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Refolding and buffer exchange at high concentration Dialysis, formulation and lyophilization Fig. 1. A novel purification strategy for the improved recovery of bioactive proteins from inclusion bodies. not interact with fully folded protein, as reported for the P22 tailspike protein (33,34). This supports the concept of pulse renaturation processes for high-throughput refolding of denatured protein. This process exercises the theory that the overall yield of purified bioactive proteins from...
Site Specific Integration for High Level Protein Production in Mammalian Cells
Calos 1. Introduction Recombinant proteins have various applications in basic and applied research, as well as in therapy. Numerous systems have been developed to produce large protein amounts (1-3). The most convenient systems are those that involve the overexpression of recombinant proteins in bacteria, mainly because they are fast and easy. However, there is a need for eukaryotic overexpression systems, because several proteins require either the proper...
Coomassie Blue Staining of Gel Proteins
Place the gel in Coomassie Blue gel stain at room temperature for 15 min with gentle agitation. 2. Pour off the stain, add destain, and leave at room temperature overnight with gentle agitation (see Note 17). 3. Obtain an image of the gel, if required, using dedicated equipment or by recording a digital image of the gel using a flatbed digital scanner (Fig. 2). Fig. 2. To obtain a digital image of a stained gel, place the gel with gloved hands onto a flatbed scanner, draw a gel-trimming tool...
Ipa400
Protein A Ceramic HyperD Prosep-A rA High Capacity Poros 50 A High Capacity UltraLink Immobilized ProtA Affi-Gel Protein A Gel Affi-Prep Protein A Support Protein A Agarose 4XL Protein A Cellthru AF-Protein A Toyopearl 650M Crosslinked agarose Crosslinked agarose Crosslinked agarose Ceramic polyacrylamide Porous glass Uppsala, Sweden Amersham Biosciences Amersham Biosciences RepliGen Corp., Waltham, MA Biosepra, Cedex, France Bioprocessing (Millipore) Watford, UK PerSeptive Biosystems, MA...
Rodney G Keck John B Briggs and Andrew J S Jones
With the growth in the number of recombinant proteins being developed for use as human therapeutics, there also exists a corresponding need for new and highly sensitive analytical techniques to characterize these new pharmaceuticals. Many new assays were developed to monitor and characterize the heterogeneity that naturally exists in these complex molecules (1,2). For glycoprotein-based therapeutics, one major source of heterogeneity arises from the oligosaccharides present on the protein (3)....
Peak Integration and Relative Percentage Calculation
After obtaining a deconvoluted mass spectrum for partially reduced MAb, the area of each mass peak can be obtained by selecting the Integration function on MassLynx Fig. 4. Deconvoluted mass spectra of partially reduced heavy chain of MAb before (A) and after (B) treatment with a-galactosidase. Fig. 4. Deconvoluted mass spectra of partially reduced heavy chain of MAb before (A) and after (B) treatment with a-galactosidase. software. The relative percentage of each oligosaccharide is calculated...
Spray Drying
SD consists of three steps of operation atomization, dehydration, and powder collection. As shown in Fig. 1A, the protein solution feed is sprayed by an atomizer into a drying chamber. Aided by the large specific surface area of the droplets and the hot air, dehydration takes place in a matter of seconds in the laboratory-scale drying chamber. Finally, the dry particles are carried into the cyclone and settle in the product collector. The most important process parameters include drying air...
Notes
Water will not flow through the PVDF membrane unless the membrane is first wetted with methanol. Therefore, water placed in the unused wells will remain throughout the procedure. The unused wells are filled with water to minimize evaporative losses during the 37 C-incubation steps and to ensure adequate vacuum is applied to those wells containing samples. 2. The routine method employs denaturation of the sample by reduction and alkylation to maximize the accessibility of the protein-glycan bond...
Stacey Ma Wendy Lau Rodney G Keck John B Briggs Andrew J S Jones Kathy Moorhouse and Wassim Nashabeh
Carbohydrates are known to play a key role in the therapeutic use of recombinant proteins (1). The fundamental understanding of the biological roles of the carbohydrate moieties demands high-performance analytical tools to accomplish both the structural characterization and routine analysis of carbohydrates. Among the various analytical techniques developed currently, nuclear magnetic resonance and mass spectrometry are indispensable tools for the structural elucidation of carbohydrates (2-4)....
