Buffers and Conditions for Use and Maintenance of CHTTM Ceramic Hydroxyapatite Julia Zhen,* Pete Gagnon,† and Mark Snyder* *Bio-Rad Laboratories, Inc

Buffers and Conditions for Use and Maintenance of CHTTM Ceramic Hydroxyapatite Julia Zhen,* Pete Gagnon,† and Mark Snyder* *Bio-Rad Laboratories, Inc. † Validated Biosystems, Inc.

Abstract Most previous studies have focused on loss of calcium as the 2. Pure buffers degrade CHT: Mass loss vs. cycle numbers 5. 5 mM PO4 stabilizes CHT w/o increasing backpressure primary mechanism for degradation of CHT. Two lines of CHT Mass Loss evidence suggest that the loss of phosphate may be equally important: 1. Phosphate stabilizes CHT in the presence of buffers that otherwise are independently known to degrade CHT, such as Tris 20 and MES. 18 16 s

2. A conductivity aberration occurs in a phosphate gradient s 14 o L

when CHT is previously equilibrated in a buffer from which s 12 s a 10 phosphate is absent. M

T 8 H

Comparison of the phosphate gradient abnormality against a C 6 gradient of sodium chloride shows that the effect is real and that % 4 CHT is absorbing phosphate from the early part of the phosphate 2 gradient. This in turn suggests that non-phosphate buffers 0 0 1 2 5 dissolve phosphate from the CHT itself. This is apparently not a 0 0 0 function of the buffers themselves but simply from the absence of Cycles in 50mM MES, pH6.5 phosphate. This further suggests that inclusion of phosphate in the equilibration buffer should prevent this loss, with the expected 6. 5 mM PO4 or less stabilizes CHT w/o sacriﬁcing IgG binding capacity result that the phosphate conductivity trace is linear from the 3. Phosphate is necessary to maintain CHT stability: A clue PO Conc. vs CHT Mass Change at pH7.5 and Effect of PO4 on CHT 40µm type I Human gamma A 4 B globulins Dynamic Binding Capacity beginning. We conﬁrmed this expectation, and as a result from conductivity slope at pH 6.5 pH8.5 After 10 Cycles Run strongly recommend that at least 5 mM phosphate be included 30.0 7 25.0 pH 7.0 A 6 not only in all CHT buffers, but also in large volume protein B n u pH 6.5 R l 10.0 10.0 s 5 m / e l g c 20.0 m

samples applied to CHT. It likewise indicates that water should y

9.0 9.0 , C r

4 h 0 / 1

m r c 8.0 8.0 e 3 0 not be used in the cleaning and maintenance of CHT. If a low t f 0 A 7.5 7.5 3 15.0

, s C 7.0 7.0 s 2 B o L ionic strength cleaning agent is to be used, then it should contain D

50 mM MES w/ 4.7 g/L NaCl linear gradient s G

s 1

6.0 6.0 g I a 50 mM MES w/ 4.7 g/L NaCl linear gradient 10.0 M T

H 5 mM phosphate at a pH not less than 6.5. T 0 C

5.0 5.0 5.0 5.0 H C

50 mM MES, 4 mM PO w/ 0.1 M PO linear gradient % -1 4.0 4.0 4 4 5.0 -2 3.0 3.0 c 0 1 2 5 0 1 2 5 o m m m m m m n 2.5 2.5 t M M M M M M ro 0.0 l Materials and Methods 2.0 2.0 0 5 10 15 20 25 30 35 40 45 50 55 PO4 Conc. (mM) PO4 Conc. (mM) 1.0 1.0 50 mM MES, 2 mM PO4 w/ 0.1 M PO4 linear gradient 50 mM MES w/ 0.1 M PO4 linear gradient · CHT column stability study. 1 ml column, 600 cm/hr, 0.6 g 0.0 0.0 0.0 0.0 -1.0 -1.0 A. Less PO4 needed to stabilize CHT with higher pH buffers CHT packed 10 CV buffer A, 24 CV linear gradient to 100% B. 5 mM PO4 can stabilize CHT without signiﬁcant reduction of IgG binding 0.00 1.00 2.00 3.00 4.00 5.00 0.00 1.00 2.00 3.00 4.00 5.00 buffer B (0.24 mM PO4, + buffer A, same pH) 5 CV 1 M NaOH, mS/cm Min.Tenth mS/cmmS/cm Min.Tenth mS/cm capacity on CHT at pH 6.5 (50 mM MES) and pH 7.0 (50 mM HEPES) 1CV 0.5 M PO , pH same as buffer A 4 A. Dent formation at conductivity slope in PO gradient for CHT column 4 Cycles 7. 5 mM PO4 prevents CHT mass loss over a wide variety of B. Dent at conductivity slope in PO gradient for CHT column disappeared Unpack, wash 3x H O, 3x IPA, vacuum dry, 42oC over night 4 buffers and pH values 2 after adding 2-4 mM PO in equilibration buffer Total weight - tray weight = dry CHT resin weight 4 pH Buffer 10 cycles 50 cycles pH Buffer 10 cycles 50 cycles

