Comparison of Maglev Centrifugal Pump and Quaternary Diaphragm Pump Effects
on mRNA encapsulated LNPs
Mark McElligott - bioX, NH, USA
E-mail: mmcelligott@bioxeng.com
Keywords: mRNA, LNP, PDI, DLS, FBRM
1. MOTIVATION
Lipid nanoparticles (LNPs) are the most developmentally advanced non-viral gene delivery
system that delivers nucleic acids. LNPs have several benefits over previous lipid-based nucleic
acid delivery systems through:• High nucleic acid encapsulation efficiency• Potent transfection capabilities• Improved tissue penetration for drug delivery• Low cytotoxicity and immunogenicity
The MFG of LNPs in a process fluid stream is critical, and there are some equipment options forconsideration that many have ignored.
2. INTRODUCTION
Messenger ribonucleic acid (mRNA) encapsulated in lipid nanoparticles (LNP) is a vaccine
modality that has found recent effectiveness and prominence within the industry. Both themRNA and LNP are susceptible to physical degradation and may present as fragmentation,
aggregation, precipitation, fusion, or leakage of mRNA from the LNP. 1
One proposed mechanism of physical degradation is processing vaccine solutions using
recirculated pumping. Some prolonged recirculated pumping involves tangential flow filtration(TFF), typically used throughout the industry to concentrate and often diafilter a product pool.
This study evaluates and compares maglev single-use centrifugal pump, and single-use
quaternary diaphragm pump and their effects on mRNA encapsulated LNPs at target flow rates,pressures, and durations.
3. MATERIALS AND METHODS
3.1. mRNA and LNP Formulation
An LNP suspension (in mainly Potassium phosphate and Sodium phosphate buffers, with
Sodium Chloride, Dibasic sodium phosphate dihydrate, sucrose, 18%EtOH, etc.) was
formulated using conventional and self-amplifying mRNA (SAM) comprised of the following
lipid profiles captured in Table 1:Page | 1

Table 1: Lipid Profiles
Profile 1

Profile 2

ALC-0315 = (4-hydroxybutyl) azanediyl)bis (hexane-6,1-diyl)bis(2-hexyldecanoate)
ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N ditetradecylacetamide
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC)
Cholesterol
DLin-MC3-DMA: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4(dimethylamino)butanoate
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC)
PEG2000-DMG = Alpha-(3′-{[1,2-di(myristyloxy)propanoxy]carbonylamino}propyl)-ω-methoxy, polyoxyethylene
CholesterolParameters and Detection Methods
The parameters monitored in this testing have been:The average particle size of the LNPs

The Polydispersity Index (PDI) represents the variation in sample particle size.
Higher PDI is represented by aggregation or fragments present in a sample

% mRNA encapsulated in LNP relative to total mRNA (free plus encapsulated). A
decreasing percentage of encapsulation over the run indicates damage to LNPs (andan increase of free mRNA).

Capillary Gel Electrophoresis and/or Dynamic Light Scattering (DLS) have been shown torelate log normal particle size distribution for small spherical particles 2. Yet another use for
DLS is to determine the Polydispersity Index (PDI) of particles. PDI indicates a variation in theparticle size of the sample per Equation 1 below. An increase in PDI may be indicative ofaggregation or fragments present in the sample.
PDI =Equation 1

LNP’s used in this experiment were analyzed using Focused Beam Reflectance Measurement(FBRM) technology and determined to exhibit the appropriate sphericity for DLS as adetection method.
RiboGreen Assay is a fluorescence based assay that has published detection down
to 1.0 ng/mL RNA and linearity with fluorescence changes verses RNA
concentration3. This assay was used to determine % encapsulation of mRNA.
Encapsulation% =𝑇𝑜𝑡𝑎𝑙 𝑚𝑅𝑁𝐴 − 𝐹𝑟𝑒𝑒 𝑚𝑅𝑁𝐴
𝑇𝑜𝑡𝑎𝑙 𝑚𝑅𝑁𝐴Page | 2