Contributors
Varsha Bhakta Canadian Blood Services, Research and Development Department, Hamilton, Ontario, Canada John R. Birch Lonza Biologics plc, Slough, UK Ilse Blumentals Merck & Co., Inc., Rahway, NJ Ineke G. A. Bos Department of Immunopathology, Sanquin Research at CLB, Amsterdam, The Netherlands Nicola Boschetti R & D Virology, ZLB Behring AG, Bern, Switzerland Sheri Bradshaw Schering Plough Research Institute, Union, NJ John B. Briggs Department of Analytical Chemistry, Genentech Inc., South...
From Waterin Oil Adjuvant Formulations Aaron P Miles and Allan Saul
Water-in-oil adjuvants are currently undergoing experimental testing in human vaccine trials (1-10). Two such adjuvants are the squalene-based Montanide ISA 720 and the mineral oil-based Montanide ISA 51 (11). Vaccines containing these adjuvants are intended to provide a lasting depot effect, with vaccine persisting at the injection site for many months (12). Because it is often convenient to formulate these vaccines well ahead of use, and because of the extended residence at the injection...
Characterization of Monopegylated IFN
IFN pegylated at histidine is sensitive to hydrolysis in the presence of either strong acid or neutral hydroxylamine. The latter reagent is highly selective for carboxy-alkylated histidines. In contrast, IFN pegylated at amino groups is resistant to these chemical stresses. Therefore, the extent of depegylation by neutral hydroxylamine provides a quantitative measure of carboxylalkylated histidine residues within a mix of positional isomers. 3.3.1. Sensitivity to Acid Hydrolysis 1. Adjust an...
Heterologous Gene Expression in Yeast
O'Callaghan, and Mick F. Tuite Saccharomyces cerevisiae and Pichia pastoris provide appealing alternatives to bacterial and mammalian protein expression systems for the production of recombinant proteins. The main advantages of using yeast include an extensive toolbox of genetic modification strategies, production of authentic eukaryotic products, and low culture costs when compared with mammalian systems. Target recombinant proteins can be produced intracellularly or...
Filtration
The evaluation of viral clearance by nanofiltration consists of numerous individual studies designed to demonstrate the effectiveness, consistency, and robustness of the manufacturing process step to reduce the level of viable viruses in the feed stream. A relatively large number of studies are performed at laboratory scale (virus challenges) to determine the appropriate filter sizing for a manufacturing scale process and to evaluate the effect of feedstream variability on nanofilter...
Mkkstlalvvmgivasasvqa
The signal sequence is composed of three domains N, H, and C. The N domains of signal sequences are shown in bold, whereas the C domains are underlined. 1. E. coli XL1-Blue, BL21(DE3), HB101, and MC4100. Competent cells can be prepared using the standard calcium chloride method. 2. Recombinant plasmids pUCOb (6), pTrcSOb4 (7), pTrcSObD (7), pTHKCSFmII (8), and pJS101AP (9 see Fig. 2 and Note 1). 3. Chromosomal DNA of E. coli W3110. 4. Luria-Bertani (LB) medium and LB agar plates sterilized by...
Chromatographic Purification
Purification by Ion-Exchange Chromatography 1. Pack 25 mL Q-Sepharose into the glass column ( 5-cm bed height). Equilibrate the column at flow rate of 2 mL min with equilibration buffer. Use the AKTA purifier for ionexchange chromatography. 2. Filter the refolded protein through a 0.45- m filter, and load onto the pre-equilibrated Q-Sepharose ion-exchange column at a flow rate of 2 mL min. Wash the column using three-column volumes of equilibration buffer containing 10 mM NaCl to remove...
NMR Tube Types
NMR tubes come in several forms, but for peptide NMR work, use either a standard NMR tube as shown in Fig. 1A or a microtube as shown in Fig. 1B,C. A 600- L sample volume is required in a standard NMR tube. Standard tubes of choice for 600 MHz NMR are either Norell-509UP or Wilmad 535-PP7 that are washed as described in Subheading 3.3.2. before use (see Note 6). You require 1.2 mg of a 2.0-kDa peptide to create a 600- L NMR sample of 1 mM concentration. If you have a NMR microtube, such as a...
