Operating Conditions for IEX

Cation exchange chromatography Preequilibration 250 mM sodium phosphate, pH 6.0 Equilibration 25 mM sodium phosphate, pH 6.0 Load to 50 g l Equilibration buffer wash Low salt wash 25 mM phosphate, 50 mM NaCl, pH 6.0 Elution 25 mM phosphate, 200 mM NaCl, pH 6.0 Strip preequilibration buffer Regeneration 0.5 N NaOH Storage 0.1 N NaOH Preequilibration 250 mM Tris, pH 8.0 Low salt wash 25 mM Tris, 50 mMNaCl, pH 8.0 Elution 25 mM Tris, 200 mM NaCl, pH 8.0 6.4.2 Hydrophobic Interaction Chromatography...

Calculation Of Log Reduction Factors In Aviral Clearance Study

The following sections provide examples of virus clearance studies performed for a typical monoclonal antibody purification process and shows how the log reduction values for each process step and the manufacturing process as a whole is estimated. Since the virus titers are normally expressed with 95 confidence intervals, the same should be done when reporting viral clearance for each process step and the production process as a whole. In the example shown in Table 14.4, the log reduction value...

Introduction

Transgenic sources (plants and animals) for biopharmaceutical production offer numerous advantages over bioreactor-based production, and the most important ones are the ease and the associated low cost for large-scale production. It has been estimated that the cost of producing a recombinant drug from transgenic plants is only 10 to 20 of the cost of using fermentation 1 . For example, depending on the scale, the total production cost of monoclonal antibodies (MAbs) via mammalian cell culture...

Insulin

In 1982, Eli Lilly made history by launching the world's first successful product of modern biotechnology for human healthcare recombinant human insulin for treatment of diabetes. In 1969, Lilly filed a patent on a novel crystallization method for pancreatic insulin 33 . This crystallization process has been used for over thirty years to manufacture insulin. This is the 8.2 process, so named because the maximum yield of crystalline insulin occurs at pH 8.2 (Table 5.4). In this process, insulin...

Buffers for IMAC

Immobilized metal affinity chromatography steps are typically operated at close to neutral pH to ensure that the His residues that interact with the metal ions are uncharged and can form coordination linkages. Since the ligands on IMAC resins are negatively charged (such as IDA), buffers for this technique usually include a moderate salt concentration (0.2 to 0.5 M NaCl or equivalent) to prevent nonspecific ionic interactions with any uncharged sites. Common buffers employed for IMAC include...

Example of Operating Conditions for an IMAC Process Step

Charging 100 mM acetate, 100 mM zinc sulfate, pH 4.0 Flush 100 mM acetate, pH 4.0 Preequilibration 25 mM sodium phosphate, 200 mM NaCl, 50 mM imidazole, pH 7.0 Equilibration 25 mM sodium phosphate, 200 mM NaCl, 2 mM imidazole, pH 7.0 Load to 50 g l Equilibration buffer wash Imidazole wash 25 mM sodium phosphate, 200 mM NaCl, 5 mM imidazole, pH 7.0 Elution 25 mM sodium phosphate, 200 mM NaCl, 20 mM imidazole, pH 7.0 Strip preequilibration buffer Regeneration 0.1 M EDTA Storage 0.1 N NaOH

Contributors

West Greenwich, Rhode Island Glen Bolton Department of Chemistry and Chemical Biology Rensselaer Polytechnic Institute Troy, New York Regulatory Affairs Chiron Corporation Emeryville, California Department of Chemical and Biological Engineering Rensselaer Polytechnic Institute Troy, New York Development Merck Research Laboratories Merck amp Co., Inc. West Point, Pennsylvania Institute for Technical Chemistry Forschungszentrum Karlsruhe Eggenstein-Leopoldshafen, Germany Department of Chemical...

Column Sanitization And Reuse Of Chromatography Resins

While the nanofiltration step is done with disposable filters and are thus used only once, the same cannot be said of the chromatography resins. Chromatography resins such as recombinant protein A affinity resins are prohibitively expensive to be used only once in a manufacturing process. Moreover, if a resin is used only once, all the process-scale chromatography columns will need to be packed and tested for each lot processed, which would make the manufacturing process very inefficient. Thus,...

