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...

Info

Then this calculated value of Tsys is used along with Equation 9.5 to fit the data by using x as the only fitted parameter value in Excel. Using this method, Tsys 10.3 and x 0.638. This result is also plotted in Figure 9.3, and is nearly identical to the first method. Frequently, rather than reporting liquid volumes directly, the volumes are normalized by dividing by the membrane volume. This makes the results dimen-sionless and independent of scale. The volumes are then referred to in terms of...

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

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...

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...

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...

Purification Optimization Strategies For Viral Vectors

Virus Purification Plants Flowchart

Optimization of the primary capture step from clarified cell culture lysate or supernatant should be performed on a scaled-down unit that is suitable for scouting the effect of loading pH, sodium chloride concentration, or conductivity in the load, wash and elution on product purity as well as yield. As the flow rate, in principle, has little effect on the dynamic binding capacity, a reasonable starting point is 10 to 20 MV min. One of the first parameters to optimize is the loading pH at which...

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...

Recent Developments In Vector Purification

Diafiltration Schematic

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...

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...