Question

Asked 11 April 2017

Mahidol University

How do you determine the purity and consistency of biological drugs?

I bought another batch of Vancomycin a while back; however, this batch has a brownish color instead of white like the previous one. According to Sigma, the range of color for vancomycin can be from white to brown, which makes the color valid. I also tried testing it out by running it through UV-vis to detect the peak, which shows up at 281 nm, the place where Vancomycin shows up.

I was confused and asked the supplier what is the reason and what I got is the following:

"Vancomycin can range in color since it is a biological product. Biological products frequently have a range of physical properties since the source material is from a stochastic reactant. I’m not sure how else to address this. Chemical synthesis yields more consistent results than biological processes."

This sparks many questions that I don't know the answer to and would appreciate any help. Mostly, it has to do with my inexperience with biological products.

How do you determine the purity of biological product (and if it is pure, what caused the color differences)? Can

Can biological product be used for testing because won't its properties be inconsistent? If yes, then how do you do it?

If I need to restock the drug, does that mean I need to redo most of the experiment as I am now in possess of a new batch of drug with possibly new properties?

Purdue University West Lafayette

Hi,

I prefer LC-MS as a preferred method to determine the purity of the drug. HPLC is also a very good option, but with MS you can get an additional support for the molecular species.

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Frank T. Edelmann

Otto-von-Guericke University Magdeburg

Yasser Fakri Mustafa

University of Mosul

Naser Jawad Kadhum

University of Kufa

Yunus Shukor

Universiti Putra Malaysia

Corentin Lefebvre

University of Picardie Jules Verne

Farshid Hossein Panahi

University of Tabriz

the determine of purity and consistency of commercial drug is difficult, please study the method of HPLC you can see more information

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1 Recommendation

Adam B Shapiro

Natural product antibiotics are often supplied with bioassay or purity information as a measure of potency. The label will say something like 900 µg/mg, meaning that the product is 90% active ingredient.

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1 Recommendation

James Keane Bashkin

University of Missouri–St. Louis

This is a thorny question. HPLC and activity, both answers given above, are good methods to use, and both are probably essential.

However, it is important to realize that biological drugs can age and go bad with time, so keeping your main supply stored at the lowest recommended temperature and making aliquots that will last a few days or a week can help a great deal with reproducibility (the half-life of the aliquots depends on the specific substance). That way, only small amounts of material are exposed to room temperature or 37 deg C or whatever your experimental conditions are.

What happens when things "go bad"? For example, glutamine and asparagine can hydrolyze to glutamate and aspartate, respectively, if you have a peptide or protein based based drug. These changes can be seen in ion exchange chromatography, capillary electrophoresis or gel electrophoresis, depending on the kind of resolution you need. Some amino acids can epimerize, giving you undesirable isomers. Some non-peptide-based drugs can also epimerize, and this can happen a lot with certain carbohydrates.

So, keeping an eye on the properties of your drug, especially its activity, throughout the course of a set of experiments is important. That's part of the role of positive controls (and why they should be run with every experiment).

The source of color in your sample could be a very tiny amount of highly-colored impurity. Of course, that is speculation on my part. It could also be a lot of moderately colored impurity. but techniques like evaporative light scattering, coupled with UV/Vis detection and HPLC, can sort that kind of problem out. You are allowed to purify purchased materials, and sometimes it is necessary. Published procedures are usually available.

Best of luck!

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1 Recommendation

William P Katt

Cornell University

In general, journals recommend/require 90-95% purity for compounds being used for biological tests, which also means people accept up to 5-10% impurity in testing results. Not that you shouldn't aim for the purest compound possible, but it's hardly necessary to redo experiments just because you got a new stock of compound, so long as the source/purity is similar.

Second, as was said, coloration can be the result of tremendously small impurities, so while it's not bad to pay attention to color, it's not something you should worry much about if the activity seems similar and the purity is similar.

But if you're worried, yes - HPLC or NMR is probably the simplest way to get a reading on purity, and if they both show a clean/pure compound, you're probably fine moving forward, whatever the compound color may be.

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1 Recommendation

Manisha Kotadiya

Parul University

LCMS,NMR,FTIR ARE THE BEST TECHIQUES.COLOUR IMPURITIES U ACN REMOVE BY ANY DECOURISING AGENTS.AND THEN U MIGHT BE GET EXACT PURITY OF DESIRED COMPOUND

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Lakshmy Ramakrishnan

King's College London

In order to determine purity of a particular drug, the best way to analyse is through HPLC - for this however, you need to keep a batch of the drug purchased from a reliable source (i.e. in its purest form - e.g. 99% pure) as the reference standard and compare it to the ones you are not certain of (e.g. another batch from the same manufacturer or from another manufacturer but of different purity).

