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Application Note: L-Glutamine Supplementation in CHO Cell Culture

Background

L-glutamine is an essential amino acid often used to support the growth of CHO cell cultures. Glutamine supports 

the growth and protein production of cells that have high energy requirements and high protein production. It is an 

essential nitrogen source for buildign up molecules such as nucleotides, amino acids and vitamins. However, L - 

glutamine is unstable at physiological pH in liquid media. That is why it is often added to the media right before the 

inoculation of cells. Alternatively, glutamine dipeptides such as Alanyl-Glutamine with higher stability (for example, 

Ajinomoto’s AminoStable™) can be used to ensure sustainability of the glutamine source. Additional consideration 

to  be  taken  into  account  when  using  an  L-glutamine  source  is  the  production  of  ammonia,  which  at  high 

concentrations can be detrimental to culture growth (See ‘Note on Toxic Byproducts’ on Pg. 3).

Effect of L-Glutamine Supplementation on Growth and Productivity

The graphs below present growth profiles in terms of viable cell density (VCD), as well as productivity (protein titer), 

for two cell lines, CHO-K1 and CHO-DG44. Significant effect of L-glutamine addition can be seen, especially in the 

case of CHO-K1. Here, even the lowest dose of 2mM L-glutamine (triangles), added on Day 0 of the culture, led to 

rapid cell growth and transition to exponential growth phase (CHO-K1, top-left graph, Day 2-4), while in the case of 

no L-glutamine addition (circle) biomass growth was extremely slow and did not reach transition to exponential 

growth  phase  within  the  two  weeks  of  culture.  Correspondingly,  the  amount  of  protein  produced  is  greatly 

influenced by the culture growth, as can be seen on the top right-side graph: protein titer at Day 9 is over 3 times 

higher when L-glutamine was supplemented compared to the case with no glutamine addition. In the case of CHO- 

DG44 cell line (bottom graphs), although the effect of L-glutamine supplementation is less substantial, it did allow 

quicker transition to exponential growth phase, and the overall protein titer was increased by ~20% (2 mM) 

compared to the case of no glutamine addition (lower-right graph). It is also important to note that supplementing 

too much L-glutamine can be detrimental to culture growth, as seen in the top-left graph for CHO-K1 line: optimal 

growth was observed for 2 mM L-glutamine, while larger doses led to earlier decrease in cell density.

Figure 1. Effect of L-glutamine supplementation on CHO culture. Growth profile (viable cell density, VCD) and
protein titer throughout fed-batch culture for CHO-K1 and CHO-DG44 cell lines (both are IgG1-producing cell lines),
at different concentrations of L-glutamine supplmentation. L- glutamine was added one time, in Day 0,
at concentrations of either 0 (circle), 2 mM (triangle), 4 mM (square), 6 mM (diamond) or 8 mM (‘x’). 
Media used: CELLiST™ BASAL3 and FEED2 (feed added on days 4, 7, 9, and 11 at 6% for CHO-K1 and 4%
for CHO-DG44 cell line). Culture was stopped when viability dropped below 70%.

CHO-GS Cell Lines (glutamine synthetase selection system)

Cell lines that incorporate the glutamine synthetase (GS) selection system (often termed ‘CHO-GS’, or CHOZN® lines) 

usually produce enough amount of endogenous L-glutamine to sustain their growth. The GS enzyme plays an 

essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form 

glutamine. In this cell line, depending on the specific characteristics of the clone (such as copy number, etc.), addition 

of exogenous L-glutamine source is usually not required. However, as can be seen in the graph below for a CHO-GS 

line, L-glutamine supplementation (2-4 mM) can lead to a marginal increase in the overall titer.

Figure 2. Effect of L-glutamine supplementation in CHO-GS cell line. Growth profile (viable cell density, VCD) and
protein titer throughout fed-batch culture for IgG-producing CHO-GS cell line, at difference concentrations of
L-glutamine supplementation. L-glutamine was added one time, in Day 0, at concentrations of either 0 (circle),
2 mM (triangle), 4 mM (square), 6 mM (diamond) or 8 mM (‘x’). Media used: CELLiST™ BASAL3 and FEED2
(feed added on days 4, 7, 9, and 11 at 4% of initial volume). Culture was stopped when viability dropped below 70%.

Conclusion

Because its importance in cell growth and function, and due to the difference in glutamine requirements 

between cell lines, it is important to optimize glutamine supplementation per cell line. In general, it is 

recommended  to  add  glutamine  for  encouraging  culture  growth,  especially  when  performing  cell 

adaptation, or when switching from one culture medium to another, as this will support the cells in their 

adaptation to the new medium. For example, when switching to CELLiST™ media, we recommend 

supplementing the cell culture with 2-4 mM of L-glutamine, to ensure smooth transition and proper 

adaptation of your cell line to the new CELLiST™ medium. As mentioned previously, consideration should 

be taken not to overfeed the culture with of L-glutamine source, as this can lead to excess in ammonia 

production, which can be detrimental to cell growth.

Note on Toxic Byproducts (Ammonia and Lactate)

Below graph shows ammonia and lactate levels for two cell lines, CHO-K1 and CHO-GS. As can be seen, ammonia 

concentrations  are  directly  correlated  with  L-glutamine  supplement  concentrations.  However,  at  L-glutamine 

concentrations of 2-4mM, increase in ammonia levels is minimal and would have little detrimental effect on culture 

growth. As for lactate (right side), it can be seen that for CHO-K1 line addition of as little as 2 mM L-glutamine would 

lead to proper growth and lactate profile, with lactate consumption taking place after Day 4. In the case of no 

glutamine addition, culture growth is slow and lactate continues to accumulate beyond Day 7.

In the case of CHO-GS, the effect of L-glutamine supplementation is minimal and does not have much effect on 

culture growth and lactate profile. However, higher concentrations of glutamine will lead to higher ammonia levels, 

which are toxic and can be detrimental to culture growth (lower-left graph). This can also be seen in Figure 2 (left 

graph), where higher doses of L-glutamine (6 and 8mM) lead to reduced cell growth and productivity. 

Figure 3. Effect of L-glutamine supplementation on ammonia (NH3) and lactate profiles in CHO-K1 and CHO-GS cell line.
Concentrations of the two byproducts are shown at difference doses of L-glutamine supplementation.
L-glutamine was added one time, in Day 0, at concentrations of either 0 mM (circle), 2 mM (triangle), 4 mM (square),
6 mM (diamond) or 8 mM (‘x’). Media used: CELLiST™ BASAL3 and FEED2 (feed added on days 4, 7, 9, and 11 at 6% for
CHO-K1 and 4% for CHO-GS cell line). Culture was stopped when viability dropped below 70%.

 

> Effect of Poloxamer 188 (P188) supplementation on mAb-producing CHO cell culture

Attached Files

• 05. CSC_technote_Glutamine Application Note.pdf (935.9K) 0회 다운로드 | DATE : 2024-05-08 21:10:07