Optimization of the Process of Chinese Hamster Ovary (CHO) Cell Fed-Batch Culture to Stabilize Monoclonal Antibody Production and Overall Quality: Effect of pH Control Strategies

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Must be improved

We have carefully revised the introduction section by citing several critical references. Please kindly refer to lines 30-101.

Is the research design appropriate?

Must be improved

The materials and methods section was thoroughly amended to improve the logic and reasonability of our study (lines 103-175).

Are the methods adequately described?

Must be improved

Similar to above response, we have carefully described the methods in the revised manuscript, including the cell line, pH control strategy, and the analytical methods.

Are the results clearly presented?

Can be improved

Thanks for your evaluation.

Are the conclusions supported by the results?

Must be improved

We have re-organized the conclusion by showing the key results obtained from our study. Please kindly refer to lines 407-423.

 3. Point-by-point response to Comments and Suggestions for Authors

Comments: Dear authors, Thank you for the interesting article. The optimization of CHO processes is an important topic, as well described in the introduction. In the process described here, very good results are already achieved with over 6 g/L product titer. And this also leads to the main problem of the article. The actual optimization is anything but impressive with a 1.07-fold increase in titer. The associated scale-up approach from the 3 to the 15 L system is not shown. The comparisons in the 3 L systems show a stronger dependence on the clone than on the pH control strategy. The process optimization therefore consists of a clone comparison (whereby relevant information on the cell line is missing and two clones are used) and the comparison of two pH setpoints, which are only controlled unilaterally using CO₂. The statistical analysis using student's t-test only shows a significant difference in G1F glycosylation and Man5 proportion, but not in titer for the same clone, aggregation and acid and basic variants.

The main finding would be that one clone is better than the other and that a pH of 7.2 (which is the classic setpoint for CHO cultures) is preferable. Unfortunately, this is not a novel discovery and is already described in the literature cited by the authors. Despite the very good experimental results, the added value for the reader is therefore very low in its current form.

Response: Thank you very much for the valuable comments on our original manuscript. We agreed with the EA5 titer was only increased from 6.1 g/L in 3 L bioreactor to 6.5 g/L in 15 L bioreactor. We would like to explain that the results obtained from 15 L bioreactor was to verify the effectiveness of the proposed pH control CHO cell fed-batch culture developed in 3 L bioreactor. The detailed descriptions of the methods of “2.2. Fed-batch culture conditions and pH control strategies” were carefully revised to highlight the research aim of our study.

In the revised manuscript, we also provide the information of the two clones, please kindly refer to lines 105-107: “A monoclonal antibody named EA5 was produced by two CHO suspension cell clones (Clone 605 and Clone 448) derived from CHO-K1 cells, which were stored and maintained by EA Therapeutics, China.” Meanwhile, we also added the statistical analysis results in Figure 2 to provide accurate comparison of the effects of different pH control strategies on EA5 production and the critical quality attributes (CQAs).

In preparation of the revised manuscript, we have taken all of the valuable comments and suggestions raised by reviewers into considerations and made the relevant modifications with red color. We hope that all of the concerns would have been properly answered or clarified.

Further questions and comments can be found in comments 1-13.

Comments 1: Lines 52-62: You mention 4 cultivation modes generally employed, but just describe fed-batch and perfusion. What are the use cases for batch and continuous culture? Also, a transition sentence to the next paragraph would be helpful.

Response 1: We greatly appreciate you for raising this important issue. In the revised manuscript, we have added some descriptions by citing critical references to discuss the features of batch and continuous cultures. Please kindly refer to lines 56-60: “In a batch culture, the final product is harvested at one time after adding the media, which often results in unsatisfactory cell density and productivity [10,20]. The cells and products are removed from the bioreactor in a continuous culture; thus, this cell culture mode is not implemented for productive processes [19].” Meanwhile, the new-added references were also attached here for your reference.

New-added references:

19. Berrios J., Altamirano C., Osses N., Gonzalez R. Continuous CHO cell cultures with improved recombinant protein productivity by using mannose as carbon source: Metabolic analysis and scale-up simulation. Chem Eng Sci. 2011, 66, 2431-2439. https://doi.org/10.1016/ j.ces.2011.03.011

20. Tihanyi B., Nyitray L. Recent advances in CHO cell line development for recombinant protein production. Drug Discovery Today: Technologies. 2020, 38, 25-34. https://doi.org/ 10.1016/j.ddtec.2021.02.003

Comments 2: Lines 85-88: Details of the cell line would certainly be interesting for the reader. If the exact details are not possible for reasons of confidentiality, it would at least be helpful to specify the cell line type (K1, DG44, CHO-S etc.). Information on genetic modification would also be interesting.

