CHROMATOGRAPHYMPC™ Ceramic Hydroxyfluoroapatite
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 igh physical andH
chemical stability
Unmatched selectivity learance of impurities
Cand aggregates ina single step apid and simple
Rcolumn packing

Separation of Impurities in a Single Step
The newest addition to Bio-Rad’s line ofceramic apatite chromatography media is MPC
ceramic hydroxyfluoroapatite. MPC is a secondgeneration CHT ™ ceramic hydroxyapatite mixedmode chromatography media, a composite ofhydroxyapatite and fluoroapatite prepared bychemically substituting 25% of the hydroxyl groupsof hydroxyapatite nanocrystals with a fluorine reagent.
Its chemical formula is Ca10(PO4)6(OH)1.5(F)0.5.
The partial fluorination confers a more chemicallystable form of the matrix during pH excursions thatare inherent to buffer exchanges. MPC has uniqueseparation properties, unmatched selectivity, andresolution similar to CHT, including binding capacity(Tables 1 and 2), protein separations (Figure 1),and clearance (Figures 2 and 3, Table 3). MPC is
40 µm and is directly comparable to CHT Type I,
40 µm (Tables 1 and 2).

Mechanism of Action and Standard
Chromatography
MPC interacts with biomolecules by multiple modes.
Cation exchange occurs when negatively charged
phosphate groups interact with protein amino groups.
Much stronger coordination complexes can form
between carboxyl clusters, phosphoryl moieties,
or both on biomolecules and the calcium sites on
MPC via metal affinity. Repulsion effects and thegeometric charge distribution on MPC provide
unique selectivity. Typically, acidic, basic, and neutral
proteins are bound to MPC using low ionic strength
phosphate buffer. Elution is accomplished throughthe use of a sodium chloride or phosphate gradientof increasing strength or via similar step elutions.
Regeneration of the support with phosphate buffers
at neutral pH is followed by sanitization with up to2 N NaOH. Regardless of greater chemical stability,
we recommend implementing pH mitigation steps,
for example, the surface neutralization system (SNS).
A pilot-scale study with MPC using SNS achieved
125 cycles. For more detailed information, refer to theCHT user’s guide at www.bio-rad.com/CHTGuide.

Table 1. Specifications.
CHT Type I, 40 µm

MPC

Functional groups

Ca 2+, PO4, OH

Ca 2+, PO4, OH, F

Observed dynamic binding capacitylysozyme (Lys)≥25 mg Lys/g CHT≥25 mg Lys/g MPC

Nominal pore diameter

600–800 Å

600–800 Å

Maximum backpressure

100 bar (1,500 psi)

100 bar (1,500 psi)

Nominal mean particle size

40 ± 4 μm

40 ± 4 μm

Tap settled density*(g/ml tap settled bed)

0.63 g/ml

0.72 g/ml* Under ideal conditions.

Table 2. Characteristics.
CHT Type I, 40 µm

MPC

Observed dynamic binding capacity IgG

25–60 mg IgG/ml CHT*

25–50 mg IgG/ml MPC**Typical linear flow rate range

50–1,000 cm/hr

50–1,000 cm/hr

pH stability

6.5–14 pH

6.5–14 pH

Base stability

At least 1 year in 0.1 N NaOH

At least 1 year in 0.1 N NaOH

Regeneration

0.4–0.5 M sodium phosphate, pH 7–7.5, is generally
sufficient. If higher concentrations are needed,
use potassium phosphate

0.4–0.5 M sodium phosphate, pH 7–7.5, is generally
sufficient. If higher concentrations are needed, usepotassium phosphate

Autoclavability (bulk)

121°C, 20 min in phosphate buffer, pH 7

121°C, 20 min in phosphate buffer, pH 7Sanitization

1–2 N NaOH

1–2 N NaOH

Recommended column storage

0.1 N NaOH

0.1 N NaOH

* 40 μm particles, 300 cm/hr, 5 mM sodium phosphate, pH 6.5.
** 40 μm particles, 300 cm/hr, 5 mM sodium phosphate, 25 mM NaCl, pH 6.5.
0.100

