Protein purification principles and practice pdf


 

Protein purification: Principles and practice, second edition. by Robert K. Scopes, Springer-Verlag, DM (xv + pages) ISBN 0 6. J.G.P. Volume , number 1. FEBS LETTERS. December Protein Purification: Principles and Practice by Robert E. Scopes. Springer- Verlag; New York, ISBN ; Digitally watermarked, DRM-free; Included format: PDF; ebooks can be used on all reading devices; Immediate eBook download after.

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Protein Purification Principles And Practice Pdf

Chapter 3 Protein Purification by Affinity Chromatography 53 Scopes, R. K. ( ) Protein Purification, Principles and Practice, 3rd ed. Springer Advanced. A biologist's interest in protein-lipid interactions arises primarily from the functional and structural significance of cellular membranes. Since the days of the. Protein purification: Principles and practice, by R Scopes. Pp Springer‐ Verlag, Berlin, Heidelberg and New York. DM ISBN 3–––2.

The publisher's final edited version of this article is available at Curr Protoc Protein Sci See other articles in PMC that cite the published article. Abstract When the first version of this unit was written in protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches many of which were described and mentioned in this unit and elsewhere in the book. In the interim there has been a shift towards an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein and whether to engineer a self cleavage system or simply leave them on. We will briefly address some of these issues. Also although this overview focuses on E. Protein Expression The expression of recombinant proteins, especially using bacterial vectors and hosts, is a mature technology. Following sequence determination of the constructs, plasmids are transformed into expression hosts, single colonies picked, and fermentation performed. With E. The problem is, of course, how to isolate it in an active form. Hence, using small-scale fermentations and laboratory-scale processing equipment, proteins or subdomains thereof can usually be produced in sufficient quantities 10 to mg to initiate most studies including detailed structural determinations. Some strategies for achieving high-level expression of genes in E. Some of the above characteristics also hold true for the production of proteins using yeast and baculovirus eukaryotic expression systems, although more effort and expertise is required to construct the vectors and, with the baculovirus system, produce cells for processing.

Some of the above characteristics also hold true for the production of proteins using yeast and baculovirus eukaryotic expression systems, although more effort and expertise is required to construct the vectors and, with the baculovirus system, produce cells for processing. A yeast expression system may be a wise choice for proteins that form insoluble inclusions in bacteria, and for the production of membrane-associated proteins Cereghino and Clegg, ; UNITS 5. The baculovirus system has proven very useful for producing phosphorylated proteins and glycoproteins Kost, ; UNITS 5.

The construction of stable mammalian protein expression vectors requires considerably more time and effort but may be the only approach for producing complex multidomain proteins UNITS 5. Alternatively, transient gene expression systems using various viral vectors e.

It is of interest to note that the large-scale transient expression systems in mammalian cells are being actively developed by biotechnology companies Wurm and Bernard, The choice of a host system for the production of recombinant proteins is discussed in unit 5. Also, there is a special issue on the production of recombinant proteins in the journal Biotechnology Advances Sanchez and Demin, In this issue there are excellent overviews of protein expression and production using E.

As mentioned by Chen , for many investigators the initial choice is often Escherichia coli which remains the preferred system for laboratory investigations and initial development in commercial activities and is a benchmark for comparison among the other various expression platforms.

This is due to such factors as ease of genetic manipulation, availability of optimized expression plasmids, and ease of growth. This unit presents an overview of recombinant protein purification with special emphasis on proteins expressed in E. Practical aspects and strategies are stressed throughout, and wherever possible, the discussion is cross-referenced to the example protocols described in the rest of Chapter 6.

The first section deals with information pertinent to protein purification that can be derived from translation of the cDNA sequence. This is followed by a brief discussion of some of the common problems associated with bacterial protein expression see also UNIT 5.

Planning a protein purification strategy requires that the solubility of the expression product be determined; it is also useful to establish the location of the protein in the cell—e. This unit includes flow charts that summarize approaches for establishing solubility and localization of bacterially produced proteins see also UNIT 5.

Purification strategies for both soluble and insoluble proteins are reviewed and summarized in flow charts see also Chapter 1. Many of the individual purification steps, especially those involving chromatography, are covered in detail in Chapters 8 and 9, and elsewhere Scopes, ; Janson, The methodologies and approaches described here are essentially suitable for laboratory-scale operations.

