K-kit Applications for Biotechnology& Nanopharmaceuticals

www.bioma-tek.com
chrischen@ma-tek.com

PAGE 1OUTLINE EM-based imaging for pharma & CRO K-kit for biotechnology & pharmaceuticals
 What is K-kit
 Conclusion

PAGE 2

EM-based imaging for pharma & CRO
Drug Manufacture (CMC)• Raw material IQC• Drug homogeneity• Uniformity between batches• Process monitoring

Drug discovery and development• Candidate drug screening• Structures and sizes• Stability test
• Pre-clinical study

Pharmacokinetic studies (PK)• In vitro stability• Bioavailability• Sizes and concentrations• Drug Interaction• Excipient effect
Early-phase clinical studies

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Protein particles in Abraxane®
PaclitaxelAlbumin(TEM images by K-kit)• Total calculated particle #: 319• Average size: 85.1 nm• Standard deviation: 27.0 nm* Scale bar: 200 nm

Size/size distribution (D10 , D50 , D90 )Parameter

Size (nm)

D 1055.6

D 50

80.1

D 90

122.2

Span: (D90 - D10 ) / D50

0.831
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NOAAs of Au Nanoparticles (NPs) in Blood
Image-based statistic analysis of particle concentration(K-kit vs. ICP-MS)( Tai etc. Anal. Chem. 2012, 84: 6312-6316 )* Scale bar: 20nm

Statistic analysis of Aggregation andagglomeration of Au NPs in blood

K-kit can be used to analyze physicochemical characteristics of NPs in blood by TEM.
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K-kit for biotechnology& pharmaceuticals
Does ANDA ≈ RLD?
Observing the morphology and size
distribution by electron microscopy

What we can do by using K-kit:⚫ In-situ EM Imaging of biological specimens and Nanopharmaceuticals inaqueous solutions⚫ Immunoelectron microscopy to identify nanogranules in liquid⚫ Image-based statistic analysis allowing nanoobjects, aggregates, andagglomerates (NOAAs) in aqueous condition to be characterized

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Isolated platelet granules in K-kit
AB

200nm

200nm

(A) α-granules are large and round or oval in shape (indicated by white arrows). A
dense granule with a high-density core was also identified (by a white arrowhead).
The small circular vesicles with low electron density are lysosomes or exosomes.
Some broken granules could also be observed. (B) The α-granules (by whitearrows) could be identified with a long axis length of about 200 nm.
PAGE 7

Immunoelectron micrographs of isolated platelet granules
AB

200nm

200nm

(A) This picture shows that the 6-nm gold particle-labeled granules are α-granules.
The other vesicles not significantly labeled by gold particles might be lysosomes
or exosomes. (B) There are abundant gold particles located on the surface of αgranules (indicated by arrows).
( Appl. Sci. 2020, 10, 4946; doi:10.3390/app10144946 )PAGE 8

Drug crystals of Doxorubicin in liposomes(with negative staining)
The enclosed drug crystals in liposomes can be well identified with K-kit.
PAGE 9

Biological specimens observed with K-kit

The nucleoid of E.coli

The nanofiber structures of collagen

PAGE 10

Nanopharmaceuticals observed with K-kit

Resovist® , which a human used MRI
T2 contrast agent with iron oxide
nanoparticles in the solution

Restasis® , cyclosporine ophthalmic
emulsion
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Suitable for most Nanopharmaceuticals⚫ Applicable particle concentration for K-kit: 1011~1014 particles/mlAuroVist® solution was directly loaded
and sealed in a K-kit in liquid form.

Oil emulsion in water was loaded
and sealed in a K-kit in liquid form.

PAGE 12Available for low-contrast sample observation
TEM images of liposomes in Doxil®

Cryo-TEM (In formula)• High cost & difficult• Cryo-holder required• Low success rate

On Cu grid (With N.S.)• Low cost & easy
• At dry state• Serious aggregates

In K-kit (With N.S.)• Low cost & easy
• In-situ liquid state( N.S. - Negative Staining )PAGE 13

The latest news about K-kit

The poster on right side was published in Juneat RCCM Banyuls 2019 meeting in France; K-kit
has been considered as an EM-based imaging
solution for new drug development by thefamous pharmaceutical company SANOFI.