50

Myoglobulin

40

Cytochrome C

3530

Ovalbumin

0.050

0.06

25200.025

15

Absorbance unit

Absorbance unit

0.075

0.07

45

Conductivity, mS/cm

a-chymotrypsinogen A–0.01060

70Volume, ml

0.0205500.04
0.03

0.01

1000.05080		51015Volume, ml

Fig. 1. Separation of protein standards. MPC separates the standard protein
mixture similarly to CHT Type I, 40 µm. Sample: 6 mg of ovalbumin, 3 mg ofmyoglobulin, 3 mg of a-chymotrypsinogen A, and 3 mg of cytochrome C in 1.5 ml
10 mM sodium phosphate (NaPi), pH 6.8. CHT column: 0.5 x 10.3 cm, packed bed
volume 2 ml; MPC column: 0.5 x 10.4 cm, packed bed volume 2 ml. Protocol: 1.55
ml/min; 400 mM NaPi, pH 6.8, 5 column volumes (CV); 10 mM NaPi, pH 6.8, 15 CV;
load volume 50 µl; linear gradient elution 0 to 75% 400 mM NaPi, pH 6.8, over 15 CV;
strip 400 mM NaPi, pH 6.8, 5 CV; sanitization 1 N NaOH, 5 CV. CHT (—); MPC (—).

Fig. 2a. MAb-S size exclusion chromatography (SEC) profile of starting material.
SEC profile shows the higher molecular weight impurities (dimers/aggregates) andmonomers in the starting material (material applied to the second step (Figure 2b)
using CHT or MPC). Sample: 2.9 mg/ml mAb-S in 10 mM NaPi, pH 7. Column:
300 x 7.8 mm, Bio-Sil® SEC 250 HPLC. Protocol: 1 ml/min; equilibration in 1 ml of
0.1 M NaPi, 0.15 M NaCl, 0.02% azide, pH 7; injection volume 0.1 ml; elution in15 ml of 0.1 M NaPi, 0.15 M NaCl, 0.02% azide, pH 7.

1.00

0.040

0.75

0.030
Absorbance unit

Absorbance unit

0.035

0.50

0.25

0.025
0.020
0.015
0.010
0.005

0.00

0020

4060
Volume, ml

80100Fig. 2b. MAb-S purification profile. Elution profile shows separation of the monomerfrom higher molecular weight impurities. Sample: 7.26 mg mAb-S/ml packed bedin 5 ml 10 mM NaPi, pH 7. Column: 0.5 x 10 cm, packed bed volume 2.1 ml ofUNOsphere SUPrA™ rProtein A media. Protocol: 0.5 ml/min; 10 mM NaPi, pH 7,5 column volumes (CV); load volume 5 ml; 10 mM NaPi, pH 7, 5 CV; linear gradientelution 0 to 100% 10 mM NaPi, 1 M NaCl, pH 7, over 40 CV; strip 10 mM NaPi,
1 M NaCl, pH 7, 5 CV; sanitization 1 N NaOH, 5 CV. CHT (—); MPC (—).

© 2013 Bio-Rad Laboratories, Inc.

051015Volume, ml

Fig. 2c. SEC profile of pooled monomer fractions. The SEC profile of the pooled
fractions confirms aggregate clearance from the monomer. Column: 300 x 7.8 mm,Bio-Sil SEC 250 HPLC. Protocol: 1 ml/min; 100 mM NaPi, 150 mM NaCl, 0.02% azide,
pH 7, 1 ml; load volume 0.1 ml; elution 100 mM NaPi, 150 mM NaCl, 0.02% azide, pH
7, 15 ml. CHT (—); MPC (—).

Bulletin 6432

0.125
2.00
0.100

1.75
1.50
Absorbance unit

Absorbance unit

0.075

0.0500.025

1.25
1.00
0.75
0.50

0.000

0.25
0–0.02551015020Volume, ml

Fig. 3a. MAb-G SEC profile of starting material. SEC profile shows the higher
molecular weight impurities (dimers/aggregates) and monomers in the starting
material (material applied to the second step (Figure 3b) using CHT or MPC).
Sample: 4.41 mg/ml mAb-S in 10 mM NaPi, pH 7. Column: 300 x 7.8 mm,
Bio-Sil SEC 250 HPLC. Protocol: 1 ml/min; equilibration in 1 ml of 0.1 M NaPi,
0.15 M NaCl, 0.02% azide, pH 7; injection volume 0.1 ml; elution in 15 ml of0.1 M NaPi, 0.15 M NaCl, 0.02% azide, pH 7.

4060
Volume, ml

80100Fig. 3b. MAb-G purification profile. Elution profile shows separation of the monomerfrom higher molecular weight impurities. Protocol as described in Figure 2b. CHT (—);
MPC (—).