Large-scale methodologies have been previously reviewed Asenjo and Patrick ; Thatcher, ; Sofer and Hagel, A section on glycoproteins produced in bacteria in the nonglycosylated state is included to emphasize that, although they may not be useful for in vivo studies, such proteins are well suited for structural studies. The present invention is based, at least in part, on the finding that both flow through and bind-elute techniques can be combined to achieve greater purification and recovery of a protein of interest, e.

HIC is often utilized in either a bind-elute mode, in which the protein of interest remains bound to HIC media until eluted during an elution phase, or a flow through mode, in which the protein of interest flows through the column while the impurity binds to the media.

Recently, a chromatographic method termed "weak partitioning mode" has been described for the purification of proteins U.

Patent No. According to U. Compared to the flow-through mode, in which the Kp for the product is typically low e. Importantly, U.

Specifically, U. In addition, U. Accordingly, U. When applied to HIC, the weak partitioning mode described in U. Patentees report that HIC performance deteriorates with respect to both contaminant reduction and product recovery at stronger binding conditions.

The present invention is based, at least in part, on the finding that both flow through and bind-elute techniques can be combined to achieve greater purification and recovery of a protein of interest. Moreover, the present invention is predicated, at least in part, on the surprising finding that such methodology can be employed under isocratic wash conditions and at stronger binding conditions than previously appreciated, for example, at a Kp greater than 10, so as to achieve greater purification and recovery.

In various embodiments, the portion of the protein of interest binds to the HIC media at a Kp of greater than 10, 15, 20, 50, 60, 70, 80, 90, , , , , , , , , , , , , or For example, in various embodiments, the portion of the protein of interest binds to the HIC media at a Kp of greater than 10, the portion of the protein of interest binds to the HIC media at a Kp of greater than 20, or the portion of the protein of interest binds to the HIC media at a Kp of greater than In a particular embodiment, the protein of interest is adalimumab.

In one embodiment, a substantial portion of the impurity bound to the HIC media remains bound upon washing with the wash buffer. In one embodiment, the at least one impurity is an aggregate of the protein of interest, for example, selected from the group consisting of a multimer, a dimer, a trimer, a tetramer, an oligomer and other high molecular weight species. In a particular embodiment, the protein of interest is adalimumab and the at least one impurity is an aggregate of adalimumab.

For example, the aggregate may be selected from the group consisting of multimer 1, multimer 2 and multimer 3. In another embodiment, the impurity is a process-related impurity or a product-related substance.

For example, the impurity may be a process-related impurity selected from the group consisting of a host cell protein, a host cell nucleic acid, a media component, and a chromatographic material. Alternatively, the impurity may be a product-related substance selected from the group consisting of a charge variant, an aggregate of the protein of interest, a fragment of the protein of interest and a modified protein. In a particular embodiment the impurity is an acidic or basic variant, for example, of adalimumab.

In a particular embodiment, the basic variant is a lysine variant species, for example, an antibody, or antigen-binding portion thereof, having heavy chains with either zero, one or two C-terminal lysines.

In another embodiment, the impurity is an acidic species AR , for example, selected from the group consisting of a charge variant, a structure variant, a fragmentation variant, a process-related impurity and a product-related impurity. In yet another embodiment, the acidic species is AR1 and the fragmentation variant is a Fab fragment variant, a C-terminal truncation variant or a variant missing a heavy chain variable domain. In a particular embodiment, the impurity is a fragment such as an Fc or a Fab fragment.

In another embodiment, the impurity is a modified protein such as a deamidated protein or glycosylated protein. In one embodiment, the protein of interest is an antibody or antigen-binding fragment thereof, a soluble protein, a membrane protein, a structural protein, a ribosomal protein, an enzyme, a zymogen, an antibody molecule, a cell surface receptor protein, a transcription regulatory protein, a translation regulatory protein, a chromatin protein, a hormone, a cell cycle regulatory protein, a G protein, a neuroactive peptide, an immunoregulatory protein, a blood component protein, an ion gate protein, a heat shock protein, an antibiotic resistance protein, a functional fragment of any of the preceding proteins, an epitope-containing fragment of any of the preceding proteins, and combinations thereof.

