Looking into the HPLC Column

while Running CIP

Imre SALLAY, PhD

OSAKA SODA CO., LTD., Osaka, Japan

imre@osaka-soda.co.jp

The big application

- By far the biggest RP silica application is

insulin purification.

- Insulin is prone to self-aggregation and

fibrillation

- The fibrillated goo has to be removed from

column, most commonly by NaOH wash

The problem with Insulin

Insulin, insulin analogs and other diabetes

treating drugs (GLP-1) are prone to self-

aggregation, FIBRILLATION

Unprotected (un-bonded) silica melts at pH 13.

Surface modification / bonding makes it

last longer.

But still…

CIP on silica???

Trouble begins with fragments falling off

NaOH at pH 13 is

hydrolyzing siloxane

bonds in the silica matrix.

First small chunks of silica

start falling off (“fines”).

With the chunks of broken off silica bonded ligands are lost.

Problem with LEACHABLES.

Silanol groups get exposed.

Negatively charged silanol groups decreasing selectivity.

The problem with silica

Game over! Did we clean it to death???

Frequent NaOH wash kills the

silica.

What we could not say so far

was whether we cleaned the

silica to death (over clean)

or we did not clean it enough!

We had no scientific tool to

monitor CIP effect on time.

min

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

-- VIRGIN

-- after use

Sample : 1. Uracil, 2. Pyridine, 3. Phenol

Pyridine-phenol test(A model for basic impurities)

Retention time of pyridine became longer.

Silanol effect is increased due to ligand loss.

The problem with degregation

Possible CIP agents:

- Acetic acid or Formic acid

- SDS (with special washing), EDTA if metal ions are sorbed

- Urea, Ammoniumhydroxide, TRIS(to suppress hydrophobic interactions on high pH)

- NaOH in combination with organic solvent(to fragment aggregates and to suppress hydrophobic interactions)

Evaluating current CIP protocols

Simple protocol from literature:

X CV 0.1 n NaOH aq./organic solvent 30/70 (pH 13) is pumped

through the column followed by pH adjustment with acid.

This step is implemented after every five number of purification

cycles.

- Is the NaOH concentration too low or too high?

- Is the frequency adequate?

- Does the silica get cleaned enough?

- Do we over-clean the silica?

0.60

0.70

0.80

0.90

1.00

0 200 400 600 800 1000

ratio to ini

tial

CV

retention time for Naphthalene

15mM 25mM 100mM 250mM

Effect of different NaOH concentration

-

Real, used silica sample analysis:

Elemental analysis

TOP MIDDLE BOTTOM

C% H% N%

VIRGIN 8.6 1.8 0

TOP 12.82 2.27 1.63

MIDDLE 8.43 1.77 0

BOTTOM 8.51 1.77 0

Real, used silica sample analysis:

Chromatographic evaluation

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

uV(x10,000)

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

uV(x100,000)

Mobile Phase : MeOH/H2O=60/40

Flow rate : 0.2mL/min

Oven temp : 40ºC

Detection : UV, 254 nm

Sample :

1. Uracil

2. Methyl benzoate

3. Toluene

4. Naphthalene

Aromatic standard

Mobile Phase : MeOH/H2O=30/70

Flow rate : 0.2mL/min

Oven temp : 40ºC

Detection : UV, 254nm

Sample :

1. Uracil

2. Pyridine

3. Phenol

Basic standard

VIRGIIN

TOP

VIRGIN

TOP

1

2

3

4

1

23

Real, used silica sample analysis:

Chromatographic evaluation

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

uV(x100,000)

Mobile Phase : MeCN/20mM Potassium Phosphate Buffer (pH=3.2)=35/65

Flow rate : 0.2mL/min

Oven temp : 40ºC

Detection : UV, 254nm

Sample :

1. Uracil

2. Benzoic acid

3. p-Toluic acid

4. Methyl benzoate

Acidic standard

VIRGIN

TOP1

2

3

4

Aromatic standard Basic standard Acidic Standard

N4 As4 k'4Pressure(MPa)

As2 As3k'2/k'

3As3 As4

k'3/k‘4

VIRGIN 1,655 1.11 1.97 1.0 1.20 1.13 0.43 1.12 1.09 0.50

TOP 1,227 0.92 2.00 1.1 1.12 1.00 0.42 0.99 0.93 0.61

MIDDLE 1,183 0.89 1.88 1.0 1.07 0.94 0.46 0.96 0.90 0.51

BOTTOM 1,344 1.02 1.87 1.0 1.20 1.08 0.48 1.10 1.05 0.51

Real, used silica sample analysis:

Summary of chromatographic evaluation

Acidic standard test is good indicator to judge how dirty silica is.N : plate number, As ; asymmetry, k’ : (t-to)/to

Elemental analysis

after CIP using alternative agents

C% H% N%

VIRGIN 8.6 1.8 0

TOP 12.82 2.27 1.63

0.1M NaOH (15%ACN)10CV

9.87 1.65 0.46

6M Guanidine hydrochloride (15%ACN) 10CV

11.08 2.00 0.98

8M Urea (15%ACN)10CV

11.11 2.06 1.05

HCOOH (15%ACN)4CV

8.67 1.75 0.18

Aromatic Standard

k’4 : Retention time for Naphtalene

The shorter it goes the more ligand we lose.

Basic Standard

k’2/k’3 : Ratio of retention time for the peaks

The number gets bigger with increasing silanol exposure.

Acidic Standard

k’3/k’4 : Ratio of retention time for the peaks

The number gets smaller with more Nitrogen removed.

Deceptive

Indicator of dirty silica

Shows damage of silica

Standard chromatographic tests

and what they can show us

SUMMARY

Classic chromatography standard tests are used to indicate the state

of the silica stationary phase providing “non-invasive” way.

Now we can “see” whether the silica is cleaned enough or not.

We can “see” how much damage has been inflected on the silica.

These most valuable new tools provide way to re-evaluate the CIP

step in the biggest RP HPLC applications.

Better CIP provides longer silica life, resulting in better API production

on more affordable way.

This way we contribute to the elimination of suffering from this planet.