Response 2: Thank you very much for your valuable comments. In the revised manuscript, we have added the cell line type information: “A monoclonal antibody named EA5 was produced by two CHO suspension cell clones (Clone 605 and Clone 448) derived from CHO-K1 cells, which were stored and maintained by EA Therapeutics, China.” (lines 105-107)

Comments 3: Line 94: You should add more details for the inoculum preparation conditions, e.g. temperature, shake flasks nominal and filling volume, shaker amplitude, dissolved CO2 level etc.

Response 3: We greatly appreciate your kind suggestion. Upon the suggestion, the inoculum preparation conditions were carefully added in the 2.1 section. Please kindly refer to lines 115-118: “Afterwards, the inoculum was expanded in a 125 mL shake flask with 30 mL of CD04 medium and 50 mg/L DS addition at an initial cell density of 0.3×10⁶ cells/mL. It was cultivated at 37 °C in a 6% CO₂ incubator at 130 rpm for 3 days.”

Comments 4: The aim of your study is process optimization based on the pH control strategy (lines 12-13). However, there is no strategy described in your methods section, just the setpoints (7.2 and 6.8, lines 106-107). What was the deadband of the pH control strategy? Did you use air/O₂ for CO₂ stripping or just a one-way control strategy? Which buffering system was used?

Response 4: Thank you very much for raising this important issue. Indeed, the aim of our study was to enhance the EA5 production by CHO cells based on an optimized pH control strategy. In order to address this issue, we have revised the title of original “2.2. Fed-batch culture conditions” to “2.2. Fed-batch culture conditions and pH control strategies”. Furthermore, some descriptions were also added in this section. The deadband of pH control strategy was 0.10 (i.e., 7.2 ± 0.1, and 6.8 ± 0.1). In our study, the pH was automatically controlled by adding either CO₂ or NaHCO₃ solution. Meanwhile, the dissolved oxygen (DO) in culture broth was controlled at 40% by sparging air. Herein, we also attached the revised contents in this response to easily follow, lines 119-144:

“2.2. Fed-batch culture conditions and pH control strategies

In this study, every fed-batch process was conducted in triplicate in the bioreactors (Applikon Biotechnology, the Netherlands). The four fed-batch processes (batches #1-#4) were conducted in a 3 L bioreactor with 1.5 L working volume to investigate the effects of pH control strategies on the EA5 titer and quality produced by Clone 605 (batches #1-#2) and Clone 448 (batches #3-#4). One fed-batch culture (batch #5) was carried out in a 15 L bioreactor with 8 L working volume to verify the effectiveness of the process control strategy using Clone 448. The CD04 medium supplemented with 50 mg/L DS and 25 mM MSX was used as the basal medium for the cell cultures in different bioreactors (batches #1-#5). At the beginning of CHO cell culture, the initial cell density was controlled at ~0.8×10⁶ cells/mL. According to a previous study [17], pH was not controlled during days 1-3. Afterwards, two pH control strategies were used. For batches #1 and #3, the pH was automatically controlled at 7.2 ± 0.1 during the fed-batch stage by adding either CO₂ or a NaHCO₃ solution (days 3-14). For batches #2 and #4, the pH level was controlled at 6.8 ± 0.1 during the fed-batch stage by sparging CO₂ or adding the NaHCO₃ solution (days 3-14). During the cell culture processes, the temperature was maintained at 36.5 °C during days 1-6, and then it was decreased to 33.0 °C. The DO level was controlled at 40% during the whole culture process by sparging air at a rate of 10-50 mL/min in the 3 L bioreactor and at a rate of 100-500 mL/min in the 15 L bioreactor. The agitation speed was kept at 280 rpm in the 3 L bioreactor and 180 rpm in the 15 L bioreactor. In batch #5, the pH level was controlled at 7.2 ± 0.1 during the fed-batch stage. The initial glucose in the basal medium was consumed by CHO cells, and its concentration was reduced gradually. When the glucose concentration in the culture broth was below 3.0-4.0 g/L, feeding medium (4.5% of Feed02A and 0.45% of FeedB02) containing 50 g/L glucose was fed into the bioreactor to control the glucose concentration within the range ~4.0-5.0 g/L during the fed-batch stage. The CHO cell fed-batch cultures were ended on day 14.”

Comments 5: How was the DO controlled? By O₂ only or by air/O₂ or air/O₂/N₂?

Response 5: Thank you for pointing this out. In this study, the DO was automatically controlled at 40% by sparging air. This description was also added in the revised 2.2 section: “The DO level was controlled at 40% during the whole culture process by sparging air at a rate of 10-50 mL/min in the 3 L bioreactor and at a rate of 100-500 mL/min in the 15 L bio-reactor.” (lines 135-137)

Comments 6: The stirring speed (280 rpm) was applied to both scales or just to the 3 L scale? What was the scale-up approach? How does this affect the volumetric power input, impeller tip speed, oxygen transfer rate, CO₂ stripping rate etc.?

