Efficient Detection of Pre-Proinsulin by Double Antibody Sandwich ELISA

Round 1

Reviewer 1 Report

The current study describes a method for the detection of pre-proinsulin, and it is an intriguing one. Of course, I suppose, it is essential for many reasons involved in the pathogenesis of diabetes mellitus the ability to detect pre-proinsulin. 

In addition, it is crucial for pharmaceutical purposes to verify insulin purification.

First, I think the authors should add a paragraph describing the role (if any) of pre-proinsulin in the pathogenetic mechanisms of type 2 diabetes and type 1.

Furthermore, as mentioned in ref 3, “Two methods have been used for insulin production in E. coli. In the first method, insulin A and B chains expressed independently in E. coli from the chemically synthesized cDNAs were purified and combined under optimal conditions for synthesizing disulphide bonds to obtain intact bioactive insulin (Chance and Frank 1993; Riggs 2021). Genetech used this method in 1978 for the production of recombinant human insulin. In the second method, human proinsulin expressed in E. coli from a single chemically synthesized cDNA was purified to excise C-peptide by proteolytic digestion with carboxypeptidase (Chance et al. 1999). The second method is more competent and handy for the large-scale production of therapeutic insulin and has been commercially used since 1986”. 

And in ref 2, “This study developed a novel and efficient biosynthetic laboratory-scale method of human insulin production using an innovative recombinant cloning strategy, pET21b-hPin vector; and an E. coli BL21 (DE3) expression system was employed using proinsulin”. 

So, it is not clear to me the role of pre-proinsulin according to previously mentioned references in insulin synthesis. Therefore, I think that the authors should clarify this issue. 

 

Overall, the experimental study is exciting and may be helpful for further investigation in the future.

Author Response

Response to reviewers

Comments and Suggestions for Authors

Reviewer #1:

The current study describes a method for the detection of pre-proinsulin, and it is an intriguing one. Of course, I suppose, it is essential for many reasons involved in the pathogenesis of diabetes mellitus the ability to detect pre-proinsulin. 

In addition, it is crucial for pharmaceutical purposes to verify insulin purification.

 

First, I think the authors should add a paragraph describing the role (if any) of pre-proinsulin in the pathogenetic mechanisms of type 2 diabetes and type 1.

Reply: Thanks for your suggestion. In addition, insulin therapy has shown significant advantages over anti-diabetic drugs in both type I and type II diabetes (Chaudhury, A.; Duvoor, C.; Dendi, V.S.R.; Kraleti, S.; Chada, A.; Ravilla, R.; Marco, A.; Shekhawat, N.S.; Montales, M.T.; Kuriakose, K.; et al. Clinical Review of Antidiabetic Drugs: Implications for Type 2 Diabetes Mellitus Management. Front Endocrinol 2017, 8, 6). Insulin, originally produced as preproinsulin, is transformed into a proinsulin by proteolytic action and finally into the active polypeptide hormone, insulin (Patil, N.H.; Devarajan, P.V. Colloid and Interface Science in Pharmaceutical Research and Development. 2014, 411–442). Recently, developments in recombinant DNA technology and protein engineering have led to the increasing availability of human insulins and modified insulin analogues with improved pharmacokinetic properties (Landgraf, W.; Germany, M.A.D.D., Sanofi-Aventis Frankfurt,; Sandow, J.; Germany, P., Centre of Pharmacology, Johann-Wolfgang-Goethe University, Frankfurt/Main, Recombinant Human Insulins – Clinical Efficacy and Safety in Diabetes Therapy. European Endocrinol 2016, 12, 12). Most of the insulins currently used in clinical trials are formed by recombinant DNA expression in E. coli or yeast to form inactive pre-proinsulin (PPI), subsequently enzymatically cleaved and purified to obtain high purity insulin. The efficient and stable expression of PPI is a prerequisite for obtaining high-yield insulin (Govender, K.; Naicker, T.; Lin, J.; Baijnath, S.; Chuturgoon, A.A.; Abdul, N.S.; Docrat, T.; Kruger, H.G.; Govender, T. A Novel and More Efficient Biosynthesis Approach for Human Insulin Production in Escherichia Coli (E. Coli). Amb Express 2020, 10, 43. Bhoria, S.; Yadav, J.; Yadav, H.; Chaudhary, D.; Jaiwal, R.; Jaiwal, P.K. Current Advances and Future Prospects in Production of Recombinant Insulin and Other Proteins to Treat Diabetes Mellitus. Biotechnol Lett 2022, 44, 643–669). PPI, although low in molecular weight (only 15kD), remains immunogenic and its prolonged presence can produce antibodies and thereby reduce the pharmacological effects of insulin (Luo, Z.; Fu, J.P.; Li, N.; Liu, Z.X.; Qin, T.; Zhang, X.L.; Nie, P. Immunogenic Proteins and Their Vaccine Development Potential Evaluation in Outer Membrane Proteins (OMPs) of Flavobacterium Columnare. Aquac Fish 2016, 1, 1–8). However, undigested and unremoved PPI seriously affects the quality and safety of insulin (Riggs, A.D. Making, Cloning and Expression of Human Insulin Genes in Bacteria: The Path to Humulin@. Endocr Rev 2020, 42, bnaa029-. Zieliński, M.; Romanik-Chruścielewska, A.; Mikiewicz, D.; Łukasiewicz, N.; Sokołowska, I.; Antosik, J.; Sobolewska-Ruta, A.; Bierczyńska-Krzysik, A.; Zaleski, P.; Płucienniczak, A. Expression and Purification of Recombinant Human Insulin from E. Coli 20 Strain. Protein Expres Purif 2019, 157, 63–69). Therefore, the accurate detection of PPI expression and residues in insulin is very critical and urgent in the quality control of insulin biosynthesis. This part of additional explanation has now been added in the introduction section of the revised manuscript.

