T able of Contents
Section 1Introduction (1)
Section 2Product Description (1)
2.1 What is CHT? Ceramic Hydroxyapatite? (1)
2.2 Specifications (2)
2.3Characteristics (2)
2.4General Handling and Packing (3)
Section 3Chromatography (3)
3.1 CHT? Ceramic Hydroxyapatite Mechanism (3)
3.1.1Buffers (4)
Table I: CHT Stability in Various Buffers (4)
3.1.2Elution (4)
3.1.3Trace Metal Contamination (5)
3.1.4Phosphate (5)
3.1.5Calcium (5)
3.1.6Chemical Compatibility/Load Preparation (5)
3.2Method Development (6)
3.2.1Protocol I: IgG Monoclonal Antibodies (8)
3.2.2Protocol II: Globular Proteins (10)
3.2.3Protocol III: Plasmids (12)
3.2.4Protocol IV: Acidic Proteins (14)
3.2.5Scouting Tips (14)
Section 4Regeneration, Sanitization, and Storage (15)
4.1Regeneration (15)
4.2Sanitization (15)
4.3Storage (15)
Section 5Column Packing Protocols (15)
5.1General Handling and Powder Preparation (15)
5.2Guidelines for Packing Low-Pressure Process Columns (15)
5.2.1Recommended Column Packing Solutions (17)
5.3Open-Column Methods (17)
5.3.1Gas-Assisted Axial Compression Packing of Open Columns with Motorized Adjustable Inlet Adaptors (17)
5.3.2Gas-Assisted Flow Packing of Open Columns With Adjustable Adaptors
at Less Than 700 cm/hr Flow Rate (19)
5.3.3Gas-Assisted Flow Packing of Open Columns With Adjustable Inlet Adaptors Capable of 700 cm/hr (22)
5.4Media Transfer Station Methods (24)
5.4.1Axial Compression Packing of Clod Columns With Motorized Adjustable Inlet Adaptors (24)
5.5Media Packing Station Methods (26)
5.6Unpacking for Disposal (28)
5.7Packed Column Qualification (28)
5.8Comments on Column Packing (29)
5.8.1Comments on Column Qualification for Columns With Adjustable Inlet Adaptors (29)
5.8.2Comments on Column Qualification for Contained Operating System Pressure-Packed
Clod Columns (30)
5.8.3Comments on Column Qualification on Columns Ud in Purification Campaigns (30)
5.8.4Conditioning the Column for the Purification Application (30)
Section 6Ca Studies (30)
6.1Packing Results —Custom GE Healthcare Chromaflow 900/200–400 (30)
6.2Packing Results —Prototype Milipore IsoPak IPP350/500 (32)
6.3Table 3: Summary for Packing CHT Type I, 40 μm (32)
6.4Table 4: Summary for Packing CHT Type I, 80 μm (33)
Section 7Appendices (33)
7.1CHT to Buffer for Packing 50 cm High Open Columns (34)
7.2CHT to Buffer for Packing 60 cm High Open Columns (35)
7.3CHT to Buffer for Packing 70 cm High Open Columns (36)
7.4 CHT to Buffer for Packing 90 cm High Open Columns (37)
7.5CHT to Buffer Guide for Contained Operating System Columns (38)
Section 8Reference (39)
Section 9Ordering Information (40)
Section 1
Introduction
CHT? ceramic hydroxyapatite is a leading purification medium of biomolecules in today’s demanding downstream process industry. Its mixed-mode support offers unique lectivities and often parates biomolecules that appear homogeneous using other chromatographic methods. The diver binding capabilities of CHT for host cell proteins, leached protein A, antibody dimmers and aggregates, nucleic acids, and virus allow its u at any stage from initial capture to final polishing.
The robust properties of CHT ceramic hydroxyapatite improve efficiency, yield, and financial value through:
?Excellent capture at elevated flow rates enabling processing at all scales
?Large capacity for higher-titer upstream feedstocks
?Exceptional lectivity allowing for a two-step chromatographic process
This manual is a guide for the u of CHT as a media support in your purification process. The manual is organized into four main topics:?Product Description
?Chromatography
?Regeneration, Sanitization, and Storage
?Column Packing Protocols
?Ca Studies
Throughout this manual, we have incorporated recommendations ranging from method scouting and optimization to column packing techniques that reprent feedback from process chromatographers globally. Should you have further questions, contact either your local Bio-Rad process chromatograp
hy sales reprentative or the Bio-Rad chromatography technical support department for further assistance at 1-510-741-6563.
Section 2
Product Description
2.1 What is CHT? Ceramic Hydroxyapatite?
Hydroxyapatite (Ca5(PO4)3OH)2is a form of calcium phosphate ud in the chromatographic paration of biomolecules. Sets of five calcium doublets (C-sites) and pairs of –OH containing phosphate triplets (P-sites) are arranged in a repeating geometric pattern. Repeating hexagonal structures can be en in electron micrographs of the material. Space-filling models and repeat structure from Raman spectroscopy have also been constructed. Hydroxyapatite has unique paration properties and unparalleled lectivity and resolution. It often parates proteins shown to be homogeneous by electrophoretic and other chromatographic techniques. Applications of hydroxyapatite chromatography include the purification of different subclass of monoclonal and polyclonal antibodies, antibodies that differ in light chain composition, antibody fragments, isozymes, supercoiled DNA from linear duplexes, and single-stranded from double-stranded DNA.