Response 6: Thanks for your critical comments. Indeed, the agitation speed of bioreactor during CHO cell culture is an important condition because it could enhance the mass transfer efficiency and guarantee the substrate supply. On the other hand, higher agitation speed often leads to severe shear force which would have negative effects on the metabolism of CHO cells and EA5 production. Therefore, in the 3 L bioreactor (batches #1-#4), the agitation speed was kept at 280 rpm. When scaling up the bioreactor from 3 L to 15 L, the agitation speed was controlled at 180 rpm. The different speeds were mainly because the following issues. (1) We would like to control the DO around 40% during CHO cells fed-batch culture process by sparging air. The working volume and aeration rate of 3 L bioreactor are 1.5 L and 10-50 mL/min, while the indexes of 15 L bioreactor are 8 L and 100-500 mL/min. Those operation factors could automatically controlled DO at 40% level. (2) In general, scaling up of bioreactors should consider comprehensive factors including mass transfer coefficients (K_La), volumetric power input (P/V), oxygen transfer rate (OTR), etc. However, in our study, we used two commercially bioreactor from Applikon Biotechnology Co., Ltd. (The Netherlands) to optimize the culture process. The volume of the two bioreactors was 3 L and 15 L. Therefore, our aim is to verify the effectiveness of proposed pH control strategy in 15 L bioreactor to provide some guidance for further optimization. (3) In general, the pilot scale of CHO cell culture is around 200-2000 L, which is much higher than our scales (3-15 L bioreactors). Meanwhile, we will use the P/V rule to scale up the CHO cell fed-batch culture in the future. Finally, we sincerely would like to thank the reviewer for raising this important issue. We hope that our response could address the concern.

Comments 7: You mention analytical devices for cell density and morphology (Vi-Cell XR), Osmolality (Osmo210) as well as glucose and lactate (Cedex Bio HT, section 2.4). How did you measure pCO₂ (e.g. depicted in Figure 1)?

Response 7: Thank you very much for pointing this out. We are sorry for the unclear description in our original manuscript. Upon the valuable suggestion, we have added the methods for measuring pCO₂ in the revised manuscript. Please kindly refer to lines 164-166: “A blood gas analyzer (RAPID Lab348 EX, Siemens, Germany) was used to measure the pCO₂ in the CHO cell cultures.”

Comments 8: You mention consistent pH trends for the first three days with subsection adjustments (lines 168-170), but you described that pH was not controlled during day 1-5 (lines 104-105). What is correct?

Response 8: We are very sorry for the unclear descriptions related to the pH control strategy and the pH trends shown in Fig. 1A of the original manuscript. As the reviewer mentioned above, in this study, our aim is to enhance the EA5 production performance with CHO cells fed-batch culture. Therefore, the pH during the batch stage (day 1-3) was not controlled in batches #1-#4 (3 L bioreactor) and batch #5 (15 L bioreactor). When the glucose concentration in the culture broth was decreased below 3.0-4.0 g/L, the feeding medium containing 50 g/L glucose was fed into the bioreactor to provide enough nutrients for product biosynthesis (i.e., fed-batch stage). During fed-batch stage, different pH control strategies were conducted. As shown in Fig. 1A, the pH changing profiles of the four batches were similar during the first three days. Afterwards, pH control strategy was carried out and the pH changing features were different in days 3-14. In order to clearly describe the pH trends, we have amended the contents in the revised manuscript. Please kindly refer to lines 129-133 and lines 178-180.

Lines 129-133: “According to a previous study [17], pH was not controlled during days 1-3. Afterwards, two pH control strategies were used. For batches #1 and #3, the pH was automatically controlled at 7.2 ± 0.1 during the fed-batch stage by adding either CO₂ or a NaHCO₃ solution (days 3-14). For batches #2 and #4, the pH level was controlled at 6.8 ± 0.1 during the fed-batch stage by sparging CO₂ or adding the NaHCO₃ solution (days 3-14).”

Lines 204-206: “The pH change trends during the first three days (batch stage) were similar among the four batches; afterwards, pH was adjusted to 7.2 (batches #1 and #3) and 6.8 (batches #2 and #4) in the fed-batch stage.”

Comments 9: Why is the VCD after 3 days higher for Clone 605 and the pH 7.2 control strategy (lines 176-178) if pH control did not start until day 3 (or 5)?