1.00

Absorbance unit

0.75

0.50

0.25

0051015Volume, ml

Fig. 3c. SEC profile of pooled monomer fractions. The SEC profile of the pooledfractions confirms aggregate clearance from the monomer. Protocol as described inFigure 2c. CHT (—); MPC (—).
Table 3. Quantified data from all assays. All pooled fractions and mAb starting material purified on MPC and CHT were analyzed for host cell protein (HCP ELISA kit,Cygnus Technologies), for dsDNA (PicoGreen dsDNA quantitation kit, Invitrogen Corporation), for protein A leachables (Protein A ELISA kit, Cygnus Technologies), and for
aggregate clearance by SEC (Bio-Sil SEC 250, Bio-Rad Laboratories, Inc).
SampleIgG Concentration,
mg/ml

Protein A, ppm

HCP, ppm

DNA, ppm

Dimer/Aggregate, %MAb-S
Protein A eluate

2.9033242121.62

CHT pool

0.40<0.6<22.6<0.03

MPC pool

0.46

<0.5

<22.8

<0.03

MAb-GProtein A eluate

4.41

17<20.82

4.39

CHT pool

0.91

<0.3

<11.4

<0.03

MPC pool

0.77

<0.3

<11.4

<0.03

© 2013 Bio-Rad Laboratories, Inc.

Bulletin 6432

BSA pl = 4.9

Apo ferritin pl = 5.0–5.5

0510Run time, min

400.010

20015005Fetuin pl = 3.3

600.015400.0100.005

200010Run time, min

Absorbance unit

80

Conductivity, mS/cm

Absorbance unit

0.025

515400.02

20000510Run time, min

15Pepsin pl = 2.90.020

010Run time, min

600.04800.030

600.020400.0102000150510Run time, min

Conductivity, mS/cm

00.020800.06

Conductivity, mS/cm

10060

Absorbance unit

200.050

800.030

Conductivity, mS/cm

300.100

Absorbance unit

40Conductivity, mS/cm

Absorbance unit

500.150

Carboxypeptidase pl = 4.6–5.20.040

15Fig. 4. Elution profiles of five acidic proteins. CHT and MPC produce similar elution characteristics for the five acidic proteins studied. Samples: porcine carboxypeptidase B(CBP), pI 4.6–5.2, pepsin pI 2.9, fetuin, pI 3.3, BSA, pI 4.9, and apoferritin, pI 5.0–5.5, each prepared to a final concentration of 2–3 mg/ml in water. Column: 0.7 x 2.6 cm,packed bed volume 1 ml, executed using a BioLogic DuoFlow™ system and software. Protocol: 300 cm/hr; load volume 100 µl; linear gradient elution 0 to 100% 0.02 M HEPES,0.01 M NaPi, pH 6.5, to 0.02 M HEPES, 0.4 M NaPi, pH 6.5, over 20 CV. CHT (—); MPC (—).
1, 20.8

Phosphate groupAdenosine (1)AMP (2)
ADP (3)
ATP (4)301235040

A 260

300.4

20Conductivity, mS/cm

40.60.2
10pH 6.8
00020

40

6080

100

Run time, minFig. 5. Nucleic acids separation profile. Elution profile shows separation of ADP andATP. Sample: 1 mg each of adenosine, AMP, ADP, and ATP mixture dissolved in 1 ml of10 mM NaPi, pH 6.8, filtered with 0.22 μm membrane. Column: 0.4 x 10 cm. Protocol:
1 ml/min; load volume 0.03 ml; wash 10 mM NaPi, pH 6.8, 2 CV; linear gradient elution
0 to 75% 400 mM NaPi, pH 6.8, over 12 CV; strip 400 mM NaPi, pH 6.8, 4 CV. CHT (—);
MPC (—).

© 2013 Bio-Rad Laboratories, Inc.