Design And Setup Of Magnetic Separator

Due to the current absence of a market, no commercially available magnetic separator systems suitable for industrial downstream processing currently exist. That said, the physical principles, wide variety of available designs from parallel large-scale industries, and inherent advantages of magnetic separation techniques per se represent a sound basis for the imminent advancement of bespoke magnetic separation methods for industrial downstream processing. Such designs will necessarily feature...

Viral Clearance Using Membrane Chromatography

The potential for contamination of therapeutic proteins produced in cell culture by viruses is a regulatory concern. Steps are included in downstream processing specifically to meet regulatory requirements redundant and complementary unit operations are included that clear any potential viral contaminant from the protein product. For viral clearance applications, performance is measured by the log reduction value LRV , which is simply LRV Log10 C . Typical LRV values for anion exchange column...

Ion Exchange Chromatography

Ion exchange IEX unit operations are believed to remove viruses from in-process intermediates by ionic binding.9 Experience from the gene therapy and vaccine fields has shown that viruses can be partitioned from protein contaminants based on charge difference. Thus, it can be surmised that a flow through anion exchange unit operation conducted in neutral, low conductivity buffers removes negatively charged viruses from positively charged mAbs by binding them with high avidity while the mAb...

Choice Of Viruses In The Viral Clearance Studies

The viruses that are used in the clearance studies fall primarily into three categories, relevant viruses, specific model and nonspecific model viruses. Relevant viruses are viruses that are either a the identified viruses, or b of the same species as the viruses that are known, or likely to contaminate the cell substrate or any other reagents or materials used in the production process. A specific model virus is a closely related to the known or suspected virus same genus or family , and b...

Virus Validation Studies

The purposes of the virus validation studies are to confirm the LRV claims for the filtration step and to verify the filter sizing established in the scale-up phase. These tests are run at a small scale, maintaining critical parameters such as pressure, flux, and loading capacity at their commercial operation values while mimicking other operating procedures such as preprocessing WFI flush-outs and buffer equilibration. The tests are typically run concurrent with the manufacturing-scale...

Critical vs Noncritical Operating Parameters

A clear understanding of critical operating parameters is required for design of a successful viral safety strategy. Operational parameters are process inputs that are directly controlled. Typically, these parameters are physical or chemical e.g., temperature, process time, column flow rate, column wash volume, reagent concentration, or buffer pH . Performance parameters are process outputs that may be monitored to ensure or confirm acceptable process performance in the context of this...

References

Modern antibody based therapeutics. Biopharm 2004 Dec. 18-25. 2. Cochlovius B, Braunagel M, and Welschof M. Therapeutics antibodies. Mod. Drug Discov. 2003 6 33-38. 3. Stockwin LH and Holmes S. Antibodies as therapeutic agents Vive la renaissance Expert Opin. Biol. Ther. 2003 3 1133-1152. 4. Djik MA van, Winkel J, and GJ van de. Human antibodies as next generation therapeutics. Curr. Opin. Chem. Biol. 2001 5 368-374. 5. Lo BKC. Review Antibody humanization by CDR grafting. Meth....

Purification Of Monoclonal Antibodies And FcFusion Proteins

Till date, monoclonal antibodies have been typically produced by mammalian cell culture to ensure proper folding and glycosylation. Efficient recovery and purification of antibodies from cell culture media is a critical part of minimizing manufacturing costs 10 . Figure 16.2 shows the typical breakdown of costs associated with an antibody production process 11 . As can be seen from Figure 16.2, a significant percentage 30 to 40 of the total manufacturing cost of therapeutic antibodies is...

Cytotoxicity And Viral Interference Testing

Prior to performing virus-spiking studies, it is essential to perform cytotoxicity and virus interference studies on the process intermediates. This is a regulatory requirement because samples generated during an actual spiking study may cause significant problems in the titration of the virus thereby obtaining an accurate estimation of the virus present in the sample. These problems may arise from the cytotoxicity of the samples. Cytotoxicity assays are performed to demonstrate whether process...