Response 9: We sincerely appreciate the reviewer for raising the valuable comments. Similar to the above Comments 8 & Response 8, the unclear description of pH control strategy in our original manuscript led to some misunderstanding. Actually, in batches #1-#4, the pH was controlled after day 3 (fed-batch stage) and it was not controlled in day 1-3 (batch stage, also shown in Fig. 1A). As shown in Fig. 2C, the VCD at day 2 in all batches including Clone 605 and Clone 448 was all around 3×10⁶ cells/mL, while it showed different trends after performing pH control. For Clone 605, controlling pH at 7.2 in day 3-14 could achieve higher VCD than that of pH 6.8. For Clone 448, the final VCD at day 14 was 9.2×10⁶ cells/mL (batch #3), while it was 8.3×10⁶ cells/mL in batch #4. The results indicate that controlling pH at 7.2 during fed-batch stage is beneficial for enhancing VCD. In the revised manuscript, we have added some descriptions to address this concern. Please kindly refer to lines 217-221: “From the variation trends of VCD in the four fed-batch cultures (Figure 1C), it could be observed that a VCD of 5.3×10⁶ cells/mL was achieved by Clone 605 after 3 days of culture. By applying the pH 7.2 control strategy at the fed-batch stage, it exhibited a faster cell growth rate compared to the other three batches during the same period (days 3-14). On day 14, the VCD reached up to 10.8×10⁶ cells/mL in batch #1.”

Comments 10: What is the benefit of the break of the y-Axis in Figure 2(D)?

Response 10: Thank you very much for your valuable comment. The Fig. 2D shows the N-glycans percentages of EA5 harvested on day 14 in batches #1-#4. Clearly, the main N-glycan is G0F which occupies around 60% in all fed-batch cultures, while G0, G1F, and Man5 are maintained below 20% (Fig. 2D). Thus, in order to clearly follow the N-glycans proportion of different cultures, we used the break y-axis to show the data. Meanwhile, the differences among the four fed-batch cultures were statistically analyzed and the results were shown in Fig. 2D as well. We hope that the revisions could address the issue raised by the reviewer.

Comments 11: What does the standard deviation in the figures refer to? The number of experiments is not explicitly stated, are they technical replicates?

Response 11: Thank you for pointing it out. In our study, the fed-batch culture process of each condition was conducted in 3 L (batches #1-#4) or 15 L (batch #5) bioreactor with triplicates. The data shown in figures were mean ± standard deviation (SD, n=3). In the revised manuscript, we have added the information of number of experiments in the Materials and methods section and Results and discussion section. For example, lines 120-121: “In this study, every fed-batch process was conducted in triplicate in the bioreactors (Applikon Biotechnology, the Netherlands).”; lines 215-216: “Each batch was conducted in the 3 L bioreactor in triplicate, and the data are shown as mean ± SD (n=3).”; lines 285-286: “The data are shown as mean ± SD (n=3). Different letters in the column for the same index represent significant differences (P < 0.05) among batches #1-#4.”, and lines 357-358: “The culture of batch #5 was conducted in the 15 L bioreactor in triplicate, and the data are shown as mean ± SD (n=3).”

 4. Response to Comments on the Quality of English Language

Point 1: Please check the use of articles or plurals (e.g. lines 14, 20 and 39) and the translation of similar words (e.g. "recently" on line 151 where "currently" would be the correct word).

Response 1: We sincerely thank you for the careful check of the language. Upon the valuable suggestions, the language including description, words and grammars of the revised manuscript have been carefully checked, modified, and corrected at our best. In addition, we also invited my colleague, an English speaker, Prof. Dr. M. Bilal, to modify our manuscript in vocabulary using and grammar. Meanwhile, the English was polished by the editing services of MDPI (https://www.mdpi.com/authors/english, English editing ID: english-81954). With the careful modification and mistakes corrections, we hope that the English level of the revised manuscript would have reached the language criteria of Fermentation publication.

Response to Reviewer 2 Comments (Please see the attachment)

1. Summary

Thank you very much for taking the time to review this manuscript. We totally accepted the criticism about the innovation of our original manuscript. In preparation of the revision, we have carefully amended the manuscript by considering all of the reviewers’ comments and suggestions. Please find the detailed point-by-point responses below and the corresponding revisions in the re-submitted files. We hope that our responses could address the issues raised by the reviewer.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Scientific papers have the purpose to show innovation. While the authors have well described their work which is of good quality, I do not detect any innovation in the paper. I am sorry to decline recommending the paper for publication. The optimisations executed, the data presented, all are standard technology since many years, even decades. With more than hundreds of processes based on Fed-Batch manufacturing for recombinant proteins, the authors should have been aware that companies have done process optimisations in a similar way as they themselves did.