 

Furthermore, as mentioned in ref 3, “Two methods have been used for insulin production in E. coli. In the first method, insulin A and B chains expressed independently in E. coli from the chemically synthesized cDNAs were purified and combined under optimal conditions for synthesizing disulphide bonds to obtain intact bioactive insulin (Chance and Frank 1993; Riggs 2021). Genetech used this method in 1978 for the production of recombinant human insulin. In the second method, human proinsulin expressed in E. coli from a single chemically synthesized cDNA was purified to excise C-peptide by proteolytic digestion with carboxypeptidase (Chance et al. 1999). The second method is more competent and handy for the large-scale production of therapeutic insulin and has been commercially used since 1986”. 

And in ref 2, “This study developed a novel and efficient biosynthetic laboratory-scale method of human insulin production using an innovative recombinant cloning strategy, pET21b-hPin vector; and an E. coli BL21 (DE3) expression system was employed using proinsulin”. 

So, it is not clear to me the role of pre-proinsulin according to previously mentioned references in insulin synthesis. Therefore, I think that the authors should clarify this issue. 

Reply: Thanks for the corrections. Pre-proinsulin (PPI), although low in molecular weight (only 15kD), remains immunogenic and its prolonged presence can produce antibodies and thereby reduce the pharmacological effects of insulin (Luo, Z.; Fu, J.P.; Li, N.; Liu, Z.X.; Qin, T.; Zhang, X.L.; Nie, P. Immunogenic Proteins and Their Vaccine Development Potential Evaluation in Outer Membrane Proteins (OMPs) of Flavobacterium Columnare. Aquac Fish 2016, 1, 1–8). However, undigested and unremoved PPI seriously affects the quality and safety of insulin (Riggs, A.D. Making, Cloning and Expression of Human Insulin Genes in Bacteria: The Path to Humulin@. Endocr Rev 2020, 42, bnaa029-. Zieliński, M.; Romanik-Chruścielewska, A.; Mikiewicz, D.; Łukasiewicz, N.; Sokołowska, I.; Antosik, J.; Sobolewska-Ruta, A.; Bierczyńska-Krzysik, A.; Zaleski, P.; Płucienniczak, A. Expression and Purification of Recombinant Human Insulin from E. Coli 20 Strain. Protein Expres Purif 2019, 157, 63–69). Therefore, the accurate detection of PPI expression and residues in insulin is very critical and urgent in the quality control of insulin biosynthesis. This part of additional correction as your suggestion has now been added in the introduction section of the revised manuscript.

Overall, the experimental study is exciting and may be helpful for further investigation in the future.

Author Response File: Author Response.pdf

Reviewer 2 Report

 

Title: Efficient detection of pre-proinsulin by double antibody sandwich ELISA

Although study looks interesting there are major issues with this manuscript.

1. English language throughout the manuscript needs to be improved.

2. Introduction:

-I would advise beginning the introduction with a brief description of diabetes, corelating with obesity, insulin resistance, type 1 & 2 diabetes. Also answer the question rising from reader behind the insulin requirement in this section.

- I would recommend  to include the currently available therapies on the market, including insulin, as well as the drawbacks  for exploring alternative therapies.

-The authors should clarify the novelty of this article in the ‘Introduction’ and ‘Conclusion’ section.

3. Materials and Methods

-Authors need to include the manufacturer, supplier, country, and city for the reagents/instruments used for these studies.

4. Results:

- These sections need to be written in the brief rather than descriptive. I would suggest to remove the methods and discussion portion from the results.

- Figures 1, 2, and 3 missing an error bar with graphs. I strongly advise that the graphs be modified by plotting the Mean±SEM.