PAGE 14What is K-kit ?
A disposable device for liquid sample analysis in TEM,SEM and FIB, free of cross contamination issue.

Observation
Window

Silicon Body

LiquidChannel

Liquid loading by capillary action

Extracellular vesicles of platelets(With negative staining in K-kit)
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Imaging at original liquid state
Aggregated as drying on Cu grid

Nanoparticles in liquid by K-kit

By using K-kit, one can observe the particles that distributed uniformly in liquid,
preventing the nano particles from dry-induced aggregation and agglomeration.
PAGE 59
16

Slurry Lotion- SiO2 Nanoparticles in CMP Slurry- TiO2 and ZnO Nanoparticles in Sunscreen

TiO2

K-kit Application

Electronics

Cosmetics

Food

Bio-Med

Aggregation/agglomeration

Beverage

Bio Sample- CaCO3 Nanoparticles in Milk

- Au Nanoparticles in Blood

ZnO

Particle concentration

PAGE 17

(Example) NOAAs of ZnO NPs in sunscreen

LotionZnO NOAAs in sunscreen

TEM images

Size and size distribution

K-kit can be used for characterizing NOAAs of cosmetics in final product forms,
to assess the safety risks of nanomaterials in cosmetic ingredients.
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Quick, easy, and budget friendly

K-kit

Copper grid(Compatible with all kinds of TEM holders)

Hitachi
H-7501 SS holder

JEOL
EM31640 STHB holder

FEI
TECNAI F20 holder

Gatan
CT3500HT holder

K-kit is disposable and with a vacuum-encapsulated channel structure so that noany cross-contamination risk and no need to do further cleaning before the use.
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Available for SEM and EDX analysis

SEM/ STEM by FEI Helios 400
100nm Polystyrene beads

10nm Au nanoparticles
HAADF

Bright Field

HAADF

Dark Field

PAGE 20

Available for TEM diffraction analysis

NBD (Nano-beam diffraction)

SAD (Selective area diffraction)

As shown the examples here, Au nanoparticles in K-kit were formed by thereduction process from AuCl4 solution in TEM.
PAGE 21

Product features( Chip size )K-kit( Shipping packages )⚫ Window length 300 𝜇𝑚, Width 25 𝜇𝑚⚫ Channel height (H):
0.2 and 2.0 standard; 0.1, 0.5, 1.0 and 5.0 available( K-kit carrier )H = 0.1, 0.2, 0.5,1, 2, 5 (𝜇𝑚)

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Quick and easy process in sample preparation
1

Channel tips, to ensure the cleannessof the channel before use.

Remove the channel tips
2
3

4

Liquid loading
GluingCopper grid
PAGE 23

K-kit Tool Box for Sample Preparation

Glass-slide pack

K-kit by tools

Gluing standSample-loading stage

Accessory box

Shipping package
K-kit holder & needle pen
PAGE 24

K-kit distributors worldwide

RowacoLabTechTed PellaNano Pioneer

Micro to Nano
EMSSPI

LFG
ANAME

Zhongjingkeyi

MA-Tek Shanghai

HitachiElminet

MA-Tekwww.bioma-tek.com

2014-2020
Microscopy
Solutions

PAGE 25

Conclusion
1. K-kit is a liquid cell designed to facilitate convenient TEM observation ofliquid samples, allowing nanoobjects, aggregates, and agglomerates(NOAAs) in liquid samples to be characterized.
2. K-kit is a Silicon chip; it fits on a 3 mm diameter of copper grid and, hence,is compatible with most existing TEM holders such like FEI, JEOL, Hitachi
and Gatan etc.; it's an affordable and the best option, bringing Liquid TEM
accessible to general researchers and industries.
3. The possible applications of K-kit for pharma & CRO are basically on bothfields in-situ EM imaging and immunoelectron microscopy, which the former
allowing Nanopharmaceuticals to be observed the morphology and size
distribution in aqueous condition and the latter providing unique insights into
biological systems (e.g., to label and identify the proteins in human cells) forthe development and future applications in biomedicine.
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Thank you!

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