동물세포 배양공정의 생산성 향상을 위한 삼투압의 이용

이균민, 류준수

한국과학기술원 생물과학과

Application of Osmotic Pressure to Animal Cell Culture for Improved Productivity

Gyun Min Lee, Joon Soo Ryu

Department of Biological Sciences, Korea Advanced Institute of

Science and Technology

INTRODUCTION

Osmolality is one of the most important physical properties of culture medium. Osmolality is a measure of concentration and is defined as the molality of particles in a solution. Most cell culture media are designed to have the osmolality of 280-320 mOsm/kg through balancing the concentration of nutrients and osmolytes1). When cells are bathed in a solution of the same osmolality as the cytoplasm, they  would neither shrink nor swell. Osmotic pressure results when concentrations of dissolved solutes on the inside and on the outside of a cell are different. In an effort to improve antibody productivity of hybridoma cells, effect of osmotic pressure on hybridoma cells have been extensively studied. As a result, the following observations have been made.

First, the response of hybridoma cells to osmotic pressure varies among cell lines2,3). Second, for most hybridomas, hyperosmotic pressure induced by either sugar or ionized salts suppresses cell growth, while it increases the specific antibody secretion rate (qAb)2-8). Third, the enhanced qAb resulting from hyperosmotic pressure is not transient and is maintained in successive passages9). Finally, hypoosmotic pressure also suppresses cell growth, but it does not enhance qAb10). Hyperostmotic pressure, which can be induced by addition of cheap salts to media, has been suggested as being an economical solution to increase qAb in hybridoma cell cultures. Despite the potential of commercial strategies based on hyperosmotic pressure, the use of hyperosmolar medium has not been popular mainly because cell growth is depressed at elevated osmolality. Hence, the enhanced qAb in a batch culture does not result in a substantial increase in the final antibody concentration2,4-6). This study deals with the responses of hybridoma cells to osmotic pressure and its application to hybridoma cell cultures for improved antibody production.

RESULTS AND DISCUSSION

Effect of Osmotic Pressure on Hybridoma Cell Growth and Antibody Production

Most commercially available media are designed to have an osmolality around 290 mOsm/kg which is close to that of animal serum. As the medium osmolality goes far form this standard range, changes in cellular growth and other metabolic activities occur. Regardless of serum concentrations, cultivation methods, and basal media, hyperosmotic pressure substantially enhanced qAb of S3H5/□2bA2 hybridoma in batch culture by depressing cell growth, although the degree of enhancement in qAb was influenced, as shown in Figure 1. If the enhanced qAb at elevated osmolality is not transient, it can be used to maximize antibody productivity in perfusion culture systems where cell growth and production are separated. As shown in Figure 2, the enhanced qAb of hybridoma at elevated osmolality decreased while the cells were adapting to hyperosmotic pressure in a long-term, repeated batch culture9). However, the cells even after adaptation could still maintained the significantly enhanced qAb when compared with the qAb of cells obtained at standard osmolality.

Response of S3H5/□2bA2 hybridomas regarding growth and antibody production to hypoosmotic pressure resulting from NaCl subtraction was shown in Figure 3. Cell growth and antibody production  at 276 mOsm/kg were comparable to those at 329 mOsm/kg (standard DMEM). Cells grew well at 219 mOsm/kg, although their growth and antibody production were slightly decreased.

Taken together, medium osmolality is one of the important factors that can be easily manipulated by addition or subtraction of inorganic salts. When subjected to hyperosmotic pressure, hybridoma cells displayed enhanced qAb by suppressing cell growth. Hypoosmotic pressure also suppresses cell growth but does not enhance qAb.

Figure 1. Cell growth and antibody production during a batch culture of S3H5/□2bA2 hybridoma cells in various basal media: (A) IMDM, Data points: (□) 281, (□) 339, (□) 390, (□) 440 mOsm/kg. (B) RPMI 1640, Data points: (□) 305, (□) 363, (□) 407, (□) 465 mOsm/kg. (C) DMEM, Data points: (□) 316, (□) 379, (□) 434, (□) 499 mOsm/kg.

