Theoretical design of a space bioprocessing system to produce recombinant proteins – npj Microgravity

The operational scenario investigated was a six person crew on a 3-year Mars mission with 600 days of surface operations³⁹. A total of ~14 g of recombinant carbonic anhydrase was estimated to be required to maintain a liquid amine CO₂ scrubber with active enzyme throughout surface operations. Affinity purification of the enzyme was assumed to be needed to reduce liquid amine side reactions with cellular debris. Sufficient carbonic anhydrase could be supplied by approximately 80 bioprocessing runs using 1 L cultures with a yield of 180 mg purified enzyme per run. Supplies and power for a total of 100 production runs were assumed to give sufficient redundancy for production runs that failed to meet expected yield.

A review of recombinant protein purification from unicellular microorganisms identified sub-process steps (Fig. 1). Essential steps for the operational scenario, such as lysis or purification, were retained. Biomass dewatering was investigated as an optional step. Non-essential steps, such as storage and drying, were eliminated to simplify potential workflows. A trade study of potential methods for each sub-process step was conducted, and non-crew time ESM was used to evaluate individual technologies for in-space application. Technologies with comparable ESM were used to develop three designs, which were analyzed using four metrics described below.

Non-crew time ESM metric

Non-crew time ESM was calculated using Eq. 1 based on guidelines from Levri, et al.²⁰.

Where, M_i is initial mass (kg), V_i is initial volume (m³), V_eq is volume equivalency (kg eq m⁻³), P is power consumed (kW), P_eq is power equivalency (kg eq kW⁻¹), C is thermal cooling (kW), C_eq is thermal cooling equivalency (kg eq kW⁻¹), D is mission duration (number of runs), M_t is time dependent mass (kg), V_t is time dependent volume (m³), CS is cold storage mass (kg), CS_eq is cold storage equivalency (kg eq kg⁻¹), W is water mass required per run (kg), W_eq is water treatment equivalency (kg eq kg⁻¹), WS is solid waste mass per run (kg), and WS_eq is solid waste storage equivalency (kg eq kg⁻¹).

Crew time was excluded from all ESM calculations due to the large uncertainty in estimating crew time requirements without empirical tests of the integrated designs. Equation 1 includes core non-crew time ESM components for mass, volume, power, and cooling as well as estimates of working mass for the cold storage, water, and waste storage systems that were determined to be externalities of all the bioprocessing designs. Parameters for volume (V^ESM), power (P^ESM), and thermal cooling (C^ESM) used equivalency factors estimated for nominal crewed surface missions to Mars from both 2015 and 2022 Baseline Values and Assumptions Documents (Table 2)^39,40.

Cold storage (CS^ESM), water (W^ESM), and waste storage (WS^ESM) used equivalency factors derived from non-crew time ESM values for baseline technologies described in nominal Mars surface missions^39,61. These working mass equivalency factor calculations for Eq. 1 are detailed in Supplementary data 1. Briefly, the cold storage equivalency (CS_eq) assumed a density of 1000 kg m⁻³ for mass requiring cold storage and a 0.614 m³ internal volume freezer analogous to the current International Space Station (ISS) system³⁹. The Mars mission ESM of this freezer system was estimated at 490 kg eq. The freezer ESM was divided by the internal volume and multiplied by the density of the stored mass to obtain a CS_eq of 0.80 kg eq kg⁻¹ for cold-stored mass.

The water equivalency (W_eq) and waste storage equivalency (WS_eq) factors were calculated from non-crew time ESM divided by the estimated total mission load using a Mars mission scenario where a crew of six used 30 kg of water per crew member per day and produced 1.5 kg solid waste per crew member per day⁶¹. The non-crew time W^ESM was calculated at 12,800 kg eq, assuming a water processor similar to the ISS water treatment system⁶¹. A non-crew time WS^ESM of 4480 kg eq was calculated from the mass, volume, power, and cooling estimates for a baseline solid waste disposal system proposed for a Mars surface habitat⁶¹. The equivalency factors of 0.12 kg eq kg⁻¹ for W_eq and 0.83 kg eq kg⁻¹ for WS_eq were derived from the calculated ESM of each system and divided by the estimates of total use during 600 days of surface operations. Detailed assumptions and ESM calculations for Figs. 2, 3 are given in Supplementary data 1, and detailed calculations for the three designs are given in Supplementary data 2.

