Week 1 HW: Final Project Ideas

HTBAA Homework #1

Name: Camila Naranjo

I would like to biomanufacture alpha-1 antitrypsin (AAT), a glycoprotein that protects the lungs from damage caused by proteolytic enzymes. The production of this glycoprotein is intended to improve the quality of life for people with alpha-1 antitrypsin deficiency, an inherited condition (1). Currently, the primary treatment for this disease involves repeated administration of donated AAT, so the biomanufacturing of alpha-1 antitrypsin could make treatment more available.

For the expression system, I would consider two options, keeping in mind that, since this is a glycoprotein, post-translational modifications—especially glycosylation—will be required. CHO cells are a good option because they support post-translational modifications. On the other hand, a cell-free system derived from HEK cells could be another option.

To produce alpha-1 antitrypsin, an expression vector must first be constructed. For CHO cells, the vector must contain, in addition to the protein-coding sequence, a fluorescent gene to monitor the growth of transfected cells, an antibiotic resistance gene, a constitutive promoter, a signal peptide that allows the protein to be secreted into the medium, and, preferably, a sequence that allows the protein to be purified in downstream processes, such as a His-tag. For the cell-free system, the construct is mixed with HEK cell lysate, an energy regeneration system, amino acids, and other components necessary for protein expression.

In the AAT biomanufacturing process, vector assembly and cloning could be performed using a liquid-handling robot to reduce pipetting errors and enable parallel testing of variants. The process of optimizing expression conditions can be automated with Cloud Lab, allowing for the testing of more conditions than could be evaluated manually and thus saving production time. Once expression conditions are optimized, continuous monitoring of the cell culture can be performed using a parallel minibioreactor system with automatic, real-time data collection; in the case of cell-free systems, reaction kinetics can be monitored with an automated plate reader. Finally, the purification process would utilize automated chromatographs to ensure reproducibility.

The biomanufactured AAT is expected to benefit patients with alpha-1 antitrypsin deficiency (AATD). To estimate the potential demand for the product, Sweden was used as a reference country. (2) reported a prevalence of diagnosed AATD of 21.04 cases per 100,000 inhabitants in Sweden. Based on a current Swedish population of 10,613,531 inhabitants (3), this prevalence corresponds to approximately 2,233 diagnosed patients. However, augmentation therapy is indicated specifically for patients with severe AAT deficiency and clinically evident emphysema. (4) reported that 66% of participants in the NHLBI Registry received augmentation therapy at some point during follow-up. Applying this proportion to the estimated Swedish AATD population results in a potential target population of approximately 1,474 patients.

The FDA-approved dosage for AAT augmentation therapy is 60 mg/kg of body weight administered intravenously once weekly (5). Assuming an average patient weight of 75 kg, each patient would require approximately 4.5 g of AAT per week (60 mg/kg × 75 kg = 4,500 mg = 4.5 g), corresponding to approximately 19.55 g of AAT per patient per month (4.5 g/week × 4.345 weeks/month). Therefore, for an estimated target population of 1,474 patients, the total monthly demand would be approximately 28.82 kg of AAT (19.55 g/patient/month × 1,474 patients ≈ 28,820 g/month).

To determine how much A1AT can be produced and how long it would take, a glycoengineered Chinese Hamster Ovary (CHO) cell line producing correctly human-glycosylated A1AT was used as a reference, achieving a titer of 1.2 g/L (6). Assuming a 2,000 L bioreactor, a single batch would yield a raw titer of 2,400 g (1.2 g/L × 2,000 L). For this preliminary production model, an overall downstream recovery of 80% was assumed (7), resulting in 1.92 kg of purified A1AT per batch.

CHO fed-batch cultures can be harvested after approximately 10 days of culture (8). Downstream processing was conservatively estimated to require 7 days under a conventional batch configuration. Previous studies have demonstrated that automation and process integration can reduce downstream processing times by approximately 50% to more than 70% (9,10). Therefore, considering the automated liquid-handling and chromatography systems proposed in this work, downstream processing is conservatively expected to be completed in approximately 5 days. This gives a total cycle time of 15 days per batch, or approximately 2 batches per bioreactor per month, and a monthly output of 3.84 kg per bioreactor (2 × 1.92 kg).