You will then need to weigh the same amount of drug from each source, run HPLC and compare the peak area between each source with that of the reference standard. There is a formula for this where you will need to plug in the purity claimed for each source. This will indicate to you how pure a particular drug is with reference to your standard.

It is not necessarily true that all the properties of the drug has changed - it is likely that it has degraded due to date of expiry or improper storage/handling - therefore, the main concern would be that the amount of active drug would be considerably less, e.g. 1 mg of drug may not contain 1 mg of active drug any more as it has degraded. This would need to be quantified, otherwise, the safest option would be to purchase a fresh batch of drug and inform the manufacturer of these discrepancies.

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Pradeep Palakshan

Purdue University West Lafayette

Hi,

I prefer LC-MS as a preferred method to determine the purity of the drug. HPLC is also a very good option, but with MS you can get an additional support for the molecular species.

Asked 6 January 2025

Munirah Faleh

Hi everyone,

I'm hoping to connect with some experts on the HepG2 cell line who can share their insights on working with these challenging cells. I'm currently trying to seed them in 6-well plates for viral infection experiments and subsequent RNA extraction.

However, I’m struggling with cell density and their attachment to the wells. I've attempted to seed various cell densities—1,000,000, 800,000, 500,000, 300,000, and 100,000 cells per well—incubating overnight at 37°C with 5% CO2. Unfortunately, all the cells ended up floating, with only a few attaching, even though they adhere well in T75 flasks.

I would greatly appreciate any help from any HepG2 cell line experts who could share strategies or methods for successfully seeding these cells in 6-well plates.

Thank you!

How to create parameter and coordinate files for ligand in amber when FATAL error (maybe due to duplicate bond specifications) happens?

Question

3 answers

Asked 18 July 2024

Marco Carnaroli

Hello,

I'm trying to create parameter and coordinate files for a drug (PRG-A01) found on the following page:

http://www.probechem.com/products_PRG-A01.html

I used the guide made by Michael Barton and Tyler Luchko at the following link: https://ambermd.org/tutorials/basic/tutorial4b/index.php

At first, I obtained the sdf structure thanks to the PDB Chemical sketch tool and converted into pdb using MOE (I simply did a quick prep and saved as pdb):

