Analytical potential of protein A for affinity chromatography of polyclonal and monoclonal antibodies

1989 ◽  
Vol 61 (13) ◽  
pp. 1314-1317 ◽  
Author(s):  
Bruce Jon. Compton ◽  
MaryAnn. Lewis ◽  
Frances. Whigham ◽  
Jennifer Shores. Gerald ◽  
George E. Countryman
Keyword(s):  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Analytical Potential

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
High Yield ◽  
Staphylococcal Protein A ◽  
Purification Step ◽  
Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

scholarly journals Attempts to obtain anti(D2 dopamine receptor) antibodies via the anti-idiotypic route

1986 ◽  
Vol 238 (3) ◽  
pp. 817-823 ◽  
Author(s):  
W M Abbott ◽  
P G Strange
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Dopamine Receptors ◽  
Dopamine Receptor ◽  
Protein A ◽  
D2 Dopamine Receptor ◽  
D2 Dopamine Receptors ◽  
Selective Ligands ◽  
Receptor Antibodies

Five stable hybridomas have been obtained that secrete monoclonal antibodies against the D2-dopamine receptor-selective drug spiperone. Each monoclonal antibody has been characterized in terms of its ability to bind a range of dopamine-receptor-selective ligands. One monoclonal antibody has been purified by Protein A affinity chromatography and used to immunize mice. Anti-idiotypic antisera and one hybridoma secreting an anti-idiotypic monoclonal antibody were obtained and shown to inhibit [3H]spiperone binding to the anti-spiperone antibody used for immunization. Neither the antisera nor the anti-idiotypic monoclonal antibody, however, inhibited binding of [3H]spiperone to D2-dopamine receptors.

scholarly journals Washing with alkaline solutions in protein A purification improves physicochemical properties of monoclonal antibodies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuichi Imura ◽  
Toshiaki Tagawa ◽  
Yuya Miyamoto ◽  
Satoshi Nonoyama ◽  
Hiroshi Sumichika ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Physicochemical Properties ◽  
Affinity Chromatography ◽  
Protein A ◽  
Physicochemical Characteristics ◽  
Alkaline Solutions ◽  
Host Cell Proteins ◽  
Irreversible Aggregation ◽  
And Storage ◽  
Purification Protocol

AbstractProtein A affinity chromatography has been widely used for both laboratory scale purification and commercial manufacturing of monoclonal antibodies and Fc-fusion proteins. Protein A purification is specific and efficient. However, there still remain several issues to be addressed, such as incomplete clearance of impurities including host cell proteins, DNA, aggregates, etc. In addition, the effects of wash buffers in protein A purification on the physicochemical characteristics of antibodies have yet to be fully understood. Here we found a new purification protocol for monoclonal antibodies that can improve physicochemical properties of monoclonal antibodies simply by inserting an additional wash step with a basic buffer after the capture step to the conventional protein A purification. The effects of the alkaline wash on monoclonal antibodies were investigated in terms of physicochemical characteristics, yields, and impurity clearance. The simple insertion of an alkaline wash step resulted in protection of antibodies from irreversible aggregation, reduction in free thiols and impurities, an improvement in colloidal and storage stability, and enhanced yields. This new procedure is widely applicable to protein A affinity chromatography of monoclonal antibodies.

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