification from host cell culture contaminants such as CHOPs and to the control of a chromatographic device based on monitoring of contaminants in the chromatographic column effluent.
• the ELISA used herein has been qualified for products produced in different CHO cell subclones, and grown under different culture conditions. The ELISA showed that the quantitative comparison of blank cell run
• CHOPs produced by these various processes were nominally identical in immunoreactivity.
• ELISA immunoreactivity was quantitatively equivalent independent of cell culture size (10 L vs. 12 kL), or use of bovine or porcine peptones in the culture media.
• the same anti-CHOPs antiserum can be used for a range of products, obviating the need for tailoring anti-CHOPs ELISAs to specific products, or requiring blank runs to be performed to scale.
• immunizations result in reagents suitable for a "generic" ELISA.
• Anti-CHOPs antibodies were immobilized on an affinity column and used to develop a Real-time
• Anti-CHOPs antibodies were prepared by immunizing goats with CHOPs produced from the culture of a non-producing CHO clone. The CHOPs were also immobilized on a solid support matrix and used to affinity purify anti-CHOPs antibodies from the immune sera. These anti-CHOP antibodies were used to prepare both a "generic CHOPs ELISA" and immobilized on a solid support matrix to make an anti-CHOPs column for an online chromatographic assay.
• Real-time Process Monitoring is an HPLC software system that allows the monitoring of a purification process for a specific analyte.
• the separation was performed for purification of C2B8 on an ion exchange column (Fig. 6B), while the presence of CHOP in the effluent was monitored on an anti-CHOP antibody on-line chromatographic assay (Fig. 6A).
• the preparative column was a 1.6 X 17.5 cm Poros 50 HS.
• the flow rate was 100 cm/hr, the load was 17 g/L of C2B8 protein A pool.
• the buffer was 20 mM MES pH 5.5.
• the procedure included the following steps: equilibrate with 2 CV 60 mM NaCl, Load, wash 1 CV 60 mM NaCl, gradient 60 - 500 mM NaCl over 5 C V, regenerate 4 C V 500 mM NaCl.
• C2B8 The purification of C2B8 involved separating an already highly purified protein from trace CHOPs. In fact, the CHOPs levels are too low to be seen by A280 detection. Fractions were also collected and submitted to the generic CHOPs ELISA, with quantitatively similar results to the RPM estimation of CHOPs. Monitoring of CHOPs levels in the final product is useful for the evaluation of product purity. Eluent collection control may be performed when monitored levels of CHOP in the eluent reach a predetermined level in order to maximize purification of the protein of interest.

Landscapes

• Physics & Mathematics (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=22093458

Family Applications (1)

Country Status (3)

Cited By (1)

Families Citing this family (8)

Family Cites Families (4)

• 1998
  • 1998-12-09 WO PCT/US1998/026172 patent/WO1999034220A2/en not_active Application Discontinuation
  • 1998-12-09 AU AU21990/99A patent/AU2199099A/en not_active Abandoned
  • 1998-12-09 EP EP98965982A patent/EP1044378A2/de not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events