In vitro / In vivo ADME (PK, LC-MS/MS Analysis)

With Kolmar BioTech’s ADME services, you can assess the drug’s metabolic stability and pharmacoki- netic (PK) properties. Our services are delivered using advanced techniques, including LC-MS/MS, microplate readers, and nephelometry.

Main Services

In vitro ADME

Aqueous solubility (Nephelometry)

CYP % Inhibition & IC50 (Microplate reader)

hERG fluorescence polarization assay IC50 (Microplate reader)

Plasma protein binding (LC-MS/MS)

Plasma stability (LC-MS/MS)

Liver microsomal stability (LC-MS/MS)

GI-PAMPA permeability assay (LC-MS/MS)

BBB-PAMPA permeability assay (LC-MS/MS)

Formulation test (Stability, homogeneity)

In vivo ADME (PK: Pharmacokinetics)

Rodent PK study (includes Cassette PK, up to 5 drugs)

Non-rodent PK study: Preliminary investigation in bioequivalence test using

Beagle dog

Formulation test using beagle dog

Dose dependent study

Bioavailability

Gender difference

Food effects

Tissue distribution (includes tumor)

BB assessment(Kp value : Brain to plasma partition coefficient)

Metabolite analysis

Urine, bile, feces analysis

LC-MS/MS Analysis

With years of experience in animal testing, Kolmar BioTech provides a customized onestop service for natural products and metabolites, quantification and qualification, method development and validation by utilizing a professional analysis service that quickly and accurately measures drug concentration in samples (blood, tissue, urine, etc.) and highperformance LC-MS/MS equipment equipped with IonDrive technology suitable for new drug development.

Key Equipment

• QTRAP 6500+ LC-MS/MS
  [AB SCIEX]
• QTRAP 5500+ LC-MS/MS
  [AB SCIEX]

• IonDrive High Energy Detector+
• Sensitivity (MRM mode)
  : Positive ESI 1 pg for reserpine
  : Negative ESI 1 pg for Chloramphenicol
• Ion source : ESI, APCl

• QTRAP 6500+ LC-MS/MS
  [AB SCIEX]
• QTRAP 5500+ LC-MS/MS
  [AB SCIEX]

• Sensitivity (MRM mode)
  : Positive ESI 5 pg for reserpine
  : Negative ESI 5 pg for chloramphenicol
• Ion source : ESI, APCl

PK Profile of "Drug A"

NMR

NMR is a powerful analytical technology that provides molecular structure information that can be used directly by customers or by means of measurement to help solve technical problems. It is applied to the qualitative and high-dimensional molecular structure analysis of various organic compounds through 1 H and 13C analysis.

Main Services

Key Equipment

• AVANCE NEO 400 MHz [BRUKER]

• Magnet field strength : 9.4 Tesla (400 MHz)
• Automation System
  : SampleCase Module
  : Sample diameters from 1 - 10 mm
  : All sample tube length (including Shigemi tubes)
• Resolution for 13 C
  : less than 0.2 Hz at 50 %
  : 2 Hz at 0.55 %, 4 Hz at 0.11 %
• Probehead
  : 5 mm X-nuclei-optimized double resonance broad
  banded probe

STD NMR – binding assay

No.	Sample Name (Product Name)	Chemical shift (ppm)		Integral[rel]	MoD (%)
1	Crosslinked Sample	BDDE	1.491	0.0437	3.28
		Hyaluronic Acid	1.890	1.0000

SPR Analysis

Since launching our SPR analysis services in 2010, Kolmar BioTech has leveraged years of expertise to provide customized consultations and deliver rapid, accurate results tailored to various material properties. All SPR systems are maintained daily, weekly, and monthly according to Kolmar BioTech’s standard operating procedures. Additionally, our systems undergo annual inspections and maintenance by Cytiva, ensuring reliable data for our clients.

Main Services

Key analytes

• Small molecules
• Biomolecules
  (DNA, RNA, lipids, cells, viruses)
• Transmembrane proteins (TPs)
• Bispecific antibody 
• Immune checkpoint inhibitors (ICIs)
• SARS-CoV-2 Spike  
• Antibody drug conjugates (ADCs) 
• PROTAC 

Key Equipment

• Biacore T200 [Cytiva]
• Reichert SR7500D
  [Reichert]

Channel : 4(either parallel or series fluid connection)
Detection: No lower limit for chemical

Sample volume : 2-350 uL
Sample Conc. Range : 1 pM - 1 mM
Sample capacity : Max. 384 (vial or plate)

• Biacore T200 [Cytiva]
• Reichert SR7500D
  [Reichert]

Channel : 2(either parallel or series fluid connection)
Detection : > 100 Da

Sample volume : 1-5000 ul
Sample Conc. Range : 1 pM – 1 mM
Sample capacity : Max. 768 (vial or plate)

Surface Plasmon Resonance (SPR) Analysis
Utilizes the optical principle of SPR to detect real-time, label-free interactions between biomolecules, enabling precise analysis of molecular affinity and binding parameter

