BACKGROUND: CAMPATH-1 Abs have been used for T-cell depletion in stem-cell transplantation since the early 1980s. During that time there has been substantial progress in manufacturing techniques and quality control procedures. This article summarizes the methods used to produce the Abs for clinical use and describes results of quality control tests on representative batches. METHODS: Rat hybridoma and recombinant CHO cells were cultured in hollow-fiber fermentors. Antibodies were purified from the culture supernatant by fractionation with ammonium sulphate, or by column chromatography. Additional steps were added to assure the removal of DNA and viruses. A range of analytical methods was used to characterize the antibodies. Samples were stored frozen at -70 degrees C and re-analyzed many years later to assess the long-term stability. RESULTS: Hollow-fiber fermentors provided a simple and reliable means for antibody production, with yields between 3-10 mg/h and a convenient concentration for further processing (0.6-2.0 mg/mL). All of the CAMPATH-1 Abs (rat IgM, rat IgG2b and human IgG1) could be purified by affinity chromatography on Protein A, but the low pH required for elution caused unacceptable aggregation of the IgM. CAMPATH-1H contained approx. 20% dimeric IgG, which could be removed by size exclusion chromatography. Antibodies were stable for at least 6 years at -70 degrees C, but there was unacceptable aggregation of CAMPATH-1M in one batch stored for 9 years. DISCUSSION: Pilot-scale production of MAbs for clinical studies is feasible in a small academic center, but regulatory requirements now demand that great attention is paid to all aspects of manufacturing and quality assurance. Although the underlying principles of cell culture and protein chemistry remain the same, the level of documentation, validation and quality control has increased greatly over the last 20 years.
BACKGROUND: CAMPATH-1 Abs have been used for T-cell depletion in stem-cell transplantation since the early 1980s. During that time there has been substantial progress in manufacturing techniques and quality control procedures. This article summarizes the methods used to produce the Abs for clinical use and describes results of quality control tests on representative batches. METHODS:Rat hybridoma and recombinant CHO cells were cultured in hollow-fiber fermentors. Antibodies were purified from the culture supernatant by fractionation with ammonium sulphate, or by column chromatography. Additional steps were added to assure the removal of DNA and viruses. A range of analytical methods was used to characterize the antibodies. Samples were stored frozen at -70 degrees C and re-analyzed many years later to assess the long-term stability. RESULTS: Hollow-fiber fermentors provided a simple and reliable means for antibody production, with yields between 3-10 mg/h and a convenient concentration for further processing (0.6-2.0 mg/mL). All of the CAMPATH-1 Abs (rat IgM, rat IgG2b and human IgG1) could be purified by affinity chromatography on Protein A, but the low pH required for elution caused unacceptable aggregation of the IgM. CAMPATH-1H contained approx. 20% dimeric IgG, which could be removed by size exclusion chromatography. Antibodies were stable for at least 6 years at -70 degrees C, but there was unacceptable aggregation of CAMPATH-1M in one batch stored for 9 years. DISCUSSION: Pilot-scale production of MAbs for clinical studies is feasible in a small academic center, but regulatory requirements now demand that great attention is paid to all aspects of manufacturing and quality assurance. Although the underlying principles of cell culture and protein chemistry remain the same, the level of documentation, validation and quality control has increased greatly over the last 20 years.