Equation 2Free mRNA: RiboGreen binds to mRNA outside the LNP• TE buffer, sample, RiboGreen dye
Total mRNA: surfactant breaks open LNP, and RiboGreen binds to all mRNA in asample• TE buffer, sample, RiboGreen dye, surfactant
Buffer Blank- adjust samples• TE buffer, RiboGreen
Standard curve Blank- adjusts the standard curve• TE buffer, RiboGreen, surfactant
Encapsulation% = (Total mRNA-Free mRNA)/(Total mRNA)
Total mRNA Concentration: Correlating total mRNA fluorescence with aconcentration on the standard curve

3.2. Experimental Setup
Figure 1: Experimental Equipment Setup
Mettler Toledo FBRM G400 andEasyViewer 400
Sample Port

Discharge Tubing Length toGenerate Backpressure

Temperature
SensorLauda
TCU
50L Single-Use
Magnetic Mixer

Pump Suction line Tubing
Test Pump

Pressure
SensorClamp-On
Flow Meter(Non-Invasive)

The physical testing system consisted of a 50L single-use magnetic mixing system (Sartorius). A
Focused Beam Reflectance Measurement (FBRM) device and an EasyViewer imaging tool wereaffixed/secured through a sanitary fitting at the top of the mixing bag. A recirculation loop ofdisposable tubing was attached to the bottom outlet of the bag and pumped to the top inlet on
the bag through a sub-surface polypropylene dip tube. The recirculation loop included thepumping technology being tested and a pressure sensor, temperature sensor, sample port, anda (non-invasive) clamp-on flow meter. It was ensured that all transitions between the different
inline components with variable inner diameters/bore sizes have smooth transitions. The SingleUse Mixer (SUM) was jacketed and cooled via a temperature control unit to counter thetemperature increase effects of speed, pressure, and duration of the recirculated pumping. ThePage | 3

primary goal of this testing was to assess the impact of different pump technologies on mRNAencapsulated LNPs; therefore – by purpose – all other devices which cause or can cause shear
have been eliminated in the test loop.
Table 2: Equipment | Material List of Test Setup
Equipment | Material
Tank/Vessel

Type
50L single-use magnetic mixing
systemTest Pump A
PuraLev-2000SU with DCP-2000.2
Test Pump B
Quattroflow QF-4400
Suction Line Tubing
1” x 1 3/8” STHT-R x 38” in Length
Discharge Line Tubing
1” x 1 3/8” STHT-R x 16” in Length
Backpressure Tubing Loop for ¾” (ID) x 1” (OD) x 182’ Length Clear
30psi @ 50 L/min
Braided Vinyl Tubing
Backpressure Tubing Loop for ¼” (ID) x ½” (OD) X 78’ Length
45psi @ 23 L/min
PharMed BPT
Single-Use Pressure Sensor
PREPS-N-1-1
Clamp-On Flow Sensor
LeviFlow LFSC-i25x
Temperature Sensor
TEMPS-N-025
Temperature Control Unit (TCU) Variocool VC3000

Manufacturer
Sartorius
Levitronix
Quattroflow
Saint-Gobain
Saint-Gobain
HydroMaxx
Saint-Gobain
Pendotech
Levitronix
Pendotech
Lauda

Table 3: Measurement Equipment
Measurement Equipment
Focused Beam Reflectance
Measurement (FBRM) device
EasyViewer
Particle Sizing (Off-Line)RNA Quantitation

TypeG400

Manufacturer
Mettler-Toledo

400
Zetasizer Pro
Quant-It

Mettler-Toledo
Malvern
ThermoFisher
Scientific3.3. Experimental Method
The following measures were taken during the experiment to minimize variation:
 A consistent volume of LNPs at a target concentration was filled into the 50L single-use
mixing system for each test.
 The single-use mixing system was actively cooled to maintain and control ambient liquid
temperatures (18-25oC) throughout all tests.
 All instrumentation used was within the appropriate calibration and standardization
periods.
 Compendial grade materials were used throughout all testing to ensure quality andconsistency.
 All testing was performed continuously at the same flow rates across pumps for eachpump condition to eliminate flow rate as a test variable. Pumps were swapped in thesystem layout, with no other changes made to the flow path.
Page | 4

The tubing inner diameter and lengths were consistent across all testing per-flow rate
and pressure.
TFF recirculation pumping conditions varied based on processing needs hence a 12-hour
duration for each test condition was used to observe any physical degradation effects.