Release of MAb
Add approx 50 J,L 0.15 formic acid aqueous solution into each sample well (see Note 3). 2. Incubate the plate for 5 min to release the antibody from the immobilized protein A resin. 3. Place a 96-well plate inside the manifold system (see Fig. 1). This plate is used to collect the filtrate, which contains the antibody of interest. 4. Apply vacuum, and collect the filtrate (see Note 4). 5. Transfer the solution into HPLC vials (see Note 5). Fig. 2. Mass spectrum (A) and deconvoluted mass...
Opportunities and Challenges John R Birch and Yemi Onakunle
Over the last 20 yr, there has been extraordinary growth in the biopharmaceutical industry based on the development of recombinant DNA and hybridoma technologies in the 1970s. Prior to this, the dependence on extraction from natural sources severely limited the range and quantity of proteins available for clinical use. Recombinant DNA technology made it possible to mass produce a wide range of natural and modified proteins for the first time. In addition, hybridoma technology introduced a new...
Virus Elimination and Validation
Biological products carry a risk of transmitting viruses (or pathogens), which may come from the source material (e.g., cell banks of human or animal origin, human blood, and human or animal tissues) or as adventitious agents introduced by the production process (e.g., the use of animal sera and growth supplements in cell culture or protein stabilizers in formulation). A low-risk level of viral contamination persists in cell culture processes that harness viral replication which also promotes...
Cleaning NMR Tubes
Clean NMR tubes are important because the only NMR signals observed should arise from your sample, not from tube contaminants. Also, such contaminants may render your peptide useless and only fit for waste. Wash all tubes (even new tubes received directly from the supplier) by following this procedure. 1. Soak your tubes in concentrated nitric acid for 1 h. 2. Carefully remove the tubes from the acid, and rinse well with high-purity water. Be rigorous in rinsing the tube, which is best achieved...
Freeze Drying to Remove 1H2O
If an NMR sample is required that has a low 1H2O content and thus a high 2H2O content, exchange the water molecules in your system. This event is particularly useful for obtaining high-resolution DQFCOSY or NOESY NMR data, as well as for ascertaining which labile protons are in solvent exchange. The following procedure will create such a solvent exchanged system. 1. Remove sample from the NMR tube with an extended-tip NMR Pasteur pipet, and place sample in a 1.5-mL microcentrifuge tube. If it...
STAT Translocation Assay
IFN-a receptor-mediated signal transduction for antiviral activity involves the JAK STAT pathway (6). A key step in the pathway is the translocation of the STAT1 homodimeric or STAT1 STAT2 heterodimeric protein complex from the cell cytoplasm to the nucleus. This translocation can be accurately quantitated in near real time using specific fluorescent antibodies and the ArrayScan II (Fig. 1). 1. Plate HuH-7 cells in a 96-well Packard black view plate at a density of 10,000 cells per well in...
Solid State Protein Formulation
Methodologies, Stability, and Excipient Effects Yuh-Fun Maa and Scott P. Sellers An important message was delivered in Chapter 20 by Sellers and Maa the protein is generally more stable in the solid state than in the liquid state. Obviously, this belief is related to protein mobility. Protein movement is restricted in the dry state, substantially prohibiting the surrounding influence on the protein. Then, the notion evolves that escalating the glass transition temperature (Tg) of the dry...
Image J
Image J, made available in the public domain by the US National Institutes of Health (NIH), is a general image analysis program that constantly evolves with new specialized plug-ins (see Note 44), being created by administrators and advanced users. Although Image J does not offer tools for a complete 2D analysis, there are many functions specifically for gel analysis. Fig. 5. Silver-stained 26-cm gel showing separation of approx 2000 proteins from Saccha-romyces cerevisiae. Separation by charge...
