Effect of Sialic Acid on Mammalian Cell Culture and Protein Expression: A Potential Productivity Enhancer for Biopharmaceutical Cell Culture Processes

Round 1

Reviewer 1 Report

This reviewer found a numbers of grammatical errors/mistakes that could easily be corrected. Overall, I find the introduction a bit lengthy. While the data on ROS reduction with SA addition seems to correlate well, it is not quite clear what benefit the ROS reduction brings to the process. The idea to use SA seems to be derived from findings on "edible births' nests", which has been shown to reduce oxidative processes in certain diseases.

The SA was purchased by a Chinese company. The authors add this component in very significant quantities to the culture. Thus it is very important to indicate the origin and purity of this component. Could it be that contaminant of SA are involved in the moderate increase in productivity shown? The authors should discuss this. 

Overall, I find the paper convincing and interesting albeit not to be a very strong one in terms of productivity impact. It would be interesting to know why cell number and viability are increased? Are there synthetic limitations in CHO cells for this component? Since CHO cells have a significant lack of diploidy, and have a strong tendency towards metabolic mutations, it would be interesting to know whether corresponding genes in CHO have been mutated or deleted (this could go into a discussion paragraph).

Please find below a few indications for typo/grammar errors, as a guidance for corrections. 

… biopharmaceutical industry, such as human embryo kidney (HEK…

… can increase the harvest titer by more than 20% …

Under a fed-batch process, a large number..

… chemically defined cell culture media are used for cell culture..

…growth, precisely due to its instability (delete”high”)

Some logic improvement of the text referring to “edible birds’ nest” which are not known in the Western world….

…, if not otherwise specified. SA is considered to be a the antioxidant component in edible “bird’s nest”. Water extracts from such edible bird’s nest can inhibit the cytotoxicity induced by H202….

… the intracellular free sialic acid levels 8 times (delete “by”)

Feary et al reported that low levels oxidation of intracellular would

Corrected:

Feary et al reported that low levels of oxidation of the intracellular environment would….

…. To maintain a proper redoc level in the intracellular environment  (environment is missing)

…..does not reduced the level of intracellular oxidation to very low levels. Levels is missing !

Author Response

Point 1: While the data on ROS reduction with SA addition seems to correlate well, it is not quite clear what benefit the ROS reduction brings to the process.

Response 1: We appreciate your comment. We speculate that the reduction in ROS leads to lower lactate levels. Therefore, we have modified the article to highlight the phenomenon of lactate reduction.

Point 2: The SA was purchased by a Chinese company. The authors add this component in very significant quantities to the culture. Thus it is very important to indicate the origin and purity of this component. Could it be that contaminant of SA are involved in the moderate increase in productivity shown? The authors should discuss this. 

Response 2:：Thank you for your valuable and thoughtful comments. According to your suggestion, we have added SA information:

1: SA (purity≥99%，it is derived from E. coli fermentation method）was purchased from Wuhan Zhongke Optics Vally Green Biotechnology Co., Ltd (Wuhan, China) (Line 298-300).

2: However, it should be noted that a large amount of SA was used in this study, so the increase in productivity could be caused by the residual impurities in SA. However, this possibility is very small, because the purity of SA is very high (purity≥99%). When 100mg/L of SA was used, the total amount of impurities introduced by SA was less than 1mg/L, which was very low level for cell culture. (Line 289-293)

Point 3：It would be interesting to know why cell number and viability are increased? Are there synthetic limitations in CHO cells for this component? Since CHO cells have a significant lack of diploidy, and have a strong tendency towards metabolic mutations, it would be interesting to know whether corresponding genes in CHO have been mutated or deleted (this could go into a discussion paragraph).

Response 3: Thank you for your very professional suggestion.

Yes, the synthesis of SA in CHO cells is limited. We have not tested the effect of SA on CHO genes, but we have done some research on SP2/0, SA did not affect SP2/0 cell genes (Data not shown).

According to your suggestion, we have a discussion paragraph:

Since CHO cells have a significant lack of diploidy, and have a strong tendency towards metabolic mutations, Therefore, it will be important to study whether SA affects CHO genes. We did not conduct this research, but we have done some research on SP2/0, the results show that SA did not affect SP2/0 cell genes (Data not shown). (Line294-297)

Point 4：Please find below a few indications for typo/grammar errors, as a guidance for corrections.