SCM metric

SCM was calculated by summing the number of components and the number of one-way interactions between components²². Components are integrated subsystems that perform one or more bioprocessing steps, such as equipment available from commercial vendors. Auxiliary parts such as valves, ordinary filters, and sensors were excluded with the rationale that integration of major components account for most of the system complexity, cost, and failure modes²². The number of one-way interactions is derived from a top-level bioprocessing system block diagram based on physical connections between components (Fig. 6).

TRL and IRL metrics

TRL and IRL metrics were assigned for each of the dewatering, cell lysis, and purification options included in the three designs using existing guidelines^32,33,34. TRL is a numerical scoring system for ranking the relative risk of advancing development of technologies from fundamental principles to flight proven hardware systems. TRL levels were assigned to each technology considered using the following scoring: TRL 1 basic principles, TRL 2 application concept formulated, TRL 3 proof of concept, TRL 4 laboratory breadboard validated, TRL 5 breadboard validated in relevant environment, TRL 6 prototype demonstrated in operational environment, TRL 7 system demonstrated in operational environment, TRL 8 system flight qualified, and TRL 9 system flight proven³².

IRL is a similar scoring metric designed to assess risks associated with integration of technologies from identifying interfaces to demonstrating integration in flight⁵⁰. IRL levels were assigned to each technology considered the following scoring: IRL 1 interfaces identified, IRL 2 interaction characterized, IRL 3 communication compatibility shown, IRL 4 integration data quality checked, IRL 5 control established, IRL 6 information exchange established, IRL 7 integration requirements validated, IRL 8 mission qualified, and IRL 9 mission proven^33,34.

Degree of crew assistance metric

The degree of crew assistance was estimated based on whether the commercially available components were integrated and easily automatable. Crew support was assumed if commercial technology was not readily available to automate a specific step in the proposed workflow of each design. The sum of crew assisted steps was the degree of crew assistance for the design.

Dewatering technologies

Tangential flow filtration, batch centrifugation, and counterflow centrifugation were three dewatering methods compared for this analysis. The dimensions and mass of the tangential flow filter were based on Xampler cartridge with 1 mm fiber diameter, 500 kDa pore size and 30 cm path length with a 3 M housing (Cytiva, Marlborough, MA, USA). To control fluid flow through the tangential flow filter, an Ismatec Reglo ICC Digital Pump with 4-Channels and 8-Rollers (Cole-Palmer, Vernon Hills, IL, USA) was included in the ESM calculations. A modified version of the Drucker model 755VES swinging bucket centrifuge (Drucker Diagnostics, Port Matilda, PA, USA) has been approved for operation on the ISS, and the specifications of the commercially available centrifuge were used to calculate the ESM. Twenty disposable 50 mL tubes were assumed to harvest the 1 L culture volume during every run. The counterflow centrifuge data was based on specifications for the CTS Rotea Counterflow Centrifugation System (Thermo Fisher Scientific, Waltham, MA, USA).