5. Discussion:

-Authors have to corelate their findings with previous studies throughout the discussion, which is one of the lacking in the discussion.

-Justification of their findings looks weaker. How is this article more informative than the previously published ones? Justify it.

6. Conclusion:

- This section does not sound good; therefore, authors have to rewrite this section again by summarizing the outcomes including their future prospective and limitations of their findings.

7. Please fix the typo error, fonts, grammar throughout the manuscript.

 

 

Author Response

Reviewer #2:

 

Title: Efficient detection of pre-proinsulin by double antibody sandwich ELISA

Although study looks interesting there are major issues with this manuscript.

1. English language throughout the manuscript needs to be improved.

Reply: Thanks for your suggestion. We have checked carefully the language such as spelling errors and made the corrections in the revised version.

 

2. Introduction:

-I would advise beginning the introduction with a brief description of diabetes, corelating with obesity, insulin resistance, type 1 & 2 diabetes. Also answer the question rising from reader behind the insulin requirement in this section.

Reply: Thanks for your suggestion. Diabetes mellitus, commonly known as diabetes, is a metabolic disease characterized by high blood glucose levels due to defective insulin secretion or impaired insulin action (Mathieu, C.; Martens, P.-J.; Vangoitsenhoven, R. One Hundred Years of Insulin Therapy. Nat Rev Endocrinol 2021, 17, 715–725). Diabetes includes mainly Type I and Type II diabetes, and is one of the major diseases that threaten human health and life. Type I diabetes is characterized by a loss of the ability of pancreatic beta cells to secrete insulin, and patients rely on insulin therapy to maintain their lives. Type II diabetes, which accounts for approximately 90% of adult-onset diabetes, is caused by a combination of insulin secretion, insulin resistance, hepatic glucose output and lipid disorders (Wondmkun, Y.T. Obesity, Insulin Resistance, and Type 2 Diabetes: Associations and Therapeutic Implications. Diabetes Metabolic Syndrome Obes Targets Ther 2020, 13, 3611–3616). This part of additional explanation has now been added in the introduction section of the revised manuscript.

- I would recommend to include the currently available therapies on the market, including insulin, as well as the drawbacks for exploring alternative therapies.

Reply: Thanks for your suggestion. Diabetes is a chronic and metabolic disease that occurs when the pancreas does not produce enough insulin or when the body is unable to use the insulin produced effectively (Association, A.D. Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 2006, 29, s43–s48, doi:10.2337/diacare.29.s1.06.s43.). When diabetes is left uncontrolled or poorly controlled over time, it can lead to increased atherosclerosis throughout the body, resulting in atherosclerotic disease. At present there is no eradication of diabetes, but there are various treatments that can be used to help control it. Medication is divided into two categories: the first is oral medication, including sulphonylureas, biguanides, alpha glucosidase inhibitors, insulin sensitizers and glargine insulin promoters; the second is insulin therapy. Insulin preparations include animal insulin, human insulin and insulin analogues (Harrigan, R.A.; Nathan, M.S.; Beattie, P. Oral Agents for the Treatment of Type 2 Diabetes Mellitus: Pharmacology, Toxicity, and Treatment. Ann Emerg Med 2001, 38, 68–78, doi:10.1067/mem.2001.114314.). The disadvantages of drug treatment, however, are the development of resistance and weight gain. In addition to the use of medication, exercise therapy is more recommended and can increase physical activity to improve the body's sensitivity to insulin. In addition, diet is the basis of treatment for all types of diabetes, and some patients with mild diabetes can be controlled with diet alone (Klein, S.; Burke, L.E.; Bray, G.A.; Blair, S.; Allison, D.B.; Pi-Sunyer, X.; Hong, Y.; Eckel, R.H.; Metabolism, A.H.A.C. on N., Physical Activity, and Clinical Implications of Obesity With Specific Focus on Cardiovascular Disease. Circulation 2004, 110, 2952–2967, doi:10.1161/01.cir.0000145546.97738.1e). When patients with uropathy are unable to control their blood sugar through diet, exercise and other medications, or when medications cannot be used, then insulin therapy is necessary. Insulin precursors are produced in the synthesis of recombinant insulin and the precursor proteins inevitably trigger immunogenic interference with the effectiveness of insulin therapy. Therefore, the detection and quality control of insulin precursors is necessary and important. To address this issue, we have developed an ELISA method for the rapid and highly sensitive detection of insulin precursors. This part of additional explanation has now been added in the discussion section of the revised manuscript.

-The authors should clarify the novelty of this article in the ‘Introduction’ and ‘Conclusion’ section.

Reply: Thanks for your suggestion. Following your good advice, we have clarified the novelty of this article in the introduction, discussion and conclusion sections of the revised manuscript.

3. Materials and Methods

-Authors need to include the manufacturer, supplier, country, and city for the reagents/instruments used for these studies.