Production of Recombinant Therapeutic Proteins by Mammalian Cells in Suspension Culture
Lily Chu, Ilse Blumentals, and Gargi Maheshwari Within the past 10 yr, 17 monoclonal antibodies (MAbs) have been approved in the United States. A survey of these approved MAbs reveals that the predominant platform is a serum-free, stirred-tank mammalian cell culture. In fact, all 17 products are grown in only four cell lines Chinese hamster ovary (CHO) cells, mouse myeloma cells (NS0 and Sp2 0), or hybridoma cells. The trend toward platform consolidation enables the possibility to quicken...
Secretory Production of Therapeutic Proteins in Escherichia coli
Sang Yup Lee, Jong Hyun Choi, and Sang Jun Lee 1. Introduction Escherichia coli has been the workhorse for the production of recombinant proteins (1,2). However, problems often occur in recovering substantial yields of correctly folded proteins. E. coli cannot produce some proteins containing complex disulfide bonds or mammalian proteins that require posttranslational modification for activity. Overexpressed proteins are often produced in the form of inclusion bodies, from which biologically...
Calcium Phosphate Technique
Detailed transfection protocols optimized for improved reproducibility are described for both adherent and suspension cultures of mammalian cells. A turbidity assay is suggested as a quality control measure that allows a quick check on the quality of the precipitate. 3.4.1. Turbidity Assay to Evaluate Precipitates It is recommended to test new batches of CaCl2 and HEPES phosphate solutions. Until recently, the only reliable test was a transfection experiment with results available within 1 d....
Case Study
Teruhisa Nakashima and Kazuhiko Tomokiyo 1. Introduction Human factor VII (FVII) is a glycoprotein with a molecular mass of 50 kDa, is synthesized in the liver as a single-chain precursor of activated FVII (FVIIa), and it circulates in the blood at a plasma concentration of 0.5 g mL. FVIIa is a serine protease, generated by limited proteolysis of zymogen FVII by activated factors Xa (FXa) or IXa (FIXa) in vivo. Upon binding to its receptor, cofactor tissue factor (TF), FVIIa gains full...
Expression of Antibody Fragments by Periplasmic Secretion in Escherichia coli
Popplewell, Mukesh Sehdev, Mariangela Spitali, and A. Neil C. Weir Antibody-based drugs are increasingly used in the clinic, and their importance is set to escalate in the coming years as more drugs in this class progress through clinical trials. Although many such drugs utilize whole antibodies, others exploit fragments, e.g., fragment antigen binding (Fab') or single-chain fragment variable (scFv), which retain the antigen-binding specificity without the fragment crystallizable (Fc)...
Purification of Recombinant Protein
The steps described in Subheadings 3.5.1.-3.5.3. include preparation of the ProBond column (2), preparation of the extract, and purification of recombinant protein. 3.5.1. Preparation of ProBond Columns (see Note 2) 1. Transfer the desired amount of Ni-resin to a centrifuge tube and centrifuge at 1500g for 2 min (see Note 3). Fig. 4. HPV16L1 protein eluted from the Ni-resin column with increasing imidazole elution buffer concentrations as analyzed by 12 SDS-PAGE. Fig. 4. HPV16L1 protein eluted...
Purification of Monopegylated IFN Positional Isomers
The products of the IFN reactions are mixes of positional isomers whose composition changes with reaction pH. The desired monopegylated products can be resolved from dipegylated and unmodified IFN by several methods, including IE and SE chro-matography. The following section describes resolving monopegylated IFN from other products by SE chromatography. Individual monopegylated positional isomers are purified from this mix by IE chromatography (Subheading 3.2.3.). 3.2.1. Purification of...
Characterization of Interferon a2B Pegylated via Carboxyalkylation
Wylie, Marcio Voloch, Seoju Lee, Yan-Hui Liu, Collette Cutler, Brittany Larkin, and Susan Cannon-Carlson The efficacy of several therapeutic cytokines in vivo is often restricted by their clearance rate, which can be retarded by the covalent attachment of polyethylene glycol (PEG) a large, highly soluble, nontoxic adduct (1-5). However, pegylation is a double-edged sword. In addition to retarding clearance, it also frequently impairs the biological activity of the modified protein....