Bulletin 6432

Storage
MPC ceramic hydroxyfluoroapatite should be stored in
0.1 N NaOH at room temperature. In dry powder form, MPC
ceramic hydroxyfluoroapatite should be stored in a secured,closed container at room temperature.
Technical AssistanceFor more detailed information on process step development,
use recommended steps as described in the CHT Applications
Guide (www.bio-rad.com/CHTGuide). Regulatory support fileis available upon request. Bio-Rad Laboratories is an ISO 9001
registered corporation. For additional information and technicalassistance, contact your local Bio-Rad office. In the USA andCanada, call 1-800-4BIORAD. You can also send an email tolsg_techserv_us@bio-rad.com. Visit us on the Web atwww.bio-rad.com for more information on Bio-Rad’s completeline of process chromatography supports.
Ordering Information
Catalog #Catalog #DescriptionCHT Ceramic Hydroxyapatite, Type II
158-2200
CHT Ceramic Hydroxyapatite, 20 μm, Type II, 10 g
157-2000
CHT Ceramic Hydroxyapatite, 20 μm, Type II, 100 g
157-2100
CHT Ceramic Hydroxyapatite, 20 μm, Type II, 1 kg
157-2500
CHT Ceramic Hydroxyapatite, 20 μm, Type II, 5 kg
158-4200
CHT Ceramic Hydroxyapatite, 40 μm, Type II, 10 g
157-4000
CHT Ceramic Hydroxyapatite, 40 μm, Type II, 100 g
157-4100
CHT Ceramic Hydroxyapatite, 40 μm, Type II, 1 kg
157-4500
CHT Ceramic Hydroxyapatite, 40 μm, Type II, 5 kg
158-8200
CHT Ceramic Hydroxyapatite, 80 μm, Type II, 10 g
157-8000
CHT Ceramic Hydroxyapatite, 80 μm, Type II, 100 g
157-8100
CHT Ceramic Hydroxyapatite, 80 μm, Type II, 1 kg
157-8500
CHT Ceramic Hydroxyapatite, 80 μm, Type II, 5 kg
Foresight Columns
732-4735
Foresight™ CHT™ Type I Column, 40 μm, 1 ml
732-4755
Foresight CHT Type I Column, 40 μm, 5 ml
732-4736
Foresight CHT Type II Column, 40 μm, 1 ml
732-4756
Foresight CHT Type II Column, 40 μm, 5 ml
Foresight Plates*732-4716
Foresight CHT Type I Plates, 40 μm, 20 μl
732-4718
Foresight CHT Type II Plates, 40 μm, 20 μl

Description

MPC Ceramic Hydroxyfluoroapatite, Type I
158-0200
MPC Ceramic Hydroxyfluoroapatite, 40 µm, Type I, 10 g
157-0200
MPC Ceramic Hydroxyfluoroapatite, 40 µm, Type I, 100 g
157-0201
MPC Ceramic Hydroxyfluoroapatite, 40 µm, Type I, 1 kg
157-0205
MPC Ceramic Hydroxyfluoroapatite, 40 µm, Type I, 5 kg
Foresight™ Columns
732-4737
Foresight™ MPC™ Type I Column, 40 μm, 1 ml
732-4757
Foresight MPC Type I Column, 40 μm, 5 ml
Foresight Plates*732-4785
Foresight MPC Type I Plates, 40 μm, 20 μl
Foresight RoboColumn Units**
732-4828
Foresight MPC Type I RoboColumn Units, 40 μm, 200 μl
732-4829
Foresight MPC Type I RoboColumn Units, 40 μm, 600 μl* Package size: 2 x 96-well plates** Package size: one row of eight columns

Foresight RoboColumn Units**
732-4822
Foresight CHT Type I RoboColumn Units, 40 μm, 200 μl
732-4823
Foresight CHT Type I RoboColumn Units, 40 μm, 600 μl
732-4825
Foresight CHT Type II RoboColumn Units, 40 μm, 200 μl732-4826
Foresight CHT Type II RoboColumn Units, 40 μm, 600 μl* Package size: 2 x 96-well plates** Package size: one row of eight columns

For More Information
Request or download bulletins 6086 and 5667

PicoGreen is a trademark of Invitrogen Corporation.

Related Items
CHT Ceramic Hydroxyapatite, Type I
158-2000
CHT Ceramic Hydroxyapatite, 20 μm, Type I, 10 g
157-0020
CHT Ceramic Hydroxyapatite, 20 μm, Type I, 100 g
157-0021
CHT Ceramic Hydroxyapatite, 20 μm, Type I, 1 kg
157-0025
CHT Ceramic Hydroxyapatite, 20 μm, Type I, 5 kg
158-4000
CHT Ceramic Hydroxyapatite, 40 μm, Type I, 10 g
157-0040
CHT Ceramic Hydroxyapatite, 40 μm, Type I, 100 g
157-0041
CHT Ceramic Hydroxyapatite, 40 μm, Type I, 1 kg
157-0045
CHT Ceramic Hydroxyapatite, 40 μm, Type I, 5 kg
158-8000
CHT Ceramic Hydroxyapatite, 80 μm, Type I, 10 g
157-0080
CHT Ceramic Hydroxyapatite, 80 μm, Type I, 100 g
157-0081
CHT Ceramic Hydroxyapatite, 80 μm, Type I, 1 kg
157-0085
CHT Ceramic Hydroxyapatite, 80 μm, Type I, 5 kg

© 2013 Bio-Rad Laboratories, Inc.

Bulletin 6432

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