Application of Hyperosmotc Pressure to Antibody Production Processes

The enhanced qAb resulting from hyperosmotic stress was successfully used for a higher volumetric antibody productivity in perfusion systems where cell growth and antibody production were separated. First, calcium-alginate-immobilized S3H5/□2bA2 hybridoma cells were grown in a standard medium to achieve high cell concentration rapidly. Second, after cell concentration reached more than 2 x 106 cells/mL, the medium was switched to the hyperosmolar medium. Gradual increase in medium osmolality allowed further cell growth while maintaining the enhanced qAb of immobilized cells as shown in Figure 4. As a result, the volumetric antibody productivity was increased by about 40% when compared with that in the control immobilized cell culture. This enhancement of volumetric antibody productivity of immobilized hybridoma cells by applying hyperosmotic stress suggests the potential of using hyperosmolar medium in other perfusion systems for improved antibody production.

Application of Hypoosmotic Pressure to Antibody Production Processes

Cell culture longevity in fed-batch culture of hybridomas is often limited by elevated medium osmolality caused by repeated nutrient feedings11,12). Shotwise feeding of 10X DMEM concentrates elevated the osmolality of medium up to 540 mOsm/kg at the end of the fed-batch culture of S3H5/□2bA2 hybridoma, which is known to be lethal to most hybridomas. S3H5/□2bA2 hybridoma has been shown to grow without significant growth depression at 219 mOsm/kg in DMEM supplemented with 10% fetal bovine serum. In order to improve culture longevity in fed-batch culture of S3H5/□2bA2 hybridoma cells, a hypoosmolar medium (223 mOsm/kg) was used as an initial basal medium. As shown in Figure 5, the use of hypoosmolar medium delayed the onset of severe cell death resulting from elevated osmolality and allowed one more addition of 10X DMEM concentrates to the culture. As a result, approximately 1.5-fold higher final antibody concentration was obtained compared with fed-batch culture using a standard medium as an initial basal medium. When compared to batch culture, a 5.2-fold increase in final antibody titer was achieved. Taken together, the use of hypoosmolar medium as an initial medium in fed-batch culture improved culture longevity of S3H5/□2bA2 hybridoma cells, resulting in substantial increase in the final antibody titer.

In conclusion, hyperosmotic pressure, which can be induced by addition of cheap salts to media, has been suggested as being an economical solution to increase qAb in hybridoma cell cultures. The utilization of osmotic pressure for improved antibody production in hybridoma cell cultures will be substantiated in future.

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Figure 2. Repeated batch culture of S3H5/□2bA2               Figure 3. Cell growth (a) and antibody production (b)

cells in a spinner flask. (a) Osmolality, (b) viable cell           during a batch culture of S3H5/□2bA2 cells in hypoo-

concentration (□) and □ (□); (c) antibody concentration        smolar media. Data points: (□) 168, (□) 219, (□) 276

(□) and qAb (□). The arrow indicates the time of              , (□) 329 mOsm/kg

switching hyperosmolar medium to standard medium

Figure 4. Immobilized culture of S3H5/□2bA2 cells                Figure 5. Fed-batch culture of S3H5/□2bA2 cells

with gradual hyperosmotic stress: (a) osmolality;                    using a hypoosmolar medium as an initial basal

(b) viable cell concentration (□) and viability (□);                 medium. (a) Changes in medium osmolality (□)

(c) glucose (□) and lactate (□) concentration, qglu                 and qAb (□). (b) Cell growth in batch (□) and

(□), and qlac (□); (d) antibody concentration (□)                  fed-batch (□) cultures. The arrows indicate the

and qAb (□). The vertical lines indicate the time of                addition of medium concentrates. (c) Antibody

elevating the osmolality of medium                               production in batch (□) and fed-batch (□) cultures.