CHT ceramic hydroxyapatite is a spherical, macroporous form of hydroxyapatite. It has been sintered at high temperatures to modify it from a crystalline to a ceramic form. The ceramic material overcomes many of the limitations of traditional crystalline hydroxyapatite that prevent its u in industrial-scale applications. The ceramic material retains the unique paration properties of crystalline hydroxyapatite, but can be ud reproducibly for many cycles at high flow rates and in large columns. Unlike most other chromatography adsorbents, CHT is both the ligand and the support matrix. Separation protocols originally developed on crystalline hydroxyapatite can often be transferred directly to the ceramic material with only minor modifications. Two types of CHT ceramic hydroxyapatite, Type I and Type II, are available in three particle sizes, 20, 40, and 80 μm. Both types have elution characteristics similar to crystalline hydroxyapatite, but also have some important differences. CHT Type I has a higher protein binding capacity and better capacity for acidic proteins. CHT Type II has a lower protein binding capacity but has better resolution of nucleic acids and certain proteins. The Type II material also has a very low affinity for albumin and is especially suitable for the purification of many species and class of immunoglobulins.
2.2 Specifications
Type I
Type II Functional groups
Ca 2+, PO 4, OH Ca 2+, PO 4, OH Obrved dynamic binding capacity
lysozyme (Lys)
≥25 mg Lys/g CHT ≥12.5 mg Lys/g CHT Nominal pore diameter
600–800 ? 800–1,000 ?Maximum backpressure
100 bar (1,500 psi)100 bar (1,500 psi)Nominal mean particle size
20 ± 2, 40 ± 4, and 80 ± 8 μm 20 ± 2, 40 ± 4, and 80 ± 8 μm Bulk density
0.63 g/ml 0.63 g/ml
2.3 Characteristics
Type I
Type II Obrved dynamic binding capacity
IgG
25–60 mg IgG/ml CHT* 15–25 mg IgG/ml CHT** Typical linear flow rate range
50–1,000 cm/hr pH stability***
6.5–14 pH Ba stability
at least 21 months in 1 N NaOH Regeneration
500 mM sodium phosphate, pH 71,000 mM trisodium phosphate, pH 11–12Autoclavability (bulk)
121°C, 20 min in phosphate buffer, pH 7Sanitization
1–2 N NaOH Recommended column storage
0.1 M NaOH Shelf life (dry, unud material) 85 months stored dry, aled, and at room temperature * 40 μm particles, 300 cm/hr, 5 mM sodium phosphate, pH 6.5
** 40 μm particles, 300 cm/hr, 5 mM sodium phosphate, pH 6.5
*** For pH 5.5–6.0, e Section 3.1.1 Buffers and Table 1
Purity
In the preparation of ceramic hydroxyapatite, u of high-purity raw materials results in low levels of contaminants as determined by ICP mass spectrometry for metal analysis and ion chromatography for anions.
Impurity
Levels Chloride 0.005%Sulfate
0.01%Carbonate
0.01%Lead
0.001%Cadmium
0.0001%Barium
0.001%Arnic 0.001%
￡￡￡￡￡￡￡
2.4 General Handling and Packing
CHT ceramic hydroxyapatite is a rigid support and can operate under high flow rates and pressures. However, excessive physical force
beyond typical operating conditions can result in bead damage. In order to optimize the chromatographic properties of CHT, avoid excessive stirring or agitation that may lead to mechanical damage and bead fracture.
See General Handling and Powder Preparation, Section 5.1, for more details.
Section 3Chromatography
3.1 CHT? Ceramic Hydroxyapatite Mechanism
Hydroxyapatite contains two types of binding sites, positively charged calcium and negatively charged phosphate groups. The sites are distributed regularly throughout the crystal structure of the matrix. Solute species dominantly interact through cation exchange via the phosphate groups and/or metal affinity via the calcium atoms.
Cation exchange occurs when protein amino groups interact ionically with the negatively charged phosphates. The amino groups are similarly repelled by the calcium sites. Binding depends upon the combined effects of the interactions. The ion exchange interactions can be disrupted by adding neutral salts such as sodium chloride or buffering species such as phosphate to the mobile pha. Cation exchange interactions also weaken with increasing pH. Hence, the addition of salt or phosphate, or an increa in pH, can be ud to
weaken the interaction. Studies with model proteins have demonstrated that anion exchange, which might be expected from interactions of negatively charged surface residues with calcium, does not make a significant contribution.
Calcium affinity occurs via interactions with carboxyl clusters and/or phosphoryl groups on proteins or other molecules (e.g., nucleic acids); the groups are simultaneously repelled by the negative charge of the CHT phosphate groups. The affinity interaction is between 15 and 60 times stronger than ionic interactions alone and, like classical metal-affinity interactions, is not affected by increasing ionic
strength using typical elution ions (e.g., chloride). Species binding through calcium affinity may adsor
b more strongly as the ionic strength increas due to ionic shielding of the charge repulsion from the CHT phosphate sites. Metal affinity interactions can be dissociated by phosphate in the mobile pha.
Most large proteins bind by a combination of mechanisms (Figure 1):
Dominantly acidic proteins , such as albumin, bind chiefly by metal affinity interactions. Sodium chloride at 1.0 M reduces retention time by approximately 10% in the prence of phosphate gradients, indicating a minor contribution by cation exchange. To elute acidic proteins,phosphate buffers are required.
Dominantly basic proteins , such as IgG, bind chiefly by cation exchange interactions. Sodium chloride reduces retention time in the prence of phosphate gradients, indicating a minor contribution by metal-affinity. Basic proteins may be lectively eluted with either phosphate or salts.
Cation exchange Metal affinity
Metal affinity carboxyl clusters phosphoryl groups
on nucleic acids
Fig. 1. Schematic Reprentation of CHT binding mechanisms.Repulsion
Attraction Metal affinity