Response 4: Thank you very much for your remarks. We have revised the article as you suggested

Author Response File: Author Response.pdf

Reviewer 2 Report

In their manuscript, Chen et al. report experiments on determining the effects of sialic acid (SA) on recombinant protein expression in HEK293 and CHO K1 cells. SA has been reported to be an antioxidant and eliminate H₂O₂ without generating ROS. At certain optimal doses (which depend upon the different cell lines used), SA is shown to reduce reactive oxygen species (ROS) in the culture system, improve cell viability and increase antibody expression. This appears to be achieved without drastically affecting antibody N-glycans/sialylation. Nevertheless, as the authors point out, SA does appear to provoke minor changes in Ab charge, which could pose problems in the long run. Although the work is interesting, it would have benefitted from a better experimental design. In fact, as it stands it is not clear how SA compares with other antioxidants that are commonly used in such experimental circumstances (N-acetylcysteine, glutathione, thiazolidine etc), and this, I think, limits the interest in the current work.

Major Comments

• If I understand correctly when 500 mg/L SA is mentioned, that means on day 1, 125 mg/L was added, followed by further 125 mg/L doses on days 2-4. Is anything known about the actual concentrations of SA throughout the experimental period? A protocol for the determination of SA is described in Materials and Methods: why was this not used to follow the SA dosing regime?
• In Figure 1, a bar should be included to indicate the days in which SA was added.
• In view of the beneficial effects of SA at day 17, why was the process not continued to determine longevity of the effect?
• Figure 1. Why are the x-axes non-linear? The graphic is presented as a time course and it is not immediately obvious that the x-axes are not linear. At what time was the data between days 12 and 15 obtained? In Materials and Methods, it is stated that VCD and metabolic parameters were determined every two days. This does not appear to be the case for Figure 1D.
• The media are described in Table 1, but Table 1 does not contain information about the media. The media contents must be described in a new table. DO these media contain antioxidants?
• The information reported in Table 1 is difficult to understand. The different column headings must be defined in more detail (what are G0-2F?). Here a figure would help. Furthermore, there are no error estimates. How many times were these experiments repeated? Are the differences significant?
• The ROS levels indicated in Figures 4A and B appear to be cellular ROS levels (Materials and Methods). Is it not possible that the Abs are desialylated by ROS in the media (Eguchi, H., et al. (2005) Glycobiology, 15: 1094)? SA in the media should therefore block Ab desialylation – this does not appear to be the case. Finally, it is not clear to me whether or not the reduced acidic charge of Ab seen after SA treatment is within the limits of what is seen in circulating human Abs
• The discussion concerning the experiments with ManNAc are difficult to follow (lines 225 – 234).I think that the messages here would benefit from a figure in which the various sugars (ManNAc and SA) and their distribution in the medium and cells is highlighted. Are there plasma membrane transporters for SA? Does it enter the cell via endocytosis (bulk flow)?

Author Response

Point 1: If I understand correctly when 500 mg/L SA is mentioned, that means on day 1, 125 mg/L was added, followed by further 125 mg/L doses on days 2-4. Is anything known about the actual concentrations of SA throughout the experimental period? A protocol for the determination of SA is described in Materials and Methods: why was this not used to follow the SA dosing regime?

Response 1: Thank you for your careful and professional revision. We have studied the effects of different concentrations, and the total amount of SA added was 0, 1, 10, 100, 500 mg/L, respectively. however, we added SA divided into four times, each time adding 1/4 of the total (1/4 of 1~500 mg/L). For HEK293 cells, SA was added on day 9, 10, 11, 12 and for CHO-K1 Cells, SA was added on day 7, 8, 9, 10. Our main purpose of the divided addition was precisely to reduce the adverse effects of excessive supplementation on the cells’ redox environment. , therefore, compared with one-time supplementation, adding SA in four times may lead to better results.

Point 2: In Figure 1, a bar should be included to indicate the days in which SA was added.

Response 2：Thank you very much for your professional suggestion. According to your suggestion, we have adjusted the figures. Please check.

Figure 1. Effect of SA on HEK293: cell growth (a), integral of viable density (IVCD) (b), viability (c), and lactate production (d). The total amount of SA added was0mg/L (closed circles), 1mg/L (closed triangles), 10mg/L (open circles), 100mg/L (open squares), 500mg/L (open triangles). Values are reported as the average ± standard deviation (n=3). The arrow indicates the addition of SA

Point 3：In view of the beneficial effects of SA at day 17, why was the process not continued to determine longevity of the effect?

Response 3：Thank you for your valuable and thoughtful comments. I think that the article will be more convincing according to your suggestion. There are two main reasons why we did not do this: 1) It will not be cultivated for too long in the actual production process. 2) When we conduct research, our main indicators are lactate and titer. In the last few days, the difference in lactate was continuously observed. Therefore, we did not extend the culture time.