HEADER

REMARK 99

REMARK 99 MOE v2022.02 (Chemical Computing Group ULC) Thu Jul 18 15:08:13 2024

HETATM 1 O1 * 0 -16.219 1.636 -1.486 1.00 0.00 O1-

HETATM 2 C2 * 0 -16.359 1.987 0.839 1.00 0.00 C

HETATM 3 C3 * 0 -15.215 1.727 1.387 1.00 0.00 C

HETATM 4 C4 * 0 -14.286 1.316 0.345 1.00 0.00 C

HETATM 5 C5 * 0 -14.732 1.265 -1.013 1.00 0.00 C

HETATM 6 O6 * 0 -18.237 2.364 -0.412 1.00 0.00 O1-

HETATM 7 C7 * 0 -13.868 0.878 -2.019 1.00 0.00 C

HETATM 8 C8 * 0 -12.967 0.968 0.644 1.00 0.00 C

HETATM 9 C9 * 0 -12.095 0.571 -0.368 1.00 0.00 C

HETATM 10 C10 * 0 -12.557 0.538 -1.694 1.00 0.00 C

HETATM 11 O11 * 0 -11.720 0.155 -2.719 1.00 0.00 O

HETATM 12 C12 * 0 -10.666 0.191 -0.042 1.00 0.00 C

HETATM 13 C13 * 0 -10.048 -0.656 -1.169 1.00 0.00 C

HETATM 14 C14 * 0 -10.295 -0.012 -2.565 1.00 0.00 C

HETATM 15 O15 * 0 -8.647 -1.082 -0.914 1.00 0.00 O

HETATM 16 C16 * 0 -7.582 -0.324 -0.570 1.00 0.00 C

HETATM 17 C17 * 0 -3.884 -0.967 0.266 1.00 0.00 C

HETATM 18 C18 * 0 -2.759 -0.142 0.425 1.00 0.00 C

HETATM 19 C19 * 0 -3.711 -2.361 0.368 1.00 0.00 C

HETATM 20 C20 * 0 -2.456 -2.913 0.611 1.00 0.00 C

HETATM 21 C21 * 0 -1.348 -2.080 0.760 1.00 0.00 C

HETATM 22 C22 * 0 -1.503 -0.685 0.667 1.00 0.00 C

HETATM 23 C23 * 0 -17.053 2.034 -0.313 1.00 0.00 C2+

HETATM 24 O24 * 0 -0.472 0.237 0.803 1.00 0.00 O

HETATM 25 C25 * 0 0.938 -0.002 1.045 1.00 0.00 C

HETATM 26 O26 * 0 -0.128 -2.662 0.996 1.00 0.00 O

HETATM 27 O27 * 0 -7.741 0.878 -0.488 1.00 0.00 O

HETATM 28 C28 * 0 -6.328 -1.001 -0.330 1.00 0.00 C

HETATM 29 C29 * 0 -5.180 -0.349 0.009 1.00 0.00 C

HETATM 30 C30 * 0 -9.657 1.367 -2.812 1.00 0.00 C

HETATM 31 C31 * 0 -9.841 -0.960 -3.684 1.00 0.00 C

HETATM 32 H1 * 0 -15.058 1.822 2.431 1.00 0.00 H

HETATM 33 H2 * 0 -14.215 0.846 -3.017 1.00 0.00 H

HETATM 34 H3 * 0 -12.658 1.015 1.655 1.00 0.00 H

HETATM 35 H4 * 0 -10.648 -0.384 0.884 1.00 0.00 H

HETATM 36 H5 * 0 -10.104 1.111 0.115 1.00 0.00 H

HETATM 37 H6 * 0 -10.621 -1.586 -1.176 1.00 0.00 H

HETATM 38 H7 * 0 -2.853 0.911 0.359 1.00 0.00 H

HETATM 39 H8 * 0 -4.540 -3.012 0.267 1.00 0.00 H

HETATM 40 H9 * 0 -2.348 -3.965 0.684 1.00 0.00 H

HETATM 41 H10 * 0 1.472 0.947 1.100 1.00 0.00 H

HETATM 42 H11 * 0 1.369 -0.595 0.238 1.00 0.00 H

HETATM 43 H12 * 0 1.079 -0.533 1.988 1.00 0.00 H

HETATM 44 H13 * 0 -0.168 -3.639 1.036 1.00 0.00 H

HETATM 45 H14 * 0 -6.360 -2.051 -0.440 1.00 0.00 H

HETATM 46 H15 * 0 -5.204 0.711 0.095 1.00 0.00 H

HETATM 47 H16 * 0 -9.951 2.073 -2.035 1.00 0.00 H

HETATM 48 H17 * 0 -10.009 1.765 -3.764 1.00 0.00 H

HETATM 49 H18 * 0 -8.571 1.298 -2.846 1.00 0.00 H

HETATM 50 H19 * 0 -8.762 -1.107 -3.654 1.00 0.00 H

HETATM 51 H20 * 0 -10.117 -0.544 -4.654 1.00 0.00 H

HETATM 52 H21 * 0 -10.340 -1.924 -3.580 1.00 0.00 H

CONECT 1 5 23

CONECT 2 3 3 23

CONECT 3 2 2 4 32

CONECT 4 3 5 5 8

CONECT 5 1 4 4 7

CONECT 6 23

CONECT 7 5 10 10 33

CONECT 8 4 9 9 34

CONECT 9 8 8 10 12

CONECT 10 7 7 9 11

CONECT 11 10 14

CONECT 12 9 13 35 36

CONECT 13 12 14 15 37

CONECT 14 11 13 30 31

CONECT 15 13 16

CONECT 16 15 27 27 28

CONECT 17 18 19 19 29

CONECT 18 17 22 22 38

CONECT 19 17 17 20 39

CONECT 20 19 21 21 40

CONECT 21 20 20 22 26

CONECT 22 18 18 21 24

CONECT 23 1 2 6

CONECT 24 22 25

CONECT 25 24 41 42 43

CONECT 26 21 44

CONECT 27 16 16

CONECT 28 16 29 29 45

CONECT 29 17 28 28 46

CONECT 30 14 47 48 49

CONECT 31 14 50 51 52

CONECT 32 3

CONECT 33 7

CONECT 34 8

CONECT 35 12

CONECT 36 12

CONECT 37 13

CONECT 38 18

CONECT 39 19

CONECT 40 20

CONECT 41 25

CONECT 42 25

CONECT 43 25

CONECT 44 26

CONECT 45 28

CONECT 46 29

CONECT 47 30

CONECT 48 30

CONECT 49 30

CONECT 50 31

CONECT 51 31

CONECT 52 31

END

Then I used the following instructions adapted from the guide:

