Cellastim S – Optimizing Antibody Production in Animal-Free Media with the Supplementation of Recombinant Human Albumin
Published on 5 September 2020
Recombinant Human Serum Albumin Expressed in Plants Improves the Productivity and Growth Kinetics of CHO
Abstract
Due to the increased risk of transmitting infectious agents, antibody production is moving away from the use of animal-derived components in bioproduction. The switch to animal-free cell culture media often results in less performance which is evident in longer doubling time, lower peak cell density and reduced antibody titer. We evaluated recombinant human albumin (rAlbumin, Cellastim S), produced in a non-animal plant host, for its effect in hybridoma cell culture. Cellastim S outperformed plasma-derived HSA (pHSA) in promoting cell growth and antibody titer. The use of Cellastim S resulted in an average 140% increase in cell growth and 92% increase in volumetric antibody productivity across several media formulations. Cellastim S improved the growth of cells in long term culture. In addition, Cellastim S improved the effect of serum withdrawal and eased the transition to chemically defined media.
A recombinant human serum albumin (rHSA) derived from a plant-based expression system was evaluated for its ability to improve the growth kinetics and productivity of CHO cells in serum-free production media. A CHO K1 line expressing a humanized monoclonal antibody was adapted to 6 different commercial serum-free CHO media. rHSA and plasma derived HSA (pHSA) was compared at 1g/L concentration. rHSA outperformed pHSA and resulted in an average 50% increase in the Integrated Viable Cell numbers (IVCN) and volumetric productivity across media formulations. rHSA improved productivity up to 75% in the two chemically-defined (CD) media examined. Dose response studies indicated that increasing rHSA concentration increased productivity and that rHSA outperformed both BSA and pHSA.
CHO passaged in a CD medium with rHSA for 46 days showed stable growth kinetics and had more population doublings. Similar increases in productivity and IVCN were seen after extended passage. The data indicate that rHSA derived from a plant-based expression system is a robust supplement for CHO culture and can improve both the growth kinetics and productivity of CHO.
Introduction and Experimental Approach
Human Serum Albumin (HSA) is a media supplement that improves the growth and productivity of cells in serum-free culture. Albumin has several activities that make it desirable in cell culture: lipid binding, waste and toxic contaminant removal, antioxidant activities, metal carrying, and membrane stabilization. However beneficial, plasma-derived HSA (pHSA) and bovine serum albumin (BSA) are undesirable in bioproduction due to the increased risk of transmitting adventitious infectious agents. Albumin has been reduced or eliminated from the majority of modern production media formulations due to these regulatory concerns.
We evaluated a recombinant albumin produced via expression in plants (Cellastim™ – InVitria) for its ability to enhance the growth and productivity of a model CHO cell line in commercially available CHO production media.
The parameters we evaluated were initial growth, the Integrated Viable Cell Number (IVCN), and volumetric productivity. rHSA was examined in a series of four experiments. First, as a broad test of applicability, we compared the effectiveness of rHSA to pHSA in a variety of commercial serum-free, protein-free and chemically defined (CD) media. Second, we performed a dose response to confirm the effectiveness and applicability of rHSA in two different CD media. Third, we compared the growth kinetics of CHO during extended passage (46 days) in a CD media supplemented with either pHSA or rHSA. Fourth, we determined whether increases in IVCN and productivity were maintained after extended passage. Cellastim increased the growth, IVCN, and productivity in a variety of serum-free formulations better than pHSA. Volumetric productivity increased up to 75% in two CD media. CHO cells grown in either rHSA or pHSA supplemented CD media showed stable growth during extended passage. However, CHO cells grown in rHSA-supplemented media produced more cell doublings. Finally, similar increases in productivity (75%) and IVCN were found after extended passage in an rHSA-supplemented CD medium. These data indicate that plant-derived rHSA albumin has a unique performance profile that typically outperforms pHSA and BSA.
Material and Methods
HSA, cells, media, and adaptation. pHSA was obtained from Seracare or Baxter. BSA fraction V (Probumin) was obtained from Celliance/Millipore. rHSA was Cellastim™ rHSA from InVitria. Adherent dhfr- CHO line DP-12 clone 1934 producing a humanized monoclonal antibody was adapted to shake culture in 6 different commercial serum- free formulations supplemented with 0.5% dFBS as shown in Table 1. During the adaptation methotrexate (MTX) was increased, stepwise, from 200nM to 5 μM. Individual cell subclones were not selected during the adaptation procedure in avoid skewing results to a particular cell growth characteristic.
