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Methods to study device induced aggregation of proteins

von Wowern, Marie Therese LU (2023) KLGM15 20231
Food Technology and Nutrition (M.Sc.)
Abstract
This work developed a method for the investigation of how medical devices, such
as syringes and needles, affect the formation of subvisible particles in a protein
drug without excipients. Protein-based pharmaceuticals are well known to be
sensitive to different kinds of stress, and exposure to stress during the use of this
device is unavoidable.
To study this phenomenon, an in-house built probe for the operation of syringes with a texture analyser was created. Mechanical stress was modulated
by varying the expulsion speed, and related forces were captured by the texture
analyser. The formation of particles was evaluated by dynamic light scattering(DLS), size exclusion chromatography(SEC) and flow imaging microscopy by
FlowCAM.
The... (More)
This work developed a method for the investigation of how medical devices, such
as syringes and needles, affect the formation of subvisible particles in a protein
drug without excipients. Protein-based pharmaceuticals are well known to be
sensitive to different kinds of stress, and exposure to stress during the use of this
device is unavoidable.
To study this phenomenon, an in-house built probe for the operation of syringes with a texture analyser was created. Mechanical stress was modulated
by varying the expulsion speed, and related forces were captured by the texture
analyser. The formation of particles was evaluated by dynamic light scattering(DLS), size exclusion chromatography(SEC) and flow imaging microscopy by
FlowCAM.
The insulin was found to be primarily present in its hexamer form, with a hydrodynamic diameter of 5.6 nm. FlowCAM showed increased concentration of particles in both siliconized and non-siliconized syringes after mechanical stress.
The increase was associated with protein aggregates in the non-siliconized syringe, while increase in the the siliconized was mainly associated to silicone oil
droplet formation. The particles were categorized by sphericity using circle fit
and aspect ratio, with spherical cutoff at 0.85. The siliconized syringe had no
increase in protein aggregates after stress and displayed a substantial loss of native protein (2.5 %) according to SEC. This concedes with findings from other studies and might be due to oil interface adsorption. Although, the size distribution from FlowCAM yielded particle counts, which were theoretically too low to account for this loss alone. (Less)
Popular Abstract (Swedish)
Läkemedel som består av skräddarsydda proteiner används idag för att behandla bland annat diabetes. De ges traditionellt som injektioner och detta misstänks vara kopplat till skapandet av skadliga partiklar. Sprutan och nålen är ofta silikoniserade för att underlätta hantering och deras användning utsätter det känsliga proteinet för mekaniska krafter. Detta tros leda till bildandet av partiklar i form av silikon droppar och protein klumpar (aggregat). Detta arbete utvecklade därför en metod för att undersöka effekten av sprutor med nål på ostabiliserat insulin.
En 3D printad spruthållare till en maskin som kan trycka och dra samtidigt som den mäter kraften (texturometer) designades för att kunna manövrera sprutan.
Effekten av mekanisk... (More)
Läkemedel som består av skräddarsydda proteiner används idag för att behandla bland annat diabetes. De ges traditionellt som injektioner och detta misstänks vara kopplat till skapandet av skadliga partiklar. Sprutan och nålen är ofta silikoniserade för att underlätta hantering och deras användning utsätter det känsliga proteinet för mekaniska krafter. Detta tros leda till bildandet av partiklar i form av silikon droppar och protein klumpar (aggregat). Detta arbete utvecklade därför en metod för att undersöka effekten av sprutor med nål på ostabiliserat insulin.
En 3D printad spruthållare till en maskin som kan trycka och dra samtidigt som den mäter kraften (texturometer) designades för att kunna manövrera sprutan.
Effekten av mekanisk påverkan undersöktes genom att variera tömningshastighastighen av sprutan. Endast hastigheter som var associerade med laminärt flöde underöktes. Osilikoniserade sprutor jämfördes mot silikoniserade.
Tre vanligt förekommande analysmetoder (dynamisk ljusspridning, storlekskromatografi (SEC) och FlowCam) användes för att mäta partikelkoncentration och storlek.
Spruthållaren i vit nylonplast presterade bra, tog tre dagar att skapa och kostade en tiondel av färdiga alternativ på marknaden.
FlowCam mätningarna visade att partikelkoncentrationerna höll sig under tillåtna nivåer och steg med ökad tömningshastighet. FlowCam tog bilder av de individuella partiklarna i proven. Dessa kunde sedan användas för att se vilka slags partiklar proven innehöll.
Silikondropparna var helt sfäriska med slät kant och hade ibland en reflektiv mitt.
Det visade sig att koncentrationsökningen i den silikoniserade sprutan berodde på ökat antal silikonoljedroppar. I den osilikoniserade sprutan berodde ökningen istället på ett ökat antal partiklar som var ojämna, mörkare och med skrovlig kant. Dessa antogs vara proteinaggregat.

Metoden som utvecklades kan i framtiden användas för att undersöka andra proteiner eller effekten av andra faktorer såsom övergångsflöde, uppdragshastighet eller luftintroduktion. (Less)
Please use this url to cite or link to this publication:
author
von Wowern, Marie Therese LU
supervisor
organization
course
KLGM15 20231
year
type
H2 - Master's Degree (Two Years)
subject
keywords
protein formulation, protein aggregation, insulin, silicone oil, syringe, shear stress, subvisible particle, size exclusion chromatography, flow imaging microscopy, pharmaceutical technology
language
English
id
9136545
date added to LUP
2023-09-07 10:39:43
date last changed
2023-09-07 10:39:43
@misc{9136545,
  abstract     = {{This work developed a method for the investigation of how medical devices, such
as syringes and needles, affect the formation of subvisible particles in a protein
drug without excipients. Protein-based pharmaceuticals are well known to be
sensitive to different kinds of stress, and exposure to stress during the use of this
device is unavoidable.
To study this phenomenon, an in-house built probe for the operation of syringes with a texture analyser was created. Mechanical stress was modulated
by varying the expulsion speed, and related forces were captured by the texture
analyser. The formation of particles was evaluated by dynamic light scattering(DLS), size exclusion chromatography(SEC) and flow imaging microscopy by
FlowCAM.
The insulin was found to be primarily present in its hexamer form, with a hydrodynamic diameter of 5.6 nm. FlowCAM showed increased concentration of particles in both siliconized and non-siliconized syringes after mechanical stress.
The increase was associated with protein aggregates in the non-siliconized syringe, while increase in the the siliconized was mainly associated to silicone oil
droplet formation. The particles were categorized by sphericity using circle fit
and aspect ratio, with spherical cutoff at 0.85. The siliconized syringe had no
increase in protein aggregates after stress and displayed a substantial loss of native protein (2.5 %) according to SEC. This concedes with findings from other studies and might be due to oil interface adsorption. Although, the size distribution from FlowCAM yielded particle counts, which were theoretically too low to account for this loss alone.}},
  author       = {{von Wowern, Marie Therese}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Methods to study device induced aggregation of proteins}},
  year         = {{2023}},
}