TY - JOUR
T1 - Selective high-performance liquid chromatographic purification of bispecific monoclonal antibodies
AU - Tarditi, Laura
AU - Camagna, Maria
AU - Parisi, Anna
AU - Vassarotto, Cristina
AU - DeMonte, Lucia B.
AU - Letarte, Michelle
AU - Malavasi, Fabio
AU - Mariani, Massimo
PY - 1992/5/22
Y1 - 1992/5/22
N2 - The recent development of improved production techniques for bispecific monoclonal antibodies (biMAbs) has significantly increased interest in specific purification procedures. In this investigation, a general high-performance liquid chromatographic (HPLC) purification method is proposed that allows highly purified biMAbs to be obtained from mouse ascites fluid containing a mixture of different antibodies, i.e., parental MAbs, active biMAb and a mixture of randomly assembled heavy and light chains. Proteins from ascites fluid were precipitated with ammonium sulphate and applied to a high-performance protein A column to separate the total immunoglobulin fraction. BiMAbs were isolated from other immunoglobulins by two subsequent passages through a high-performance hydroxyapatite (HPHT) column. This purification protocol combines specificity of protein A for immunoglobulin G (IgG) and high selectivity of hydroxyapatite for different IgG idiotypes. All purification steps were performed rapidly and reliably by HPLC. This method was applied to the purification of six different biMAbs with consistently high yields, purity and homogeneity. This general purification method may prove extremely valuable when highly pure preparation of biMAbs is required, as for in vivo use.
AB - The recent development of improved production techniques for bispecific monoclonal antibodies (biMAbs) has significantly increased interest in specific purification procedures. In this investigation, a general high-performance liquid chromatographic (HPLC) purification method is proposed that allows highly purified biMAbs to be obtained from mouse ascites fluid containing a mixture of different antibodies, i.e., parental MAbs, active biMAb and a mixture of randomly assembled heavy and light chains. Proteins from ascites fluid were precipitated with ammonium sulphate and applied to a high-performance protein A column to separate the total immunoglobulin fraction. BiMAbs were isolated from other immunoglobulins by two subsequent passages through a high-performance hydroxyapatite (HPHT) column. This purification protocol combines specificity of protein A for immunoglobulin G (IgG) and high selectivity of hydroxyapatite for different IgG idiotypes. All purification steps were performed rapidly and reliably by HPLC. This method was applied to the purification of six different biMAbs with consistently high yields, purity and homogeneity. This general purification method may prove extremely valuable when highly pure preparation of biMAbs is required, as for in vivo use.
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U2 - 10.1016/0021-9673(92)85453-Z
DO - 10.1016/0021-9673(92)85453-Z
M3 - Article
C2 - 1618986
AN - SCOPUS:0026572365
VL - 599
SP - 13
EP - 20
JO - Journal of Chromatography A
JF - Journal of Chromatography A
SN - 0021-9673
IS - 1-2
ER -