Assays for IFNa Immune Response
Major Histocompatibility Complex (MHC) Class I Expression Assay 1. Molt-4 (ATCC). Molt-4 culture medium see Subheading 2.2., item 2. Consult the ATCC catalog for information on how to maintain Molt-4 cell culture. 2. Recombinant human IFN IFN-a2b and PEG-IFN-a2b. 3. R-Phycoerythrin (R-PE)-conjugated mouse anti-human human leukocyte antigen (HLA)-A, B, C monoclonal antibody (Becton Dickinson). The antibody should be titrated to obtain optimal results, and 20 J,L of the titrated antibody...
By Slot Blot and Scanning Laser Densitometry
Miles, Daming Zhu, and Allan Saul The production of recombinant proteins for therapeutic and vaccine uses necessitates the analysis of purified products for residual host cell antigens. Various assays to quantify these antigens in both in-process streams and purified bulk solutions have been described (1-3). Process-specific assays utilize those host cell antigens most likely to copurify with the protein of interest, whereas generic assays use all recoverable host cell antigens,...
Using Sdspage and Scanning Laser Densitometry
Numerous methods are widely used for the quantitation of proteins and their degradation products. Gel electrophoresis, followed by scanning laser densitometry, offers an accurate, sensitive, and reproducible technique that can be used at any stage during the production and purification of recombinant proteins. This technique offers many advantages over others for two main reasons (1) proteolytic degradation is common among these proteins, and this method is able to accurately quantify...
Pharmaceutical Proteins From Methylotrophic Yeasts
Duitman, Arjo L. de Boer, Marten Veenhuis, Ineke G. A. Bos, and C. Erik Hack Because of their favorable properties, methylotrophic yeasts have become increasingly important as cell factories for the production of biomaterials, therapeutic proteins, and vaccines. As a eukaryote, yeast can perform most of the posttranslational modifications that are required to ensure the functionality and or stability of recombinant human proteins, such as N- and O-linked...
Spray Freeze Drying
SFD is a relatively new method for biopharmaceutical powder preparation. This method combines the atomization and FD processes to present a potential advantage for fine-powder preparation. The principle is to atomize the protein solution into a cryogenic medium, such as liquid nitrogen, to quench freeze the droplets. The frozen droplets are then dried by lyophilization (Fig. 4). This process involves no heat for drying so that heat denaturation associated with the SD process can be avoided....
Using the Pichia pastoris Expression System
McCurdy, and Varsha Bhakta Of the numerous strategies that have been tested to slow the clearance of injected protein drugs from the body and circulation (1), fusion to albumin offers several advantages. Albumin is the most abundant protein in mammalian plasma and one of the longest lived. It lacks posttranslational modifications, with the exception of extensive disulfide bonding (2). If albumin can be fused in-frame with a therapeutic protein as a single-chain...
Stabilization of Therapeutic Proteins by Chemical and Physical Methods
Proteins are complex molecules composed of numerous reactive chemical groups and delicate three-dimensional structures. The chemical (oxidation, deamidation, hydrolysis) and physical (unfolding, aggregation) changes of proteins during the formulation process and storage not only reduce biological activity, but they can cause adverse reactions, such as immune responses, even when the total protein population is at a low level (Table 1) (1-5). Developing new formulations that maintain the...
Retention time min
HPLC elution profile of MAb purification on HA chromatography. The second purification step performed on a hydroxyapatite column at a flow rate of 4 mL min. MAb is eluted with a 10 cv of phosphate gradient (10-400 mM), pH 6.7. 10. For CIP column and storage, wash sequentially with 5 cv sterile, pyrogen-free P10, 5 cv 0.1 M NaOH, and 5 cv sterile, pyrogen-free P10, and store at 4 C. 11. The results of column packing are evaluated by protein analysis of the nonretained peak, i.e., the...
For High Resolution Nuclear Magnetic Resonance Studies of Aqueous and Stabilized Solutions of Therapeutic Peptides
This chapter covers the preparation of a purified nuclear magnetic resonance (NMR) peptide sample for analysis considering purity, pi, oxidation status, and solubility of the sample, along with the choice and preparation of the NMR sample tube. The NMR analysis of therapeutic peptides is an area that is still underdeveloped, but it offers both great promise and utility in drug screening and design (1-3). Ligand-target NMR-screening methods, such as saturation transfer difference NMR (STD-NMR 4)...