Point 4: Figure 1. Why are the x-axes non-linear? The graphic is presented as a time course and it is not immediately obvious that the x-axes are not linear. At what time was the data between days 12 and 15 obtained? In Materials and Methods, it is stated that VCD and metabolic parameters were determined every two days. This does not appear to be the case for Figure 1D.

Response 4：Thank you for your comment.

VCD and metabolic parameters were determined at least once every two days, but because there are too many data, we omit part of the data to make the graph clearer.

we feel sorry for our wrong description and we have revised this description according to your suggestion. Please check.

• Cell culture sampling was performed to monitor VCD and metabolic parameters, including glucose and lactate levels, every two days during the entire culture period at least (Materials and Methods, Line 332-333).
•  

Point 5：The media are described in Table 1, but Table 1 does not contain information about the media. The media contents must be described in a new table. DO these media contain antioxidants?

Response 5: Thank you very much for your professional questions and suggestions. We have added a new Table according to your suggestion. These media contain antioxidants, but the level is unknown.

Table 2. Summary of experimental condition for this study

Point 6：The information reported in Table 1 is difficult to understand. The different column headings must be defined in more detail (what are G0-2F?). Here a figure would help. Furthermore, there are no error estimates. How many times were these experiments repeated? Are the differences significant?

Response 6：Thank you very much for your professional questions and suggestions. Each experiment is tested three times, and the average value (n=3) is shown here and these differences are not significant. we have revised this description according to your suggestions. Please check.

Figure 7. Glycan profiles. The abscissa indicates the peak time (min).

Table 1. Addition of SA yields comparable antibody product quality

¹ The Day 14 harvest material was purified via protein A chromatography. Protein A eluates from the fed-batch shake flask experiments were tested for impurities via SEC, charge heterogeneity via CEX, N-glycan profile via HILIC-UPLC, and SA via HPLC-FLD. Values of each parameter are reported as average ± standard deviation

Point 7： The ROS levels indicated in Figures 4A and B appear to be cellular ROS levels (Materials and Methods). Is it not possible that the Abs are desialylated by ROS in the media (Eguchi, H., et al. (2005) Glycobiology, 15: 1094)? SA in the media should therefore block Ab desialylation – this does not appear to be the case. Finally, it is not clear to me whether or not the reduced acidic charge of Ab seen after SA treatment is within the limits of what is seen in circulating human Abs

Response 8: Thank you very much for your professional comments.

1) Abs will not be desialylated by ROS. Due to the limiting transport of CMP-sialic acid into the Golgi, the sialic acid levels of antibodies produced by CHO are generally very low. And from the experimental results, SA has no significant effect on the level of antibody sialylation, so we think the SA in the media have no effect on Ab desialylation.
2) The increase of SA has little effect on the acidic charge of Ab, which is within the acceptable range.

Point 8：The discussion concerning the experiments with ManNAc are difficult to follow (lines 225 – 234).I think that the messages here would benefit from a figure in which the various sugars (ManNAc and SA) and their distribution in the medium and cells is highlighted. Are there plasma membrane transporters for SA? Does it enter the cell via endocytosis (bulk flow)?

Response 8: Thank you very much for your professional questions and suggestions.，In fact, we have not seen detailed reports about SA entering cells, so we have deleted this paragraph according to your suggestions.

We tried our best to improve the manuscript and made some changes in the manuscript.  These changes will not influence the result and framework of the paper. And here we marked in red in revised paper. We appreciate for your warm work earnestly, and hope that the correction will meet with approval.
Once again, thank you very much for your comments and suggestions.

Kind regards

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript from Chen and collaborators is a concise, well written report with a clearly defined objective. It successfully evaluated the impact of sialic acid supplementation in cell culture toward the improvement of recombinant protein production. The work plan is clear and the experimental strategy is sound. I would recommend few minor modifications prior to acceptation:

- Ln 68: Although sialic acid is indeed very common in vertebrates it is also very common in bacteria as Neu5Ac but also as and a number of nonulosonic acids and in many invertebrates. The authors should be less categorical on this issue and make reference to appropriate studies.

- Ln71: NeuAc is wrongly considered as the main member of SA, due to the fact that it is the major one in Human. NeuGc is quantitatively more important than NeuAc in vertebrates. Authors should rephrase the sentence.

- Ln 214 and Table 1: authors should explain better what does “SA profile” refers to? Is it the % of sialylated NG?.