Lysis technologies

The methods considered for lysis were bead beating, enzymatic lysis, a flow-through sonicator, and a large-volume probe sonicator. The bead beater analysis was based on the Claremont Biosolutions LLC (Upland, CA, USA) OmniLyse HL beadbeater flow-through lysis device. Although originally developed for small volumes, the OmniLyse HL unit was assumed to lyse large volumes with equal efficiency using extended processing times. The enzyme lysis protocol assumed that the biomass was incubated for 30 min at ambient temperature with 0.25 mg mL⁻¹ lysozyme, 0.1 mL mL⁻¹ Pierce universal nuclease (Thermo Fisher Scientific, Waltham, MA, USA), and 0.1% weight per volume Triton X at final concentration. Mass and volumes for ESM were calculated assuming the solid reagents had a density equal to NaCl (2.17 g cm⁻³), and liquid reagents had a density equal to water (1 g cm⁻³). The QSonica (Newton, CT, USA) ultrasound generator was modeled assuming a 1-inch replaceable tip for a large volume batch method and a Q500 FloCell unit for the flow through method.

Protein purification technologies

Five His-tag affinity purification methods were compared using public domain product information available from commercial vendors. The Sigma Aldrich (St. Louis, MO, USA) His-Select Ni affinity gel was used to represent affinity columns requiring clarified lysates. The Cytiva (Marlborough, MA, USA) HisTrap FF was an exemplar of a crude lysate column that does not require clarification before sample loading. Batch resin purification specifications were estimated for the resin “teabag” method from Castaldo, et. al.⁶⁰ Millipore Sigma (Burlington, MA, USA) HIS-Select® Nickel Magnetic Agarose Beads specifications were used to estimate the amount of resin required for batch purification with magnetic beads, while mass and volume of the beads were assumed to be equivalent to affinity resin. For affinity membrane purification, Capturem large volume filters (Takara Bio USA Inc., San Jose, CA, USA) were used to estimate the quantity of membrane required, while mass and volume of the membrane was assumed to be equivalent to Whatman filter paper (200 g cm⁻²).

The buffer composition was 20 mM HEPES, 500 mM NaCl and 20 mM imidazole for the binding buffer, and 20 mM HEPES, 500 mM NaCl and 200 mM imidazole for the elution buffer for all the methods. Buffer volumes were modeled using manufacturer protocols or the resin “teabag” method⁶⁰.

Bioprocessing system designs

All three post-growth bioprocessing system designs assumed a Claremont Biosolutions LLC (Upland, CA, USA) OmniLyse® HL disposable bead beater for in-line cell lysis from the biomass reservoir. The first design included a Thermo Fisher Scientific (Waltham, MA, USA) CTS Rotea Counterflow Centrifugation System with disposable, single use kits for processing. This counterflow centrifuge includes integrated pinch-valves, a peristaltic pump, and a controller for automation. QIAexpressionist Ni-NTA resin (Qiagen Inc., Valencia, CA, USA) was included for affinity purification.

The second design uses a MidGee ultrafiltration cartridge UFP-5-C-MM01A (Cytiva, Marlborough, MA, USA) and an Applikon Biotechnology (JG Delft, Netherlands) “my-control” with built in peristaltic pumps for biomass dewatering using tangential flow filtration and subsequent cell lysis. For protein purification, an Automate Scientific (Berkeley, CA, USA) Perfusion System and ValveLink8.2 Perfusion Controller were assumed to integrate with an Masterflex (Gelsenkirchen, Germany) L/S® Digital Drive peristaltic pump with an Easy-Load® 3 Pump Head for Precision Tubing. This fluid control system was assumed to automate affinity purification in a Cytiva 5 mL HisTrap FF crude lysate column (Marlborough, MA, USA).

Dewatering and lysis components in design 3 were identical to design 2. Protein purification was based on a non-commercial affinity belt system. Rollers were proposed to move an affinity membrane belt continuously through chambers containing the lysate, wash buffers, and elution buffers. The total volume of the system as assumed to be 0.005 m³ including the chambers, walls, and rollers. The mass was assumed to be 500 g. One Transmotec Inc. (Burlington, MA, USA) 12 V, 2 A DC motor was included to operate the rollers.

Disposable materials such as bags, and sterile filters for all three designs were estimated for the system based on commercial options and material properties, while the additional mass of tubing and luers were assumed to be 0.1 kg for all designs.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41526-023-00324-w