Response 1: First of all, we thank you very much for your valuable comments on our original manuscript. Frankly, we also totally agreed with the point of “Scientific papers have the purpose to show innovation”. In preparation of the revised manuscript, we have taken all of the valuable comments and suggestions raised by the reviewers into considerations and made the relevant revisions. For the innovation of our study, we would like to address by following issues: 1) The selected mAb (EA5) that could act against complement C5 for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). EA5 has a high affinity and longer half-life period compared with Eculizumab. The EA5 was developed by EA Therapeutics, China. Therefore, from an industrial perspective, it is still important to increase the production performance of EA5 with higher titers and stabilized quality via process optimization technologies. 2) Although hundreds of processes based on fed-batch manufacturing for recombinant proteins are developed by pharmaceutical companies, due to the consideration of commercial benefits, the public results from industrial fields still unclear enough. Therefore, in this study, we would like to provide some guidance of mAb production using CHO cells for researches in universities and institutes, though only using a relatively simple process optimization approach. Finally, we sincerely hope you would accept our responses.

Comments 2: 1) Do you consider the topic original or relevant in the field? > No, it is not. Process Development for high titer in Fed Batch processes have been done since the 1990s or earlier. 2) Does it address a specific gap in the field? > There is no gap, we know since decades that pH, osmolarity and other parameters have effects on quality and quantity of products that are produced by CHO cells.

Response 2: We greatly appreciate the criticism from the reviewer. Similar to above Response 1, we used a commercial example, a monoclonal antibody EA5, to investigate the effects of pH control strategy in CHO cell fed-batch culture on EA5 production and critical quality attributes (CQAs). The two clones (Clone 605 and Clone 448) are derived from CHO-K1 cells, and stored and maintained by EA Therapeutics, China. The development of an effective process control method is indispensable for EA5 production by CHO cells. Furthermore, the results obtained in this study could provide some directions for industrial production of EA5. In the revised manuscript, we also carefully added some descriptions to highlight the logic of our study. Please kindly accept our responses and revisions.

Comments 3: 1) What does it add to the subject area compared with other published material? > It does not add anything new, since it uses a protein of interest as an example. 2) What specific improvements should the authors consider regarding the methodology? What further controls should be considered? > The authors are clearly not aware of the industries history and or progress over the last 30 years.

Response 3: We totally accept the criticism from the reviewer and we also admitted that the innovation of our original manuscript was insufficient. Different CHO cell lines for producing mAbs still have various process control strategies. We used EA5 as an example to investigate the fed-batch culture and development of an optimized method for highly efficient production of EA5. Our group believed that the results could help the company (EA Therapeutics, China) to prompt industrially development process of EA5. In addition, the introduction section was carefully re-organized by citing more critical and newly published papers, which could largely improve the quality of our manuscript. Once again, thank you very much for your effort and time on reviewing our study. We hope that our revisions could address your concerns.

Comments 4: > Are the conclusions consistent with the evidence and arguments presented and do they address the main question posed? > The conclusions are correct, i.e. modification of process etc. has impact… Yet this is not new.

Response 4: We totally accept the criticism of our original conclusion. By carefully revising the manuscript, we believed that the logic and quality of this work was largely improved. Also, the conclusion section was amended carefully. Please kindly refer to lines 408-423:

“In this study, CHO cell fed-batch culture was optimized for the highly efficient production of an mAb (EA5) that could act against complement C5 for the treatment of PNH. The two CHO cell lines of Clone 605 and Clone 448 were selected for analysis. Different pH control strategies were performed during the fed-batch stage in a 3 L bioreactor. The results indicate that controlling the pH at 7.2 during the fed-batch stage could maintain the VCD at a higher level of ~10×10⁶ cells/mL with a viability of above 85%. Compared to Clone 605, Clone 448 exhibited preferable performance in terms of the EA5 titer and product quality (EA5 titer of 6.1 g/L, 4.2% of aggregates, and 2.2% of Man5). Subsequently, the optimized pH-controlled fed-batch culture process was further conducted in a 15 L bioreactor to verify its effectiveness. As a result, a higher E5A titer of 6.5 g/L and a lower Man5 ratio (2.4%) were achieved by day 14, which would be beneficial for large-scale EA5 production. Finally, we also evaluated the clarification process by using the CHO cell culture broth from the 3 L and 15 L bioreactors under the optimized process to facilitate the subsequent EA5 purification process. Overall, our results show the relationship between bioprocess parameters and EA5 production performance, and they could also provide guidance for the effective biomanufacturing process of other mAbs using CHO cells.”

Comments 5: > Are the references appropriate? > They did not reference papers on high yield from CHO, and/or have never been on conferences during which these data are shared.