- Ln 303-310: Relative to this issue, authors should give more details and the exact experimental protocol for the analysis of N-glycans SA. Also, authors should indicate what is the starting material: purified Antibodies, purified N-glycans, cell supernatant?

Author Response

Thank you for your comments on our manuscript entitled “Effect of Sialic acid on mammalian cell culture and protein expression: a potential productivity enhancer for biopharmaceutical cell culture processes” (ID: processes-959810). The comments were very helpful for the revision and improvement of our paper; importantly, they were also essential to highlight the significance of other research. We have studied the comments carefully and made the appropriate corrections; hopefully, they will meet the reviewers’ approval. Please check the corrections in the manuscript file and the responses to your comments below (highlighted in red).

The manuscript from Chen and collaborators is a concise, well written report with a clearly defined objective. It successfully evaluated the impact of sialic acid supplementation in cell culture toward the improvement of recombinant protein production. The work plan is clear and the experimental strategy is sound. I would recommend few minor modifications prior to acceptation:

Point 1: Ln 68: Although sialic acid is indeed very common in vertebrates it is also very common in bacteria as Neu5Ac but also as and a number of nonulosonic acids and in many invertebrates. The authors should be less categorical on this issue and make reference to appropriate studies.

Response 1: Thank you for your valuable and thoughtful comments. The categorical was deleted according to your suggestion. (Line69-71)

Point 2: Ln71: NeuAc is wrongly considered as the main member of SA, due to the fact that it is the major one in Human. NeuGc is quantitatively more important than NeuAc in vertebrates. Authors should rephrase the sentence.

Response 2: Thank you very much for your professional suggestions. Modify as follows: N-acetylneuraminic acid (Neu5Ac) is the only sialic acid that can be synthesized in the human body. Thus, SA usually refers to Neu5Ac, if not otherwise specified. (Line72-73)

.

Point 3: Ln 214 and Table 1: authors should explain better what does “SA profile” refers to? Is it the % of sialylated NG?.

Response 3: Thank you very much for your professional suggestions. According to your suggestions, we have revised the "SA profile" to "SA content of the antibodies" and added a Figure to explain the "Glycan profile".

Figure 7. N-glycan profiles. The abscissa indicates the peak time (min)

Point 4: Ln 303-310: Relative to this issue, authors should give more details and the exact experimental protocol for the analysis of N-glycans SA. Also, authors should indicate what is the starting material: purified Antibodies, purified N-glycans, cell supernatant?

Response 4: Thank you very much for your professional suggestions. According to your suggestions, we have added information on detection methods:

The purified antibody was acid hydrolyzed with 0.1mol/L trifluoroacetic acid at 80℃ for 1 hour. The released SA was labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) (Sigma, USA) at 50℃ for 3 hours. The DMB-labeled SA was separated by Reverse-phase high performance liquid chromatographic (RP-HPLC) with fluorescent detector (Waters, USA).

The chromatograph column is ACQUITY UPLC BEH C₁₈ 130Å, 2.1*50mm,1.7μm. The mobile phase is acetonitrile: methanol: water (9:7:84). The excitation wavelength is 373nm, the emission wavelength is 448nm. The SA content was quantified based on external standard method. The results were reported as molar ratios. (Ln 363-370)

We tried our best to improve the manuscript and made some changes in the manuscript.  These changes will not influence the result and framework of the paper. And here we marked in red in revised paper. We appreciate for your warm work earnestly, and hope that the correction will meet with approval.

Once again, thank you very much for your constructive comments and suggestions which would help us in depth to improve the quality of the paper.

Kind regards

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The manuscript is much improved. However, there are still many small typographic errors throughout the manuscript that need to be corrected.

Author Response

Thank you for your comments on our manuscript entitled “Effect of Sialic acid on mammalian cell culture and protein expression: a potential productivity enhancer for biopharmaceutical cell culture processes” (ID: processes-959810). The comments were very helpful for the revision and improvement of our paper; importantly, they were also essential to highlight the significance of other research. We have studied the comments carefully and made the appropriate corrections; hopefully, they will meet the reviewers’ approval. Please check the corrections in the manuscript file

We tried our best to improve the manuscript and made some changes in the manuscript.  These changes will not influence the result and framework of the paper. And here we marked in red in revised paper. We appreciate for your warm work earnestly, and hope that the correction will meet with approval.

Once again, thank you very much for your constructive comments and suggestions which would help us in depth to improve the quality of the paper.

Kind regards

Author Response File: Author Response.pdf