In a particular embodiment, the protein of interest is an antibody or antigen-binding fragment thereof such as a humanized antibody or antigen-binding portion thereof, a human antibody or antigen-binding portion thereof, a chimeric antibody or antigen-binding portion thereof, or a multivalent antibody. In another embodiment, the antibody, or antigen-binding fragment thereof is selected from the group consisting of a Fab fragment, a F ab' 2 fragment, a single chain Fv fragment, an SMIP, an affibody, an avimer, a nanobody, and a single domain antibody.

In one embodiment, the methods of the invention further include repeating steps a - d at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 times using the flow through fraction, wash fraction, or combination thereof having a reduced level of the at least one impurity.

In certain embodiments, the flow through fraction and the wash fraction are combined. In certain embodiments, the at least one impurity binds to the HIC media at a Kp of greater than , greater than , greater than , greater than , greater than , greater than , greater than , greater than , or greater than In certain embodiments, the protein of interest and the at least one impurity have a Kp ratio less than 1: 10, , , , , , , or Alternatively or in combination, the Kd for the binding of the at least one impurity to the HIC media is less than or equal to about 0.

In particular embodiments, the Kd for the binding of the protein of interest to the HIC media is less than 50, 45, 40, 35, 30, 25, 20, 15, 10 or 5 times the Kd for the binding of the at least one impurity to the HIC media.

In certain embodiments, the protein of interest has a Qmax of at least about 20, at least about 30, at least about 40, at least about 50, at least about 60 or at least about In certain embodiments, the at least one impurity has a Qmax of at least about 2, at least about 5, at least about 10, at least about 20, at least about 30 or at least about In certain embodiments, the HIC media comprises at least one hydrophobic ligand.

For example, the HIC media may be selected from the group consisting of alkyl-, aryl-ligands, and combinations thereof. For example, the HIC media may be selected from the group consisting of butyl, hexyl, phenyl, octyl, or polypropylene glycol ligands.

In one embodiment, the HIC media is a column. In a particular embodiment, the load buffer and the wash buffer include a sulfate salt, a citrate salt, or a combination thereof.

For example, the sulfate salt may be ammonium sulfate or sodium sulfate. In certain embodiments, the citrate salt is sodium citrate. In one embodiment, the salt has a concentration of between about 50 mM and mM.

In a particular embodiment, the load buffer and the wash buffer have a pH between about 4. In certain embodiments, the load buffer and the wash buffer have a pH of about 4. In one embodiment, the load buffer and the wash buffer are the same. In one embodiment, the load buffer and the wash buffer are substantially the same. In certain embodiments, about g to about g of the sample are contacted per one liter of HIC media.

Alternatively or in combination, about 0. Alternatively or in combination, the concentration of the at least one impurity in the sample is about 0. In one embodiment, the at least one impurity is a host cell protein. For example, the host cell protein may be reduced by at least 0. In one embodiment, the HIC media has a dynamic binding capacity of at least about 2 g, at least about 5 g, at least about 10 g, at least about 20 g, at least about 30 g, at least about 40 g, at least about 50 g, at least about 60 g, at least about 70 g, at least about 90 g, or at least about g of sample per one liter of media.

In one embodiment, a precursor sample including the protein of interest has been subjected to affinity chromatography to generate the sample.

Alternatively or in combination, the preparation including a protein of interest and having a reduced level of one impurity is subjected to affinity chromatography. In one embodiment, a precursor sample including the protein of interest has been subjected to ion exchange chromatography to generate the sample. Alternatively or in combination, the preparation including a protein of interest and having a reduced level of one impurity is subjected to ion exchange chromatography.

In such embodiments, ion exchange chromatography may be performed using ion exchange chromatography media selected from the group consisting of i a cation exchange media, for example, comprising carboxymethyl CM , sulfoethyl SE , sulfopropyl SP , phosphate P or sulfonate S ligands, and ii an anion exchange media, for example, comprising diethylaminoethyl DEAE , quaternary aminoethyl QAE or quaternary amine Q group ligands.

In one embodiment, a precursor sample including the protein of interest has been subjected to mixed mode chromatography to generate the sample. Alternatively or in combination, the method involves subjecting the preparation including a protein of interest and having a reduced level of one impurity to mixed mode chromatography, for example, using CaptoAdhere resin. In one embodiment, a precursor sample including the protein of interest has been subjected to a filtration step to generate the sample.