Response 5: We are very sorry for the lacking citations of the critical papers on high yield of mAbs from CHO cells in our original manuscript. First of all, we would like to explain that our research group mainly focus on the process optimization for the production of mAbs from CHO cells and bioproducts from microorganisms. In 2023, we published a paper related to perfusion culture for anti-PD-1 mAb production by CHO cells, with a final titer of up to 16.79 g/L (Liang K., et al. Front Bioeng Biotechnol. 2023, 11, 1112349). Meanwhile, to address this issue, we have carefully referred to the recent published articles about mAbs production by CHO cells via process engineering strategies. Some descriptions were also added in the introduction section which was marked as red. Please kindly refer to lines 55-78:

“The CHO cell culture modes employed for mAb production are generally categorized into four types, namely, batch, continuous, perfusion, and fed-batch culture [17-19]. In a batch culture, the final product is harvested at one time after adding the media, which often results in unsatisfactory cell density and productivity [10,20]. The cells and products are removed from the bioreactor in a continuous culture; thus, this cell culture mode is not implemented for productive processes [19]. A perfusion culture involves the continuous removal of the cell supernatant from the bioreactor, and due to the large volume of perfusion media, it enables high cell density and elevated protein productivity [21]. Schwarz et al. (2023) developed a process intensification mode based on a microbioreactor (MBR) system to enhance the CHO cell density by up to 60×10⁶ cells/mL in the perfusion mode [22]. Recently, our study reported that higher titers of 8.7-9.8 g/L of anti-PD-1 mAb were produced from CHO cells under perfusion culture conditions in a 3 L bioreactor [17]. Besides the above cultures, the fed-batch mode involves the incremental addition of nutrients at different time points, aiming to enhance cell density, maintain cell viability, and prolong the cultivation duration. To date, the predominant production process for mAbs in large-scale bioreactors is the fed-batch mode [23,24]. In recent years, mAb titers obtained with fed-batch cultures have reached more than 10 g/L [10]. For example, Mahé et al. (2022) developed an intensified fed-batch process that produced 11.4 g/L of human monoclonal IgG1 antibody [25]. In general, a fed-batch culture could enhance the expression and production of mAbs, while traditional substrate feeding strategies might also cause the accumulation of metabolic byproducts such as lactate [10]. Therefore, to achieve efficient mAb manufacturing, an effective and robust fed-batch control strategy is essential to maintain high protein titers and ensure that the critical quality attributes (CQAs) remain within permissible limits.”

New-added references:

18. Shirahata H., Diab S., Sugiyama H., Gerogiorgis D.I. Dynamic modelling, simulation and economic evaluation of two CHO cell-based production modes towards developing biopharmaceutical manufacturing processes. Chem Eng Res Des. 2019, 150, 218-233. https://doi.org/10.1016/j.cherd.2019.07.016

19. Berrios J., Altamirano C., Osses N., Gonzalez R. Continuous CHO cell cultures with improved recombinant protein productivity by using mannose as carbon source: Metabolic analysis and scale-up simulation. Chem Eng Sci. 2011, 66, 2431-2439. https://doi.org/10.1016/j.ces.2011.03.011

20. Tihanyi B., Nyitray L. Recent advances in CHO cell line development for recombinant protein production. Drug Discovery Today: Technologies. 2020, 38, 25-34. https://doi.org/10.1016/j.ddtec.2021.02.003

21. Maria S., Bonneau L., Fould B., Ferry G., Boutin J.A., Cabanne C., Santarelli X., Joucla G. Perfusion process for CHO cell producing monoclonal antibody: Comparison of methods to determine optimum cell specific perfusion rate. Biochem Eng J. 2023, 191, 108779. https://doi.org/10.1016/j.bej.2022.108779

22. Schwarz H., Lee K., Castan A., Chotteau V. Optimization of medium with perfusion microbioreactors for high density CHO cell cultures at very low renewal rate aided by design of experiments. Biotechnol Bioeng. 2023, 120, 2523-2541. https://doi.org/10.1002/bit.28397

24. Luo Y., Kurian V., Song L., Wells E.A., Robinson A.S., Ogunnaike B.A. Model-based control of titer and glycosylation in fed-batch mAb production: Modeling and control system development. AlChE J. 2023, 69, e18075. https://doi.org/10.1002/aic.18075

25. Mahé A., Martiné A., Fagète S., Girod P.-A. Exploring the limits of conventional small-scale CHO fed-batch for accelerated on demand monoclonal antibody production. Bioprocess Biosyst Eng. 2022, 45, 297-307. https://doi.org/10.1007/s00449-021-02657-w

Comments 6: > Please include any additional comments on the tables and figures. > It is not worth it, since it will not enhance the paper.

Response 6: Upon the reviewers’ valuable suggestions, the original manuscript was carefully revised and some indepth discussions were added in the results and discussion section with red color. In addition, the Figure 2 was also revised to provide the statistical analysis results which could be beneficial for the comparison between different process control strategies. We hope that our revisions could address the critical concerns raised by the reviewer. Please kindly accept our responses and revisions. Once again, thank you very much for your effort and time on reviewing our study.