To meet the monthly demand of 28.82 kg, approximately 8 bioreactors of 2,000 L would need to operate in parallel (28.82 kg / 3.84 kg ≈ 7.5, rounded up to 8), producing around 30.72 kg per month, a supply that comfortably covers demand. The first purified batch would be available approximately 15 days after production begins, with continuous monthly output sustained thereafter through the parallel bioreactor line.

References

(1) Statistics Sweden. (2026, October 7). Population Statistics.

(2) Amann, T., Hansen, A. H., Kol, S., Hansen, H. G., Arnsdorf, J., Nallapareddy, S., Voldborg, B., Lee, G. M., Andersen, M. R., & Kildegaard, H. F. (2019). Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles. Metabolic Engineering, 52, 143–152. https://doi.org/10.1016/j.ymben.2018.11.014

(3) Borlido, L., Azevedo, A. M., Sousa, A. G., Oliveira, P. H., Roque, A. C. A., & Aires-Barros, M. R. (2012). Fishing human monoclonal antibodies from a CHO cell supernatant with boronic acid magnetic particles. Journal of Chromatography B, 903, 163–170. http://www.sciencedirect.com/science/article/pii/S1570023212004151

(4) Campos, M. A., Geraghty, P., Holt, G., Mendes, E., Newby, P. R., Ma, S., Luna-Diaz, L. V., Turino, G. M., & Stockley, R. A. (2019). The Biological Effects of Double-Dose Alpha-1 Antitrypsin Augmentation Therapy. A Pilot Clinical Trial. American Journal of Respiratory and Critical Care Medicine, 200(3), 318–326. https://doi.org/10.1164/rccm.201901-0010OC

(5) Chatterjee. (2022). (12) United States Patent.

(6) Chen, R., Chen, X., Shi, C., Jiao, B., Shi, Y., Yao, B., Lin, D., Gong, W., & Hsu, S. (2022). Converting a mAb downstream process from batch to continuous using process modeling and process analytical technology. Biotechnology Journal, 17(11). https://doi.org/10.1002/biot.202100351

(7) Dasí, F. (2024). Alpha-1 antitrypsin deficiency. Medicina Clínica, 162(7), 336–342. https://doi.org/10.1016/j.medcli.2023.10.014

(8) Gomis-Fons, J., Löfgren, A., Andersson, N., Nilsson, B., Berghard, L., & Wood, S. (2019). Integration of a complete downstream process for the automated lab-scale production of a recombinant protein. Journal of Biotechnology, 301, 45–51. https://doi.org/10.1016/j.jbiotec.2019.05.013

(9) Stoller, J. K., Fallat, R., Schluchter, M. D., O’Brien, R. G., Connor, J. T., Gross, N., O’Neil, K., Sandhaus, R., & Crystal, R. G. (2003). Augmentation Therapy With α1-Antitrypsin. Chest, 123(5), 1425–1434. https://doi.org/10.1378/chest.123.5.1425

(10) Wahlin, S., Widman, L., & Hagström, H. (2025). Epidemiology and outcomes of alpha-1 antitrypsin deficiency in Sweden 2002-2020: A population-based cohort study of 2286 individuals. Journal of Internal Medicine, 297(3), 300–311. https://doi.org/10.1111/joim.20058

AI Client: Claude (Anthropic)

Prompts

  • I’m going to switch to molecular biology. Can you name an enzyme or protein that can be produced to compensate for a genetic deficiency?

  • I need to know how much alpha-1 antitrypsin I would need to produce and how long it would take to produce it. To perform the calculations, assume that the goal is to reach the entire population of a country, and use this article—which estimated the incidence and prevalence of alpha-1 antitrypsin deficiency in Sweden—as a reference. Consider quantities such as production rate, yield, volume, number of parallel experiments or reactors, automation throughput, or whatever parameters are most appropriate for your system. Show and explain calculations, including units, assumptions, and sources for important numbers (if it is needed, search other references).

*AI Client: Gemini Notebook

Prompts

  • Hi, I need you to give me the main Ideas I need to know to be able to solve Homework #1. It would be better if the ideas are classified by each question.

  • I need you to make a zoom-in “automation” since I have no idea about this topic, and the same for scale calculation. Is there any formula for that?