Common Impurities of rDNADerived Protein Pharmaceuticals

Host cell proteins Western Blots, Immunoassays Other protein impurities media SDS-PAGEb, HPLCc, Immunoassays DNA DNA hybridization, Total DNA byThreshold, qPCRd Protein mutants Peptide mapping Aggregates SECe, Light scattering, Oxidized methionines Amino acid analysis, Peptide mapping, Edman degradation Proteolytic clips IEFf, SDS-PAGE reduced , HPLC, Edman degradation Deamidation IEF standard comparison , HPLC Monoclonal antibodies SDS-PAGE, immunoassays Amino acid substitutions Amino acid...

Infliximab Manufacturing Process Overview

The infliximab drug substance is manufactured by continuous perfusion cell culture. The expansion of the antibody secreting cells and production of the chimeric monoclonal antibody occur in the first two manufacturing stages preculture and expansion Stage 1 and large-scale cell culture production by continuous perfusion Stage 2 . REMICADE is purified and formulated to pre-formulated bulk PFB from cell supernatant harvest in Stages 3 through 9 of the manufacturing process as shown in Figure...

Recent Developments In Vector Purification

Plasmid Flow Chart

Adenoviral, AAV, and retroviral vectors are produced in mammalian cells. One way to release Ad and AAV vectors from cell pellets is by applying multiple freeze-thaw cycles. Retroviruses such as LV on the other hand are released into the supernatant. The viral vectors may be separated from the cellular debris by either centrifugation followed by filtration or by using a series of filters with decreasing porosity. A classical method of Ad vector purification has involved cesium chloride CsCl...

Process Changes And Product Comparability For Commercial Manufacturing

Process validation of the first commercial REMICADE manufacturing process in Leiden, The Netherlands was completed with the successful execution of five consistency batches. These were used to demonstrate reliability of the process and comparability of the product to that used in clinical trials. The results of process validation were used to support the initial licensure of REM-ICADE around the world. Not long after process validation was complete, post-approval process changes were pursued to...

Table 174

Process Chromatography Media for Polishing Monoclonal IgG UNOsphere Q Fractogel EMD TMAE Q Sepharose Fast Flow Q Sepharose XL Mustang Q Toyopearl Super Q-650 Cation exchange UNOsphere S Fractogel EMD SO3 S Sepharose Fast Flow S Sepharose XL Toyopearl S-650 Bio-Rad E. Merck GE Healthcare GE Healthcare High capacity High capacity Low capacity High capacity Pall Corporation Flow-through Tosoh Bioscience High capacity E. Merck E. Merck GE Healthcare GE Healthcare Tosoh Bioscience Tosoh Bioscience...

Qspr As A Bioprocess Development Tool

The above example demonstrates the utility of the QSPR modeling approach for the a priori prediction of chromatographic separations of proteins. The practical application of this technique in a typical downstream bioprocessing setup would involve the generation of models to predict the chromatographic behavior of the product of interest and the key impurities in a given biological mixture. Using these models, computational experiments may then be carried out by varying different operational...

Tangential Flow Filtration

Tangential Flow Filtration Principle

Tangential filtration, like NFF, is also a pressure-driven separation process. Fluid flows across membrane surface and only a small fraction of solvent and permeable materials penetrate through the membrane. The fluid circulation minimizes the formation of the filtered solids on the membrane, consequently maintaining flux without increasing pressure. The comparison of TFF vs. NFF is illustrated in Figure 1.10. TFF can be further divided into microfiltration MF and UF according to the pore size...

Ad Capture By Anion Exchange Membrane Chromatography

A rapid, simple, and scalable process was developed in our laboratory with a minimum number of sample handling steps for chromatographic capture FIGURE 20.3 Flow diagram for capture of Ad from lysate. FIGURE 20.3 Flow diagram for capture of Ad from lysate. of intact, infectious Ad viral particles using Mustang Q membranes Figure 20.3 . To measure the dynamic binding capacity of anion exchange membranes for Ad, CsCl gradient-purified Ad 1.24 x 1012 virus particles total was loaded at various...