Typical SPR sensorgram

Binding Level Screening test

Competitive Inhibition Assay

Protein-protein interactions (PPIs) Inhibitor의 IC50 Values

Fc Receptor Binding Assay

Determination of the Binding of IgGs to FcγR by SPR

Simultaneous Binding of Bispecific Antibody

Reference supported by Kolmar BioTech

• Front Pharmacol, Allergic Inflammation Caused by Dimerized Translationally Controlled Tumor Protein is Attenuated by Cardamonin. Oct 6, 2021
• Front J Hematol Oncol, KAI1(CD82) is a key molecule to control angiogenesis and switch angiogenic milieu to quiescent state. Sep 16, 2021
• Front Oncol Lett, MED28 increases the colony-forming ability of breast cancer cells by stabilizing the ZNF224 protein upon DNA damage. December 29, 2017
• FrontJ Hematol Oncol, Evaluation of polyphenols from Broussonetia papyrifera as coronavirus protease inhibitors. Jan 22, 2017
• FrontBioorg Med Chem, Dieckol, a SARS-CoV 3CL(pro) inhibitor, isolated from the edible brown algae Ecklonia cava. April 22, 2013
• FrontNature communications, Structural basis for recognition of autophagic receptor NDP52 by the sugar receptor galectin-8. Published 19 Mar, 2013
• FrontNature communications, Protein L-isoaspartyl methyltransferase regulates p53 activity. Published 26 jun, 2012
• FrontAnalytical Biochemistry, Harnessing aptamers for electrochemical detection of endotoxin. 25 February, 2012

Equipment List

NL-3000 [CYTEK™]

Laser : 405nm, 488nm, 640nm Violet(420 - 829nm), Blue(498 - 829nm), Red(652 - 829nm)  detector module Max color 9 color blue laser panel, 24 colors with three lasers Standard optical configuration Fluorescence linearity FITC R2 ≥ 0.995 / PE R2 ≥ 0.995 Resolution Capable of resolving 0.2 μm beads from noise 12 x 75 mm polystyrene and polypropylene tubes Without  Plate Loader Option Temperature 4 - 30 ℃

EPOCH™2 [BioTEK]

Wavelength range 200 - 999 nm Photometric resolution 0.0001 OD Microplate types 6 - 384 well plates Temperature control 4-Zone incubation to 65 °C Shaking Linear, orbital, double-orbital Detector Photodiode Read methods Endpoint, kinetic, well area scanning

405TS [BioTEK]

Compatible microplates 96 - and 384 - well Shake options 3 speeds Up to 600 sec Precision < 3 % CV @ 300 μL (96-well)≤ 4 % CV @ 80 μL (384-well) Wash volume 25 - 3000 μL

Maximum solvent : 4

System pressure Up to 600 bar Flow range 0.2 to 10 ml/min with G7111A Maximum solve 4 Pump Isocratic, Quaternary, Gradient Well plate MAX 132 Sample Load Detecter Photodiode array

DS-11 [Denovix]

Minimum Sample Size 0.5 μL Detector Type 2048 element CCD Wavelength Range 190 - 840 nm Wavelength Accuracy 0.5 nm Spectral Resolution 1.50 FWHM @253.65 nm Detection Limit 0.04 mg/mL BSA; 0.75 ng/μL dsDNA

YLZ-1248R [Labogene]

Max RPM/RCF Fixed angle 12,000/16,582 xg, Swing out 5,000/5,394 xg Max Capacity Fiixed angle 6*85 ml, Swing out 4*250 ml/250 ml conical Temp range -20 ℃ - 40 ℃ Pulse, timed < 10 hr or continuous

NovoCyte 1000 [ACEA Bioscience]

Laser 488 nm Detector Blue(530/30 nm, 572/28 nm, 675/30 nm) Cell size Blue(530/30 nm, 572/28 nm, 675/30 nm) Fluorescence linearity FITC R2 ≥ 0.995 / PE R2 ≥ 0.995 Resolution Capable of resolving 0.2 μm beads from noise 12 x 75 mm polystyrene and polypropylene tubes Without  Plate Loader Option Temperature 4 - 30 ℃

SpectraMax M2 [Molecular Devices]

Wavelength range 200 - 1000 nm Photometric resolution 0.001 OD Plate formats Cuvette or 6 - 384 well plates Temperature control 4 - 45 °C Shaking 0 to 999 seconds Detector Absorbance(Photomultiplier tube(PMT)) Dual monochromators

NEPHELOsterPlus [BMG Labtech]

Detection modes Nephelometry (light scattering) Measurement modes Endpoint and kinetic Microplate formats 6 - 384 well plates Wavelength Selection Photodiode Wavelength 635±10 nm Incubation +4 °C above ambient up to 45 °C or 60 °C

SpectrumTwo [Perkin Elmer] / MIRacle [PIKE]

Wavelength range 8,300 - 350 cm-1 (KBr), 6,500 - 550 cm-1 (ZnSe) Spectral Resolution 0.5 cm-1 standard S/N Ratio 9,300:1 peak-peak, 5 sec / 32,000:1 peak-peak, 1 min Wavelength Precision 0.1 cm-1 at 3,000 cm-1 Wavelength Accuracy Better than 0.01 cm-1 at 3,000 cm-1 Detector Type DTGS(deuterated triglycine sulfate) ATR Sampling

LightCycler 96 System [Roche]

Range of excitation/emission wavelengths (nm) 470/514 (SYBR, FAM, ResoLight dye) 533/572 (VIC, Hex, Yellow555) 577/620 (Red610, Texas Red), 645/697 (Cy5) Sample format 96-well plates, 8-tube strips Reaction volumes 10 – 50 ul Run time < 40 min for 3-step 40 cycles PCR Programmable temperature range 37 – 98 °C Detecter CCD camera

TurboVap LV [Biotage]

Vial capacity 24 or 48 Sample volume 1.5 - 60 ml Operating Tem 18 - 32 °C Heating Duration 40 min Maximum Pressure 9 bar (0.9 MPa, 130 PSI)