Large Scale Transient Expression of Therapeutic Proteins in Mammalian Cells
Sabine Geisse, Martin Jordan, and Florian M. Wurm 1. Introduction Research of the molecular and cellular biology of mammalian cells would be highly restricted if not for the development of methods to deliver exogenous DNA into cultivated cells. Transient transfection of mammalian cells became a routine research tool following a landmark publication by Graham and Van der Eb, who presented the calcium phosphate method as an assay to test the infectivity of purified viral DNA in 1973 (1). For the...
Recovery of Expressed Protein
Extraction of Intracellular Proteins With Glass Beads in Nondenaturing Buffer 1. Cool the culture on ice, and pellet the cells by centrifugation at 3000 rpm for 10 min at 4 C. 2. Wash the cells in 5 mL sterile H2O, centrifuge (3000 rpm at 4 C), discard the supernatant, and place the cell pellet on ice. 3. Resuspend the cell pellet in 500 J,L cold nondenaturing lysis buffer, pH 6.8, and transfer to a 1.5-mL microfuge tube on ice. 4. Add cold acid-washed (Note 17) glass beads ( 0.5-mm...
Gel Electrophoretic Methodology for the Analysis of Yeast Proteins
Byrne, and Mick F. Tuite 1. Introduction Gel electrophoretic analysis of proteins is a well-established method for separating, quantifying, identifying, comparing, and characterizing proteins. This form of analysis consists of a diverse class of methods, with new approaches and improvements being frequently added, including computer software dedicated to the analysis of protein gels. Protein electrophoresis involves four principal steps protein harvesting, in-gel...
Formulation Development of Proteins
Although a major goal of successful formulation development is to provide an adequately stable product, an understanding of the mechanisms of protein degradation is essential. Degradation mechanisms can be divided into two main categories chemical and physical. Chemical is defined as those mechanisms in which existing chemical bonds are broken and or new chemical bonds are formed e.g., hydrolysis, racemiza-tion, and oxidation , and physical is defined as no covalent bonds being broken or formed...
High Throughput Recovery of Therapeutic Proteins from the Inclusion Bodies of Escherichia coli
Escherichia coli is widely used for the expression of recombinant proteins that do not require glycosylation for their bioactivity 1,2 . Many commercially available recombinant hormones and the majority of interleukins and interferons are all expressed and produced in E. coli systems 3 . The advantages of using E. coli as an expression system include the enormous volume of data on its cell biology, its fermentation process development, and its ability to produce relatively large and inexpensive...
From Inclusion Bodies of Escherichia coli Cells
Eshwari, Lalit C. Garg, and Amulya K. Panda 1. Introduction 1.1. Isolation of Human Growth Hormone Inclusion Bodies From E. coli Cells Inclusion bodies produced in Escherichia coli are composed of densely packed denatured protein molecules in the form of particles 1,2 . In addition to the recombinant protein of interest, inclusion bodies contain small amounts of host protein, ribo-somal components, and DNA RNA fragments 3 . It is advisable to purify the inclusion...
Purification of Clinical Grade Monoclonal Antibodies by Chromatographic Methods
Horenstein, Ilaria Durelli, and Fabio Malavasi 1. Introduction Monoclonal antibodies MAbs reagents mass-produced in the laboratory to recognize a specific molecular target 1 are currently the most widely used protein-based therapeutic and diagnostic molecules in clinical trials 2 . This chapter presents the most effective ready-to-use protocol for MAb purification of clinical-grade standard. Samples of high purity are often produced via a highly selective single step. However, an...
Stabilizing Excipients
Protein stability, solubility, and pharmaceutical acceptance low-irritative stimulation at the administration site are the major factors in selecting buffer and salt components. The salts and buffers have significant effects on protein stability, especially in frozen solutions and freeze-dried solids because of the increased concentrations and possible pH changes. For example, the freezing of neutral sodium phosphate buffer and potassium phosphate buffer often results in acidification and...
