Despite the existence of effective vaccines against Hepatitis B virus, the infection with it remains an important problem worldwide due to its association with hepatocellular carcinoma. Several procedures have been used to purify the Hepatitis B surface antigen (HBsAg) for immunization purposes. Immuno-purification using HBsAg-specific murine monoclonal antibodies (MAbs) has been one of the most successful strategies for such a purpose due to the high antigen selectivity (high affinity) of MAbs…
Tag: <span>purification</span>
In order to unravel new protein activities and functions, we have expressed and purified a large number of human proteins. We have chosen to study secreted proteins and the extra-cellular domains of putative single transmembrane domain-containing proteins. In order to retain the natural protein characteristics as far as possible, we have used a mammalian expression system. Human embryonic kidney (HEK293) cells were chosen as they have been shown to possess a high protein-secretory potential. The secreted proteins were expressed with a carboxy-terminal tag and purified by affinity chromatography. Each protein was produced at a routine scale from 500 ml cell cultures, and the secreted protein was purified from the culture supernatant…
The aim in process filtrations is to purify the liquid preparation by removing particulate impurities while obtaining as large a throughput as possible under practical conditions. The rate of flow should be expeditious enough to meet time constraints when necessary. This places a focus on the applied differential pressure level that motivates the liquid flow. A balance must be sought. Higher differential pressures increase the flow rates but may decrease throughputs by compaction of the filter cake. Also, higher applied pressures may minimize the adsorptive retention of particles. Deciding which is the proper filter involves small-scale filtration trials. The choice of the filter having been made, its size, in terms of the area necessary for the processing of the production batch, is arrived at by extrapolations from the small-scale tests that were performed…
The diversity of the antibody-antigen interaction and our ability to manipulate this interaction has created an enormous potential for the discovery and development of IgG therapeutics and diagnostics. Along with the expanding clinical pipeline of antibody products, increasing efforts have been devoted to improving antibody production and purification procedures. In order to meet drug discovery needs with limited resources, the so called “flexible generic purification scheme” approach has been adopted to develop a robust manufacturing process that allows the application of similar operational conditions to different monoclonal antibody molecules…
Optimal process development creates unit operations that effectively generate, separate, and concentrate a broad array of products. Historically, tangential flow filtration (TFF) process capabilities have been limited by technological and flow restrictions. Recent innovations in TFF module design have dramatically increased the capabilities of TFF to better achieve processing objectives. NCSRT has established a best practices protocol for developing clarification, fractionation, and concentration processes for mammalian, bacteria, yeast, insect, and virus based production systems. This article presents the development platform, supplemented with application-specific expertise…
Poliovirus is a small (28-30 nm diameter) non-enveloped RNA virus belonging to the family Picornaviridae. The ability of poliovirus to cross the blood-brain barrier and its natural infectivity of central nervous system (CNS) tissue via the CD155 receptor, found exclusively in primates, has promoted the investigation of an attenuated poliovirus for the treatment of malignant gliomas. However, use of the virus in clinical testing is limited due to low yields obtained from conventional purification methodologies…
Short Monolithic Columns — An Enabling Technology for the Purification of Proteins, DNA, and Viruses
The last 30 years have seen rapid and dramatic developments in recombinant DNA technology and the related biological sciences. In 1972, Paul Berg’s group used restriction enzymes to cut DNA in half and then used ligases to stick the pieces of the DNA back together. By doing this, they produced the first recombinant DNA. Within a year, the first genetically engineered bacterium existed. A little more than ten years later, recombinant human insulin was approved for diabetic patients and became the first recombinant healthcare product. Before the end of the 1980s, the first gene therapy trial had occurred. Today, a large number of recombinant proteins are used as marketed drugs and even more are in clinical trials targeting a wide range of diseases…