Alternatively or in combination, the method involves subjecting the preparation including a protein of interest and having a reduced level of one impurity to a filtration step, for example, a depth filtration step, a nanofiltration step, an ultrafiltration step, and an absolute filtration step, or a combination thereof. In one aspect, the present invention is directed to a pharmaceutical composition including the preparation produced by any of the foregoing methods.

In another aspect, the present invention is directed to a method for producing a preparation including adalimumab and having a reduced level of at least one aggregate, by a contacting a sample of adalimumab and at least one aggregate, to a HIC media, in the presence of a load buffer such that i a portion of the adalimumab in the sample binds to the HIC media and ii a substantial portion of the at least one aggregate binds to the HIC media; b collecting a flow through fraction of the adalimumab unbound to the HIC media; c washing the HIC media with a wash buffer that is substantially the same as the load buffer such that a substantial portion of the adalimumab bound to the HIC media is released from the media; and d collecting a wash fraction of the adalimumab released from the HIC media, wherein each of the flow through and wash fractions comprise adalimumab and have a reduced level of the at least one aggregate.

In one embodiment of the foregoing method, adalimumab binds to the HIC media at a Kp of greater than 10, 15, 20, 50, 60, 70, 80, 90, , , , , , , , , , , , , or For example, adalimumab binds to the HIC media at a Kp of greater than Alternatively, adalimumab binds to the HIC media at a Kp of greater than In a particular embodiment, the aggregate is multimer 1, multimer 2 or multimer 3.

In various embodiments, the sample includes between g and g protein per liter of HIC media. In certain embodiments, the load buffer and the wash buffer include ammonium sulfate, sodium sulfate, sodium citrate, or a combination thereof.

Alternatively or in combination, the pH of the load buffer and the wash buffer is between 5 and 7. Alternatively or in combination, the salt concentration of the load buffer and the wash buffer is between about mM and mM. In another aspect, the present invention provides a pharmaceutical composition comprising a low-aggregate composition and a pharmaceutical acceptable carrier.

In one aspect, the present invention provides a pharmaceutical composition comprising a preparation of adalimumab produced by the foregoing methods and a pharmaceutically acceptable carrier. In another aspect, the present invention provides a pharmaceutical composition comprising a low-aggregate composition of adalimumab and a pharmaceutically acceptable carrier.

In combination with multi-angle light scattering MALS analysis data not shown , the apparent molecular weight of each peak was determined and identified as a multimer or the reference standard as indicated.

Figures 2A-2B depicts schematic chromatograms for two modes of chromatographic operation: bind-elute mode Figure 2A and flow-through mode Figure 2B. In the bind-elute mode, there is strong binding of the protein of interest and the impurity.

Elution conditions are chosen to selectively elutes the protein of interest. In the flow-through mode there is weak binding of the product and strong binding of the impurity. Figure 3 depicts selection of operating conditions appropriate for an antibody:media:buffer combination.

The salt concentration at or near the elution peak of the monomer is the concentration at which the monomer is eluted from the HIC media.

The column was then washed with 10 CVs of the equilibration buffer and a linear gradient from 1. See Example 1. Finally, the column was washed with 7 CVs of the equilibration buffer. The results indicate the impact that the concentration of loaded protein can have on aggregate reduction. See Example 7. Figure 7 depicts the effect of aggregate load concentration on dynamic binding capacity and aggregate clearance.

The column is conditioned and loaded at different sample load concentrations.

Protein Purification | SpringerLink

The flow-through is fractionated to determine the product quality at different times during the load and breakthrough. Using protein mass and product quality for each of the collected fractions, the accumulative impurity e. The accumulative impurity of the preparation is reduced when the concentration of the aggregate in the load is reduced, even when the total load is unchanged e.

See Example Figures 8A-8C depicts the effect of overall load protein concentration in the sample. The flow through is fractionated to determine the product quality at different times during the load and breakthrough Figure 8A.

Using protein mass and product quality for each of the collected factions, the accumulative aggregate impurity can be calculated. The accumulative aggregate impurity of the preparation is reduced when the protein concentration of the sample is reduced.

Protein purification

The Equilibrium Binding Isotherms for both the monomer and aggregate show that for all of the loading conditions Figure 8B and Figure 8C , the monomer was in the non-linear part of its binding isotherm e. Figure 9 depicts the modulation of the recovery-yield for a given target impurity clearance by diluting the load material to a specific range.