obabel PRG_A01.pdb -O PRG_A01_h.pdb -h

antechamber -i PRG_A01_h.pdb -fi pdb -o PRG_A01.mol2 -fo mol2 -c bcc -s 2

parmchk2 -i PRG_A01.mol2 -f mol2 -o PRG_A01.frcmod

tleap

source leaprc.protein.ff19SB

source leaprc.gaff

PRG = loadmol2 PRG_A01.mol2

loadamberparams PRG_A01.frcmod

saveoff PRG PRG_A01.lib

saveamberparm PRG PRG_A01.prmtop PRG_A01.rst7

The PRG_A01.frcmod file when opened gives:

"Remark line goes here

MASS

BOND

ANGLE

DIHE

IMPROPER

NONBON"

So I don't know if the line of instruction "parmchk2 -i PRG_A01.mol2 -f mol2 -o PRG_A01.frcmod" did properly its job.

Furthermore this is what each instruction returned.

[marco99@narval2 PRG_A01 preparation]$ obabel PRG_A01.pdb -O PRG_A01_h.pdb -h

[mii] Please select a module to run obabel:

MODULE PARENT(S)

1 openbabel/3.1.1 StdEnv/2023 gcc/12.3

2 openbabel-omp/3.1.1 StdEnv/2023 gcc/12.3

3 openbabel/3.1.1 StdEnv/2020 intel/2020.1.217

4 openbabel-omp/3.1.1 StdEnv/2020 intel/2020.1.217

5 gnina/1.0.1 StdEnv/2020 gcc/9.3.0 cuda/11.0

6 openbabel/3.1.1 StdEnv/2020 gcc/9.3.0

7 openbabel-omp/3.1.1 StdEnv/2020 gcc/9.3.0

Make a selection (1-7, q aborts) [1]: 1

[mii] loading StdEnv/2023 gcc/12.3 openbabel/3.1.1 ...

Lmod is automatically replacing "boost-mpi/1.82.0" with "boost/1.82.0".

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

The following dependent module(s) are not currently loaded: boost-mpi/1.82.0 (required by: amber/22.5-23.5)

1 molecule converted

[marco99@narval2 PRG_A01 preparation]$ antechamber -i PRG_A01_h.pdb -fi pdb -o PRG_A01.mol2 -fo mol2 -c bcc -s 2

Welcome to antechamber 22.0: molecular input file processor.

Info: acdoctor mode is on: check and diagnose problems in the input file.

Info: The atom type is set to gaff; the options available to the -at flag are

gaff, gaff2, amber, bcc, and sybyl.

-- Check Format for pdb File --

Status: pass

Info: Determining atomic numbers from atomic symbols which are case sensitive.

-- Check Unusual Elements --

Status: pass

-- Check Open Valences --

Status: pass

-- Check Geometry --

for those bonded

for those not bonded

Status: pass

-- Check Weird Bonds --

Status: pass

-- Check Number of Units --

Status: pass

acdoctor mode has completed checking the input file.

Running: /cvmfs/restricted.computecanada.ca/easybuild/software/2023/x86-64-v3/CUDA/gcc12/openmpi4/cuda12.2/amber/22.5-23.5/bin/bondtype -j full -i ANTECHAMBER_BOND_TYPE.AC0 -o ANTECHAMBER_BOND_TYPE.AC -f ac

Running: /cvmfs/restricted.computecanada.ca/easybuild/software/2023/x86-64-v3/CUDA/gcc12/openmpi4/cuda12.2/amber/22.5-23.5/bin/atomtype -i ANTECHAMBER_AC.AC0 -o ANTECHAMBER_AC.AC -p gaff

Info: Total number of electrons: 222; net charge: 0

Running: /cvmfs/restricted.computecanada.ca/easybuild/software/2023/x86-64-v3/CUDA/gcc12/openmpi4/cuda12.2/amber/22.5-23.5/bin/sqm -O -i sqm.in -o sqm.out

Info: The number of path atoms exceeded MAXPATHATOMNUM for atom (ID: 0, Name: O1).