Development Of An Early Clinical Phase Process

Development of a robust, consistent and optimized TFF step may be achieved by following a systematic process development methodology. The rigor and Optimize operating Membrane device selection Parameters flux, Q, C Process robustness Cleaning validation Processing plan FIGURE 10.15 Process development timeline. detail required in the method is influenced by (1) phase of drug development, (2) volume of product available, (3) relative novelty of the product, (4) development timelines. Figure...

Experiment Protocol for Developing an Optimized ATPE System for Protein Recovery from Transgenic Tobacco

Although it has been reported that alfalfa leaves may be stored twelve weeks after drying before processing without experiencing significant target protein degradation 38 , for large-scale recombinant protein production from a leafy crop, fresh leaves need to be processed. A common flow of unit operations for protein recovery using ATPE is illustrated in Figure 12.3. The freshly cut leaves first need to be rinsed with deionized water and blown dry. The dried leaves are then cut to smaller...

Protein Descriptor Generation

Several different types of descriptors have been employed in the example presented in the following discussion. These include traditional 2D and 3D descriptors obtained using the commercially available molecular operating environment (MOE, CCG, Inc., Montreal, Canada) software package, as well as some modern electron density-derived descriptors such as those from transferable atom equivalent (TAE) calculations. Details of these descriptor types are provided in the following sections. The MOE...

Multivirusspiking Approach For Virus Clearance Studies

Virus clearance studies are typically performed by spiking one model virus at a time into the load of an unit operation step and then evaluating the clearance of the virus across this step. However, the specificity of the Q-PCR method, that is the ability to quantify multiple types of target sequences associated with different viruses in a single sample without cross interference, opens up the possibility of spiking multiple viruses in the load sample of an unit operation step. This...

Appendix Use Ofthe Poisson Distribution To Determine Virustiters

An understanding of the use of Poisson distribution is useful when trying to design viral clearance studies for steps that usually clear viruses to the limit of detection, such as a low pH inactivation step and a nanofiltration step. When a sample contains a very low concentration of virus, there is a discrete possibility that if only a small fraction of the sample is tested for virus, that fraction will test negative due to the random distribution of the virus in the total sample. The...

Emea Chromatography 04 May 2012

Process Validation Studies Supporting the New REMICADE Production Facility Stage 2 Production by continuous perfusion Stage 3 Direct product capture by protein A chromatography and ultrafiltration Stage 7 Primary anion exchange chromatography Viability at thaw and accumulated generations must be comparable to the validated process during cell expansion Accumulated generations must be comparable to the validated process in the seed bioreactor Number of generations, peak-specific productivity,...

Protein A Leaching

Protein A leaching is yet another problem associated with the use of this mode of chromatography. This is a serious concern for the drug industry because Protein A is known to cause immunogenic responses in humans and has been proven toxic in a number of clinical trials 68 . One should pay special attention during development of the polishing steps to reduce leached Protein A to safe and acceptable levels. Leaching can occur by three different pathways breakdown of the support matrix, breakdown...

Protein A Affinity Chromatography

Staphylococcal Protein A, or SpA, is a type I membrane protein from the bacterium Staphylococcus aureus. SpA has high specificity for the Fc region of antibodies which has led to its widespread use as a powerful affinity ligand for several immunological and purification applications. Protein A is a 42 kDa protein consisting of a single polypeptide chain. The chain is made up of five homologous IgG binding domains followed by a C-terminal region necessary for cell wall attachment 15 . The...

Chromatographie Multicycling

FIGURE 3.3 The pilot-scale HGMF protein purification system (filtration volume 1.2 l) operated at the University of Stuttgart's Institute for Bioprocess Engineering. FIGURE 3.3 The pilot-scale HGMF protein purification system (filtration volume 1.2 l) operated at the University of Stuttgart's Institute for Bioprocess Engineering. the separation matrix and added back into the mixing vessel (G1) together with a fresh batch of crude bioprocess feedstock (G4, Figure 3.3) to begin a new cycle. All...

Exchange Membrane Chromatography

For the purification of LV vectors, Marino and coworkers 70 reported a precon-centration step that involved PEG precipitation of LV vector particles before capture on a small Mustang Q strong anion exchange membrane chromato-graphy unit. Slepushkin and coworkers 40 showed that a VSV-G pseudotyped HIV-1 based lentiviral vectors could be directly captured and eluted from clarified cell culture supernatants on a 60 ml membrane bed volume Mustang Q strong anion exchange membrane chromatography...

Aav Purification By Membrane Ion Exchange Chromatography

Davidoff and coworkers 68 reported the first chromatographic purification method for AAV-8 vector particles based on membrane ion exchange chroma-tography involving Mustang Q units that generated vector stocks with > 90 purity. The average yield of purified AAV-8 from five different vector preparations was 41 with an average dynamic binding capacity of approximately 113 VP ml of membrane. Electron microscopy of these purified stocks revealed typical icosohedral virions with < 10 empty...

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

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....

Hiac

Not suitable for crude samples Labor intensive For monoclonal antibodies For microbial expression Dipstick technology attractive Host cell proteins, Protein A Supplement to ELISA for HCP High throughput, accurate, host cell-DNA specific Not host cell-DNA specific Chromatographic leachables Simple method for multimers and clips High resolution and quantitative aggregates clipped forms detected under denaturing conditions High resolution MW profiling Hydrodynamic properties in formulation buffer...

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...

Functional Groups for Ion Exchange

Diethyl aminoethyl DEAE 9-9.5 Quaternary aminoethyl Q gt 9.5 Trimethyl aminoethyl TMAE gt 13 Table 6.2 lists some of the more established cation exchange CEX and anion exchange AEX resins, their manufacturers, the functional group, particle-size ranges, and information about the base matrix, were available. Cross-linked agarose based IEX resins have historically been amongst the first IEX resins to be marketed commercially by Pharmacia LKB now part of GE Healthcare . These are available in a...

Buffers for Ion Exchange Chromatography

Common buffers used for CEX chromatography include citrate, phosphate, acetate, and MES all of which buffer between pH 5 to 7 which is commonly used for CEX, phosphate can be used above pH 7 as well . Common buffers for anion exchange chromatography include HEPES, Tris, and borate. It is advisable to use a buffering species that does not bind to the IEX resin being employed. For this reason, the use of Tris buffers is avoided on cation exchangers even if they are being operated at pH 7 to 8 in...

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

Pod Viral Filtration Skid

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...

Sanitization Sterilization

In a typical downstream purification process, virus clearance filters are employed downstream of a chromatography column and upstream of an ultrafiltration diafiltration step. Neither of these steps is considered an aseptic unit operation. However, there appears to be an industry trend to sterilize the virus filter to reduce the bioburden, if not to render it aseptic. Some end users have adopted chemical sanitization as a means of reducing bioburden. Some of the virus filters are available...