Reply: Thanks for the constructive comments. Following your kind advice, we have added details of the instrumentation used in the experiments to the material methods in the revised manuscript.

 

4. Results:

- These sections need to be written in the brief rather than descriptive. I would suggest to remove the methods and discussion portion from the results.

Reply: Thanks for the critical and constructive comments. In line with your suggestions, we have made changes to the results section of the revised draft.

 

- Figures 1, 2, and 3 missing an error bar with graphs. I strongly advise that the graphs be modified by plotting the Mean±SEM.

Reply: Thanks for your suggestion. We have redrawn Figures 1, 2 and 3 in the revised manuscript.

Figure 1. Titers of monoclonal antibodies against PPI

Figure 2. Purification of monoclonal antibodies against PPI. (A) Reduced SDS-PAGE showing purification of monoclonal antibodies P1 and P2. 1: 10 μL of P1 ; 2: 5 μL of P1; 3: 10 μL of P2 ; 4: 5 μL of P2; (B) P3 and P4. 1: 10 μL of P3 ; 2: 5 μL of P3; 3: 10 μL of P4 ; 4: 5 μL of P4; (C) P5. 1: 10 μL of P5 ; 2: 5 μL of P5; (D) P6. 1: 10 μL of P6 ; 2: 5 μL of P6; M: molecular weight mass standards (kDa).

 

Figure 3. Specificity evaluation of paired antibody. (A) Plating antibodies are listed below the X-axis, detection antibodie is P5. (B) Plating antibodie is P5, detection antibodies are listed below the X-axis.

 

5. Discussion:

-Authors have to corelate their findings with previous studies throughout the discussion, which is one of the lacking in the discussion.

-Justification of their findings looks weaker. How is this article more informative than the previously published ones? Justify it.

Reply: Thanks for your suggestion. We have redrawn Figures 1, 2 and 3 in the revised manuscript.

Both insulin and insulin analogues are recombinantly expressed as insulin precursors and are obtained by enzymatic purification. For the determination of insulin precursors, the US Pharmacopoeia sets a limit of detection (10 ng/mg) but does not give a specific method, while the European Pharmacopoeia recommends the use of radioimmunoassay (10 ppm), but this method requires high instrumentation and radioactive contamination. Sulena et al. used the RP-HPlC method for the determination of insulin precursors, but the low sensitivity clearly does not meet the requirements of the relevant (Polez, S.; Origi, D.; Zahariev, S.; Guarnaccia, C.; Tisminetzky, S.G.; Skoko, N.; Baralle, M. A Simplified and Efficient Process for Insulin Production in Pichia Pastoris. Plos One 2016, 11, e0167207, doi:10.1371/journal.pone.0167207). This part of additional explanation has now been added in the discussion section of the revised manuscript.

 

6. Conclusion:

- This section does not sound good; therefore, authors have to rewrite this section again by summarizing the outcomes including their future prospective and limitations of their findings.

 Reply: Thanks for your suggestion. In summary, to address a series of problems such as the difficulty of detecting insulin precursors in the current production of bio-recombinant human insulin and the incon-sistency and poor sensitivity of the method, we developed a double antibody sandwich ELISA method for the efficient detection of human insulin precursors by preparing high quality and high purity antibodies to insulin precursor proteins. Our results demonstrate that this method can not only improve the sensitivity of the assay, but also precisely exclude the interference of insulin on the accuracy of the assay, and can be used as a more ideal assay in the quality control of human recombinant insulin production. The estab-lishment of this method not only ensures the quality and safety of insulin production, but also improves the accurate monitoring and control of insulin precursors in the insulin production process. The establishment of the method is also expected to provide a more effective reference for the quality control of human insulin production industry and improve the quality level of insulin production. This part of additional explanation has now been added in the Conclusions section of the revised manuscript.

 

7. Please fix the typo error, fonts, grammar throughout the manuscript.

 Reply: Thanks for your suggestion. We have checked carefully the language such as spelling errors and made the corrections in the revised version.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Conclusion:

1. Please read lines 342 to 350. There is some text written twice; please rewrite it to eliminate redundancy.

Author Response

Reply: Thanks for your suggestion. We have abbreviated and revised the conclusions section.

In summary, to address a series of problems such as difficulties in the detection of insulin precursors, unstable methods and poor sensitivity in the current production of bio-recombinant human insulin, we developed a double antibody sandwich ELISA method for the efficient detection of human insulin precursors by preparing high quality and high purity antibodies to insulin precursor proteins. Our results show that the method can not only improve the sensitivity of the assay, but also precisely exclude the interference of insulin on the accuracy of the assay, which can be used as a more ideal assay in the quality control of human recombinant insulin manufacturing, providing assurance for the efficacy and safety of human recombinant insulin products.