Cells were maintained in media, 0.5% dFBS, MTX 5 μM in 125 ml shake flasks. Growth curves were performed in duplicate 4 ml shake-batch cultures with washed cells seeded at 1.0 x105 viable cells/ml and subsequently cultured for 14 days.
Viable cell determination, IVCN, and volumetric productivity. The viable cell concentration was determined daily by a Guava PCA cell counter. IVCN was calculated as the sum of viable cell counts over the 14 day culture. The concentration of antibody produced was determined by quantitative ELISA (Bethyl).
Activity of rHSA in a Variety of Serum-free Media
The effect of rHSA supplementation was determined in a variety of serum-free formulations. Six media were chosen to represent a diverse collection of popular CHO production media. The media chosen include serum-free, protein-free, and chemically-defined formulations. In addition, two of the media contain hydrolysates in their formulations.
The model CHO cell line used in this study was adapted to each medium for 3 months as described. Following adaptation, we compared the effect of supplementing each medium with plasma derived HSA (pHSA) or recombinant HSA (rHSA) on cell growth and productivity. Cells were seeded with 1.0 x 105 cells/ml in 14-day shake-batch cultures. pHSA and rHSA were tested at 1 g/L concentration. The titer of viable cells was determined daily. At the end of the 14day culture, the IVCN (Integral of Viable Cell Number) and antibody concentration was determined. IVCN is the integrated area under the growth curve and is considered a measure of the total mass of cells generated in a culture system.
CHO cells were adapted to 6 (A-F) different serum-free commercial formulations. The formulations include serum-free (A), protein free (B,C,E) and chemically defined (D, F) media. Media B and E contain hydrolysates. Adaptation was performed in each media supplemented with 0.5% FBS (without HSA).
Dose response of rHSA chemically-defined media
The activity of BSA, pHSA, and rHSA at different concentrations in chemically-defined media was determined. The dose response of 2 different pHSAs, BSA and 2 lots of rHSA were compared at 0.5, 1, 2, and 3 g/L concentration. The experiment was performed twice to evaluate the dose response in both CD media D and F.
Stock CHO cells were washed in medium and seeded at 1.0 x 105 cells/ml in 14 day shake-batch cultures. The number of viable cells was determined daily. IVCN and volumetric productivity was compared.
are normalized where unsupplemented base medium (pink) is set to a value of 100. Error bars denote SD.
46 Day Passage of CHO in rHSA Supplemented Media
CHO was passaged for 46 days in chemically-defined medium D supplemented with two lots of pHSA or rHSA at 1g/L concentration. Comparative cultures were grown in unsupplemented CD medium D or medium D supplemented with 1g/L pHSA or 10% FBS. Cells were seeded at 4.0 x105 cells/ml and passaged twice a week. The number of cumulative populations doublings was calculated with each passage.
The purpose of the extended passage to examine the stability of growth with supplementation. Another purpose was to generate stocks of cells adapted to media supplemented with pHSA or rHSA.
Growth Kinetics and Productivity of CHO after Extended Passage with rHSA
CHO cells that had been passaged for 46 days in chemically-defined medium D supplemented with either pHSA or rHSA were compared in a dose response study. The dose response was determined at 0.5,1,2, or 3 g/L pHSA or rHSA. Cells were washed and reseeded at 1.0 x105 cells/ml. The number of viable cells was determined daily. After the run, the IVCN was calculated and the antibody titer was determined.
The results indicate that rHSA supplementation post-passage increased IVCN and productivity. The magnitude of the increase was similar to that observed before the extended passage. Productivity increased up to 75% when CD medium D was supplemented with rHSA.
Conclusions
Animal-free Cellastim™ rHSA from a plant-based expression system was evaluated for its ability to improve CHO growth kinetics and productivity. When tested in a variety of media formulations rHSA improved IVCN and productivity by 50%. The performance profile of rHSA was better than pHSA in each of the formulations.
A dose response study in two chemically defined media showed that productivity increased with increasing rHSA concentration. Productivity increased up 75% compared to the unsupplemented or pHSA-supplemented media.
CHO cells passaged in media supplemented with pHSA or rHSA showed stable growth kinetics during 46 days of extended passage. After extended passage, CHO cells showed similar increases in IVCN and productivity upon rHSA supplementation to that observed before the extended passage.
rHSA derived from a plant based expression system was found to be a robust supplement for CHO culture.
Footnotes
Steve Pettit, Mary Ann Santos, Tanya Tanner, and Ning Huang
Email: Info@InVitria.com
Phone: 1-800-916-8311