Good Practices for CHT™

Ceramic Hydroxyapatite Media UsageCHT Ceramic Hydroxyapatite, Ca10(PO4)6 (OH)2, is an easy-to-use mixed-mode chromatography media. It offers cation exchange and affinity interactions. Unlike otherchromatography media, CHT is both the ligand and the support matrix. Two types of CHT, Type I and Type II, are available in three particle sizes — 20, 40, and 80 μm.
It is used for the purification of numerous types of biomolecules and is considered the gold standard for aggregate removal. In this wall chart, we list guidelines
for the proper use of CHT in biomolecule purification.

THE GOLDSTANDARD

What Can CHT Purify?
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Golden Rules for CHT UseMonoclonal and polyclonal antibodies andantibody fragments
Antibodies that differ in light chain composition
Recombinant proteins and isozymes
Viruses, viral particles, and vaccines
Supercoiled DNA from linear duplexes
Single-stranded from double-stranded DNA

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CHT Packing

Do not compress
Keep pH >6.5 throughout purification
Add phosphate and calcium to all buffers 		
(unless otherwise noted)

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CHT is composed of incompressible particles,
so avoid compression during packing
Compatible with axial (without compression),
flow, and pressure packing
Upflow after packing is not recommended

Binding – Chemical Compatibility

ElutionOptimal binding of biomolecules on CHT depends on the feedstock/buffer components.

Elutions can be performed with phosphate-based buffers to desorb metal affinity
interactions and/or with NaCl-based buffers to desorb cation exchange interactions.

Feed loading

Elution buffer

Commonly used binding buffers
ProteinOH
CHT

Stationary phase

Phosphate-, Tris-, HEPES-, and MES-basedbuffers containing CHT compatible chemicals,such as:POO-POO-

Guanidine-HCl

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Urea

■■+P■■+

H3 NProtein

H3 NOO-■■■■

Acetonitrile■■

Ethanol/methanol■■

SDS and other 			surfactants
Neutral salts■■

CaGlycine■■

Unbound biomoleculesin flowthroughOH■■

POO-

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PP

OOOO-

■■Binding■■■■

H3 NProtein+

H3 N

 cidic proteins and small DNA fragments:A
phosphate gradient
Basic proteins: salt or pH gradient ammalian viruses: up to 600 mM 			M
phosphate buffer
Chromosomal DNA: 0.2– 0.4 M
phosphate buffer (higher if NaCl is present)

Some conditions will cause a drop in pH.1
Elution

EDTA, citrate, and other chelating agents

General Guidelines for Method Development
Proteins withUnknown pI

Protein■■+Chemicals not recommended
in the binding buffer

Buffer

-OOC
-OOC

+

CHT

Ca+

-OOC
-OOC

Commonly used elution buffers

Ca+

Stationary phase

Ca+

Example Method

At pH 6.5–7.5
pH increase stabilizes CHT
Add phosphate ≥5 mM

Elution■■■■■■With phosphate and/or NaCl
Gradient or step
Add phosphate ≥5 mM

Ca+

Load and wash: 5 mM phosphatebuffer, pH 6.5 (buffer A)

Basic Proteins

Binding

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Acidic Proteins

Binding■■■■

Bind primarily to phosphatesites via cation exchange
Calcium affinity may also playa role
Consider flowthroughfor nonbinding targets

Bind primarily to calcium sites
via metal affinity
May be able to bind in high salt
Consider flowthrough for
nonbinding targets

Regeneration■■■■

Elution■■■■■■Elution■■■■

NaCl elution may provide more selectivity
than phosphate
Try 0–1 M NaCl gradient and convert to step
Low levels of phosphate eliminate weak Ca2+
interactions. Try 5 mM, 10 mM, and then 15 mM

Phosphate by itself will elute target from
calcium as well as phosphate sites
Try 0–400 mM phosphate gradient and convert
to step
May be achieved with or without NaCl

Storage

Regenerate with 3–5 CV of 500 mM potassium or
sodium phosphate buffer at ~pH 7.5 or 400 mM
trisodium phosphate at pH 11–12

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OHCHT

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Stationary phase

Ca+

Store unused CHT in the original container atambient temperature
Store regenerated and sanitized CHT in 0.1–1.0 N
NaOH at or below ambient temperature

POO-

POO-

POO-

Gradient 1: Buffer A + 1 M NaCl,then re-equilibrate with buffer A
Gradient 2: Buffer A + 0.40 M
sodium phosphateSanitization■■

 HT Columns can be sanitized in up to 2 N NaOH
Cand stored in 0.1–1.0 N NaOH

Importance of maintaining pH >6.5: increasing the solution concentration of cations displaces protons bound to CHT phosphate groups into solution, which can lead to a drop in pH. Exposure to acidic conditions (pH <6.5) can have a deleterious		effect on the lifetime of CHT.
Changes in pH can be minimized by:■■
Using co-buffers such as MES, MOPS, PIPES, HEPES, ACES, MOPSO, Tris, histidine, lysine, and arginine■■
Performing a surface neutralization right before elution. Typically a wash with 7–8 CV 25 mM Tris, 25 mM NaCl, 5 mM phosphate at pH 7.75■■
Using hydrated buffer salts. Do not use anhydrous sodium phosphate or dodecahydrates as these two salt types can cause irreproducible results.
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Visit bio-rad.com/ProcessResins to get detailed information about CHT Ceramic Hydroxyapatite.
Visit bio-rad.com/ProteinPurification to learn about the different applications for CHT Ceramic Hydroxyapatite.

Bulletin 6932 Ver A 17-0389 0517

Good Practices for CHT™

Ceramic Hydroxyapatite Media Usage

GoodPracticesfor CHT Ceramic Hydroxyapatite™

Media Usage