5.0 Acetate + 5 mM PO4 ++ 7.0 acetate + 5 mM PO4 - · Test buffers and conditions. Buffers: MES, calcium, 5.5 Acetate + 5 mM PO4 + ++ 7.0 Imidazole + 5 mM PO4 - - phosphate and other commonly used CHT buffers). 4. Calcium prevents the CHT mass loss, however, with side 6.0 Acetate + 5 mM PO4 - + 6.0 Succinate + 5 mM PO4 + ++ Concentration of phosphate buffer: 0-50 mM. Range effect: high backpressure 7.0 glycine + 5 mM PO4 - 7.0 arginine + 5 mM PO4 - of pH: 6.0-8.5. Gradients: sodium chloride, phosphate. 6.5 Succinate + 5 mM PO4 +/- -

Ca EDTA PO Effect on CHT Mass Change 7.0 HEPES + 5 mM PO4 - A CaCl2 PO4 Effect on CHT Mass Change B 2 4 10 cycles, 50mM MES, pH 6.5 6.5 Phosphate (5 mM) - 10 cycles, 50mmM MES, pH 6.5 7.0 Tris + 5 mM PO4 - 6.5 Acetate + 5 mM PO4 - - 7.5 Phosphate (5 mM) - 12 Results 12 6.5 MES + 5 mM PO4 - - 7.5 MES + 5 mM PO4 - 6.5 Imidazole + 5 mM PO4 - +/- n 10 10 w/o PO u w/o PO 4

1. CHT column degradation after certain conditions R 4 4 1. CHT column degradation after certain conditions 7.5 Imidazole + 5 mM PO - - 1 mM PO n s

4 u e l R

c s y 8 8 6.5 glycine + 5 mM PO4 - - e C l

0 1 mM PO

y 4 1 7.5 acetate + 5 mM PO4 -

C r A B 6.5 arginine + 5 mM PO4 - - e 0

t 6 6 1 f

r A

t f e 7.5 HEPES + 5 mM PO4 - g A

n e 4 4 a 4 6.5 Tris + 5 mM PO - g h n C a

h s 7.5 Tris + 5 mM PO4 - - C s 7.0 Phosphate (5 mM) - -

a 2 2 s s M

T 8.5 Tris + 5 mM PO4 - M

H 7.0 MES + 5 mM PO4 - T C

0 H

% 0 C

% -2 Definition of mass loss symbols: “-”, no statistically significant mass loss; “+/-”, slight (0- -2

C 0 0 0 1 2 4 0 0 1 . . m m m . . m 2 5 2 5 5 M M 5 m 1%) loss; “+”, small (1-2%) loss; “++”, significant (>2%) loss m M M m m M M -4 M M C 0 0 0 1 2 4 0 0 1 2 . . m m m . . m m C D 2 5 2 5 5 m M M M 5 m M M m m CaCl2 Conc.(mM) M M M M

Ca2EDTA conc.(mM) Conclusions

A. CaCl2 can stabilize CHT w/ or w/o PO4. Side effect: high backpressure · It is necessary to add 5 mM PO4 to CHT buffers and sample solutions B. Ca2 EDTA can stabilize CHT w/ or w/o PO4. Side effect: high backpressure to prevent CHT mass loss, especially at pH 6.5 or below · Higher pH conditions may need less PO4 to stabilize CHT A. Cycled CHT column. Buffer: 50 mM MES, pH 6.5 · Some chelating agents still harm the CHT column even when 5 mM PO4 B. SEM picture for new CHT resins C. SEM picture for cycled CHT column top. is present (such as acetate or succinate at pH <6.0 ) D. SEM picture for cycled CHT column bottom. · 5 mM PO4 does not inhibit IgG (human g-globulin) binding on the column at pH 6.5 or 7 · 10 and 50 cycle studies of CHT mass loss have been done for commonly used CHT buffers w/ 5 mM PO4 Bulletin 5604 Rev A 08-0117 0208