 4. Response to Comments on the Quality of English Language

Point 1: Good quality of English

Response 1: We greatly appreciate for the positive evaluation on the language of our original manuscript. During responding the reviewers’ comments, we have added some contents in the revised manuscript. Furthermore, the language including description, words and grammars of the revised manuscript have been carefully checked, modified, and corrected at our best. In addition, we also invited my colleague, an English speaker, Prof. Dr. M. Bilal, to modify our manuscript in vocabulary using and grammar. Meanwhile, upon the editor and reviewers’ suggestions, the English was also polished by the editing services of MDPI (https://www.mdpi.com/authors/english, English editing ID: english-81954). With the careful modification and mistakes corrections, we hope that the English level of the revised manuscript would have reached the language criteria of Fermentation publication.

Response to Reviewer 3 Comments (Please see the attachment)

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

We have carefully revised the introduction section by citing several critical references. Please kindly refer to lines 30-101.

Is the research design appropriate?

Must be improved

Thanks for your suggestion. In the revised manuscript, the materials and methods section were thoroughly amended to improve the logic and reasonability of our study (lines 103-175).

Are the methods adequately described?

Must be improved

Similar to above response, we have carefully described the methods in the revised manuscript, including the cell line, pH control strategy, and the analytical methods.

Are the results clearly presented?

Must be improved

Thanks for your valuable suggestion. The results and discussion section were carefully revised and some contents were marked as red in the revised manuscript.

Are the conclusions supported by the results?

Must be improved

We have re-organized the conclusion by showing the key results obtained from our study. Please kindly refer to lines 407-423.

3. Point-by-point response to Comments and Suggestions for Authors

Through a lot of papers have been published in this area, it is still an interesting study to investigate the pH control strategy on the cell culture performance and product quality in CHO cells.

Comments 1: Are biological duplicates conducted in this study? Batch 1 and 2 are the same clone but different pH conditions, Batch 3 and 4 same clone & different pH conditions too. I also saw the error bar in some figures. So the authors need to clarify.

Response 1: We greatly appreciate the positive evaluation on our manuscript, especially “… it is still an interesting study to investigate the pH control strategy on the cell culture performance …” Meanwhile, we thank the reviewer for raising this valuable issue. In our study, each CHO cells fed-batch culture process was conducted in 3 L or 15 L bioreactor with triplicates (n=3). The data shown in figures were mean ± SD (n=3). We have added the description in the revised manuscript. Please kindly refer to lines 120-121: “In this study, every fed-batch process was conducted in triplicate in the bioreactors (Applikon Biotechnology, the Netherlands).”; lines 215-216: “Each batch was conducted in the 3 L bioreactor in triplicate, and the data are shown as mean ± SD (n=3).”; lines 285-286: “The data are shown as mean ± SD (n=3). Different letters in the column for the same index represent significant differences (P < 0.05) among batches #1-#4.”, and lines 357-358: “The culture of batch #5 was conducted in the 15 L bioreactor in triplicate, and the data are shown as mean ± SD (n=3).”

Comments 2: In the method section, the details of pH control strategy are not clear. Do you only use CO₂ to control the pH? The glucose feeding strategy is very confusing. Do you mean if the glucose level is 3-4 g/L, then the glucose supplement will be needed?

Response 2: Thank you very much for your comments. Honestly, in our original manuscript, the pH control strategy was not described in details which led to some difficult to follow. To address this issue, we have carefully revised the contents related to pH control strategy. The pH was controlled by adding either CO₂ or NaHCO₃ solution. The descriptions were located in lines 129-133: “According to a previous study [17], pH was not controlled during days 1-3. Afterwards, two pH control strategies were used. For batches #1 and #3, the pH was automatically controlled at 7.2 ± 0.1 during the fed-batch stage by adding either CO₂ or a NaHCO₃ solution (days 3-14). For batches #2 and #4, the pH level was controlled at 6.8 ± 0.1 during the fed-batch stage by sparging CO₂ or adding the NaHCO₃ solution (days 3-14).”

For the glucose feeding strategy, we are very sorry for the unclear descriptions in our original manuscript. The initial glucose (~6 g/L) in basal medium was consumed by CHO cells and its concentration was decreased to 3-4 g/L at day 3. In order to provide enough carbon source for CHO cells metabolism, the feeding medium containing 50 g/L glucose was fed into bioreactors to control glucose concentration ranging ~4.0-5.0 g/L during fed-batch stage (days 3-14). The descriptions were also added in the revised manuscript, please kindly refer to lines 139-143: “The initial glucose in the basal medium was consumed by CHO cells, and its concentration was reduced gradually. When the glucose concentration in the culture broth was below 3.0-4.0 g/L, feeding medium (4.5% of Feed02A and 0.45% of FeedB02) containing 50 g/L glucose was fed into the bioreactor to control the glucose concentration within the range ~4.0-5.0 g/L during the fed-batch stage.”