The single-use maglev centrifugal pump and single-use quaternary diaphragm (4-piston) pumpwere both tested under the following two conditions:
Table 4: Testing Conditions
Flow Rate
Test Condition(LPM)
Lipid Profile 1
Lipid Profile 2

50
23Pressure(psig)
3045

Volume per Test
Run [L]
50-70
50-70

Run Time(hours]1212Fluid
Temperature [C]
18-25
18-25

4. RESULTS
Particle Size (see Figure 2)
The maglev centrifugal pump exhibited a consistent particle-size profile with minimal variation
over the 12-hour testing period with both Lipid profiles and flow rate/pressure conditions. The
diaphragm (4-piston) pump consistently exhibited a significant particle size profile shiftdownwards.
Figure 2: Particle Size Test Results
Lipid Profile 1

Lipid Profile 2

Page | 5

Polydispersity Index (PDI) (see Figure 3)
The maglev centrifugal pump exhibited a consistent PDI profile with minimal variation over the12-hour testing period with both Lipid profiles and flow rate/pressure conditions. However, thediaphragm (4-piston) pump exhibited a significant PDI profile shift upwards.
Figure 3: PDI Test Results for Lipid Profile 1
Lipid Profile 1

Lipid Profile 2% Encapsulation (see Figure 4)
The maglev centrifugal pump exhibited a consistent % encapsulation profile with minimalvariation over the 12-hour testing period with both Lipid profiles and flow rate/pressure
conditions. Of note, the first 1.5 hours of data exhibited slight variation but within +/-5%.
The diaphragm (4-piston) pump exhibited a significant % encapsulation profile shift downwards.
Figure 4: LNP Encapsulation Test Results
Lipid Profile 1

Lipid Profile 2

Page | 6

Figure 5: Liquid Temperature
Lipid Profile 1

Lipid Profile 2

The temperature fluctuations have been in a similar range across all test conditions.
5. CONCLUSIONS
The maglev centrifugal pump technology exhibited consistent results and did not impact theLNP fluid stream during the 12-hour test execution under the conditions investigated.
The diaphragm (4-Piston) pump technology under the same conditions yielded a statisticallyrelevant impact on each of the LNP parameters measured. The particle size shift (smallerparticles), PDI upward shift, and % encapsulation downward shift are all consistent withphysical degradation. The PDI shift indicates that either the mean particle size is shifting, thestandard deviation is varying, or a combination of both is occurring. The experimental results
are evidence of fragmentation and leakage of mRNA from the LNP under the conditions
investigated.
In conclusion, pump choice and operating conditions need further attention to avoid the failure
mechanism of physical degradation of mRNA encapsulated LNPs.
6. REFERENCES
1L Schoenmaker, D Witzigmann, JA Kulkarni, R Verbeke, G Kersten, W Jiskoot, DJA Crommelin(2021) mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Elsevier Inc.
International Journal of Pharmaceutics, Volume 601, 15 May 2021.
2JC Thomas (1987) The determination of log normal particle size distributions by dynamic lightscattering. Elsevier Inc. Journal of Colloid and Interface Science, Volume 117, Issue 1, May 1987,
pages 187-1923LJ Jones, ST Yue, CY Cheung, VL Singer (1998) RNA quantitation by fluorescence-based solution

assay: RiboGreen reagent characterization. Academic Press. Analytical Biochemistry, 265(2),Dec 15 1998. Pages 368-374.
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