Figures 13AB depict the results of experiments wherein aliquots of resin are incubated with a load covering a range of protein concentrations at room temperature for 3 hours, after which the protein solution is then removed, and replaced with equilibration buffer Wash simulation and incubated at room temperature for 3 hours repeated, Wash II.

After each incubation, the concentration of the protein solution is measured and used to calculated the amount of protein Figure 13A monomer D2E7, i. However, after the protein solution is replaced with equilibration buffer see arrow , the monomer desorbs from the resin and back into solution, whereas the aggregate remains bound.

Figure 16 depicts the comparison of Apparent and Actual bound protein under flow conditions partial partitioning as a function of salt concentration. The mass balance of the impurity demonstrates irreversible binding. Moreover, the present invention is predicated, at least in part, on the surprising finding that such methodology can be employed under isocratic wash conditions and at stronger binding conditions than previously appreciated, for example, at a Kp greater than 10 or at a Kp greater than 20, so as to achieve greater purification and recovery.

In one aspect, the present invention provides a method for producing a preparation including a protein of interest, e. In a particular embodiment, the portion of the protein of interest binds to the HIC media at a Kp of greater than In another embodiment, the portion of the protein of interest binds to the HIC media at a Kp of greater than In addition, in certain embodiments, the present invention is directed toward pharmaceutical compositions comprising one or more proteins of interest purified by methods described herein.

In a particular embodiment, the present invention is directed to a pharmaceutical composition comprising adalimumab and having a reduced level of aggregates. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.

Further, unless otherwise required by context, singular terms, for example, those characterized by "a" or "an", shall include pluralities, e. Furthermore, the use of the term "including," as well as other forms of the term, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one unit unless specifically stated otherwise.

As used herein, the term "sample", refers to a liquid composition including the protein of interest and one or more impurities.

In a particular embodiment, the sample is a "clarified harvest", referring to a liquid material containing a protein of interest, for example, an antibody of interest such as adalimumab, that has been extracted from cell culture, for example, a fermentation bioreactor, after undergoing centrifugation to remove large solid particles and subsequent filtration to remove finer solid particles and impurities from the material. In various embodiments, the sample may be partially purified.

For example, the sample may have already been subjected to any of a variety of art recognized purification techniques, such as chromatography, e. The term "precursor sample", as used herein refers to a liquid composition containing the protein of interest and, optionally, one or more impurities, either derived from the clarified harvest, or a partially purified intermediate sample that is subject to a purification or treatment step prior to being subjected to HIC. Impurities in a precursor sample may be derived from the production, purification or treatment of the protein of interest prior to subjecting the resulting sample to HIC.

The term "protein of interest", as used herein refers to a target protein present in a sample, purification of which is desired.

In various embodiment, the protein of interest is an antibody or antigen-binding fragment thereof, a soluble protein, a membrane protein, a structural protein, a ribosomal protein, an enzyme, a zymogen, an antibody molecule, a cell surface receptor protein, a transcription regulatory protein, a translation regulatory protein, a chromatin protein, a hormone, a cell cycle regulatory protein, a G protein, a neuroactive peptide, an immunoregulatory protein, a blood component protein, an ion gate protein, a heat shock protein, an antibiotic resistance protein, a functional fragment of any of the preceding proteins, an epitope-containing fragment of any of the preceding proteins, and combinations thereof.

In a particular embodiment, the protein of interest is a monomer. In a particular embodiment, the protein of interest is an antibody, or an antigen binding portion thereof.

The term "antibody" includes an immunoglobulin molecule comprised of four polypeptide chains, two heavy H chains and two light L chains inter-connected by disulfide bonds.

Protein purification : principles and practice

Each light chain is comprised of a light chain variable region abbreviated herein as LCVR or VL and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions CDRs , interspersed with regions that are more conserved, termed framework regions FR.

The term "antibody", as used herein, also includes alternative antibody and antibody-like structures, such as, but not limited to, dual variable domain antibodies DVD-Ig. The term "antigen-binding portion" of an antibody or "antibody portion" includes fragments of an antibody that retain the ability to specifically bind to an antigen e. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include i a Fab fragment, a monovalent fragment comprising the VL, VH, CL and CHI domains; ii a F ab' 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; iii a Fd fragment comprising the VH and CHI domains; iv a Fv fragment comprising the VL and VH domains of a single arm of an antibody, v a dAb fragment Ward et al.

Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv scFv ; see, e.

USA , the entire teachings of which are incorporated herein by reference.

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