Comments 3: The last objective of this study in the introduction need to be rephrased. It seems like the 15L is only used for validation by the development from 3L. Also curious if the membrane part is relevant in this manuscript. The authors presented this results in the results section but did not summarize them in both abstract and conclusions.

Response 3: Let’s explain the issues raised by the reviewer. Upon the valuable suggestion, the last paragraph in the introduction section was carefully revised, please refer to lines 92-101.

“In this study, to enhance and stabilize EA5 production by CHO cells, two clones (Clone 605 and Clone 448) derived from the CHO-K1 cell line were used. Firstly, a fed-batch culture was performed in a 3 L bioreactor under different pH control strategies to select the suitable clone and pH control method. Based on this, the effectiveness of the proposed CHO cell fed-batch culture on EA5 biosynthesis and the overall product quality, including aggregation, charge variation, and glycosylation, was carefully investigated in a 15 L bioreactor. Subsequently, the loading capacity of the primary membrane during the clarification process based on the CHO cell fed-batch cultures of the 3 L and 15 L bioreactors was analyzed. Overall, the results provide some guidance for highly efficient mAb production by CHO cells through process optimization.”

Upon the valuable suggestion, we also added some descriptions in the abstract and conclusion sections. Lines 22-24: “In addition, different cell clarification processes were evaluated using the CHO cell culture broth from the 3 L and 15 L bioreactors to further improve productivity and economic performance.” Lines 419-421: “Finally, we also evaluated the clarification process by using the CHO cell culture broth from the 3 L and 15 L bioreactors under the optimized process to facilitate the subsequent EA5 purification process.”

Comments 4: Discussion should be needed on why pH impacts the cell culture performance.

Response 4: We greatly appreciate the reviewer for raising this valuable suggestion. pH is one of the most important parameters in the large-scale production of therapeutic proteins; it is also the easiest parameter to regulate. pH affects both protein production and protein quality, which determines pharmaceutical safety and efficacy. Thus, some discussions were added in the revised manuscript, lines 192-201: “It has been reported that the pH of a cell culture significantly influences various aspects, including cell growth, cellular metabolism, and the titer and quality of mAbs in CHO cells [28,33,34]. Lee et al. (2008) found that pH significantly influences the expression rate and glycosylation of a recombinant chimeric antibody in CHO cells [34]. In addition, the metabolism of amino acids, the intermediates of the TCA cycle, and nucleosides in CHO cells are largely influenced by pH change [35]. It should be noted that the culture conditions of different CHO cell lines for producing mAbs generally need to be confirmed. To the best of our knowledge, the effects of culture pH on recombinant CHO cell growth (Clone 605 and Clone 448) and EA5 quality still remain unclear.”

New-added references:

34. Lee J.C., Kim D.Y., Oh D.J., Chang H.N. Long-term operation of depth filter perfusion systems (DFPS) for monoclonal antibody production using recombinant CHO cells: Effect of temperature, pH, and dissolved oxygen. Biotechnol Bioprocess Eng. 2008, 13, 401-409. https://doi.org/10.1007/s12257-008-0155-8

35. Shen L., Yan X., Nie L., Xu W., Miao S., Wang H., Poon H.F., Qu H. Chemometric identification of canonical metabolites linking critical process parameters to monoclonal antibody production during bioprocess development. Chin J Chem Eng. 2019, 27, 1171-1176. https://doi.org/10.1016/j.cjche.2018.10.009

Comments 5: The authors mentioned this study would provide some guidance for manufacturing. More discussion will be needed for this statement.

Response 5: We totally agree with the suggestion. To this end, we have added some contents in the revised manuscript, please kindly refer to lines 403-406: “The obtained results could be used to effectively avoid risks in the CHO cell culture process for EA5 production, and they could also provide the basic conditions for process intensification of industrial production through CHO cell fed-batch culture.”; and lines 421-423: “Overall, our results show the relationship between bioprocess parameters and EA5 production performance, and they could also provide guidance for the effective biomanufacturing process of other mAbs using CHO cells.”

 4. Response to Comments on the Quality of English Language

Point 1: N/A

Response 1: During responding the reviewers’ comments, we have added some contents in the revised manuscript. Furthermore, the language including description, words and grammars of the revised manuscript have been carefully checked, modified, and corrected at our best. In addition, we also invited my colleague, an English speaker, Prof. Dr. M. Bilal, to modify our manuscript in vocabulary using and grammar. Meanwhile, upon the editor and reviewers’ suggestions, the English was also polished by the editing services of MDPI (https://www.mdpi.com/authors/english, English editing ID: english-81954). With the careful modification and mistakes corrections, we hope that the English level of the revised manuscript would have reached the language criteria of Fermentation publication.