Application Tips and Optimization Strategies for Hydroxyapatite

Hydroxyapatite is an inorganic pure-phase chromatography medium that is calcined at high temperature into spheres with stable spherical structure and relatively uniform particle size. There are multiple modes of interactions between hydroxyapatite and biomolecules, which can simultaneously achieve two separation mechanisms: cation exchange and calcium metal affinity. Cation exchange mainly relies on the negatively charged phosphate groups on hydroxyapatite. At the same time, carboxyl clusters, phosphorus clusters and others on biomolecules can form with calcium on hydroxyapatite through metal affinity. Stronger bond. Due to these unique separation mechanisms, hydroxyapatite has become an indispensable chromatography medium in today's extremely demanding downstream processes, such as antibodies, vaccines (bacterial polysaccharides, pneumonia polysaccharides, viruses, etc.), nucleic acids, enzymes and recombinant proteins. play an important role in the field of biopharmaceuticals. It can be used for the separation of host proteins (HCP), shed ProteinA, antibody dimers and aggregates, nucleic acids and viruses, and is often used in various purification stages of capture, crude purification or purification.

BioLink currently has two types of hydroxyapatite chromatography media: BaronCHT type I and BaronCHT type II. The two models are mainly generated by different burning temperatures and times. Type I is 400°C and type II is 700°C. The main particle size of the two CHT currently on the market is 40um.

Figure 1: Electron microscope image of BioLink hydroxyapatite

CHT type I has a relatively high protein loading capacity and is suitable for purifying single and double antibodies, recombinant proteins and other projects, and has a higher loading capacity for acidic proteins. CHT type II has a lower capacity than type I, has better resolution for nucleic acids and certain proteins, and is suitable for purifying plasmids, mRNA and other types of projects. CHT type II has low affinity for albumin and is more suitable for purifying various immunoglobulins and their subtypes.

Because hydroxyapatite contains calcium and phosphate, it can be stably used in pH 6.5-14 solutions and is compatible with the following reagents:

There are currently two main problems in the use of hydroxyapatite: one is lifespan. Usually CHT lifespan is generally around 40 Cycles, but it is closely related to specific usage conditions; the other is how to optimize the process.

Common factors affecting the lifespan of CHT

➣ A thicker liquid with more impurities will seriously affect the life of the filler.

➣ During use, long-term exposure to acidic conditions of pH 6.5 and below will cause damage to CHT and reduce the number of uses of CHT.

➣ All buffers used when using hydroxyapatite must not contain citric acid or EDTA. These integrators will degrade hydroxyapatite and reduce its service life.

Tips for improving CHT lifespan

➣ Adding phosphate or calcium ions to the solution can also improve the structural stability of hydroxyapatite and maintain its service life and performance (calcium ions Ca2+ will cause a precipitation reaction with phosphate, and the amount added should not exceed 2mM).

➣ During experiments, adding a weak base elution step before elution can greatly improve the column life, such as using surface neutralization system (SNS) 25mM Tris-HCl, 25mM NaCl, 5mM phosphate pH7.75 to run 6 -8CV. This SNS solution can effectively reduce the pH drift during the elution process and the increase in column back pressure during use, and can effectively reduce the loss of calcium hydroxyapatite ions to help extend the life of the packing.

➣ At the same time, the addition of Arginie, lysine, histidine, PIPES, HEPES, MES, and MOPS can also significantly reduce the proton release in CHT, greatly reducing the drop in pH and reducing the time that CHT is exposed to a low pH environment. Number of times CHT is used.

Figure 2: Effect of adding MES to the solution on pH drift

Hydroxyapatite

Principles of use and optimization strategies

Hydroxyapatite contains two binding sites: Ca and PO₄, which are distributed according to its own crystal structure. PO₄ ions are ionically bonded to positively charged proteins and have cation exchange properties; the Ca ions are combined with the free carboxyl clusters of negatively charged proteins in a metal chelation manner.

Usage strategies under different combination modes

✦ Cation exchange: When the protein is positively charged, it can combine with PO₄, but is mutually exclusive with Ca. This effect is mainly cation exchange. This cation exchange can be affected by the neutral salt concentration (such as NaCl) in the elution buffer or the phosphate concentration in the equilibration solution. This effect also weakens as the pH increases. Therefore, increasing the concentration of salt or phosphate or increasing the pH will weaken the interaction between the target protein and PO₄.

✦ Calcium metal chelation: Ca can have metal integration with carboxyl clusters or phosphate groups on the surface of proteins or other molecules (such as nucleic acids), while repelling PO₄ at the same time. The force of this binding method is 15-60 times that of general ion exchange. Therefore, this binding method is less affected by ions and is not sensitive to NaCl. Moreover, this force will increase the calcium metal integration force with the ionic strength, because the increase in ionic strength shields the mutual repulsion between the functional groups on the molecular surface and PO₄. This binding pattern can be eluted with sodium phosphate.

Figure 3: Schematic diagram of the process of hydroxyapatite

Therefore, based on the above two mechanisms of action, the conventional buffer solutions used for hydroxyapatite are generally the first: balancing solution 10mM PB PH6.8, eluent 400mM PB PH6.8; the second: balancing solution 10mM PB PH6.8, eluent 10mM PB+1M NaCl PH6.8.

If the bond is weak, easy to flow through, and difficult to elute, you can use the following methods:

1. For samples with weak binding force, the phosphate concentration in the equilibrium solution can be reduced to improve the binding ability.

2. For samples that are difficult to elute, high-concentration potassium phosphate solution can be used for elution.

Common regeneration methods for hydroxyapatite:

a. For the regeneration of hydroxyapatite, 500mM neutral phosphate or potassium phosphate solution or 400mM sodium phosphate cleaning is generally used for 3-5CV to complete the regeneration.

b. You can also clean the hydroxyapatite column with neutral KCl, NaCl, 6M urea or 8M guanidine hydrochloride containing 5mM phosphate.

c. Since hydroxyapatite itself has extremely high alkali resistance, up to 2M NaOH can be used for cleaning and disinfection (conventionally 0.5M is enough), and its storage solution can also directly choose 0.1~1M NaOH. If you want to use less than 0.1M NaOH for storage, you need to add 10mM phosphate to the storage solution.

Column packing techniques for hydroxyapatite:

1) When packing a column, hydroxyapatite is not compressible due to its own material problems.

2) Use flow velocity to pack the column rather than gravity settling.

3) During the column packing process, shear force can easily damage the particles. In order to avoid damage, a homogenization ratio of 30% to 50% should be selected when packing the column, and mix gently with minimal force.

4) For 40um CHT, generally use 10um sieve plate.

5) Do not touch the packing when the column head is finally lowered. When the column is installed, keep the column bed stable. Please refer to the column packing steps in the Baron CHT packing manual for better column packing assistance.

Based on the special principle of action of CHT, it usually brings effects that conventional medium cannot achieve. It is also often used for the purification of double antibodies, the purification of recombinant proteins, and the purification of some polysaccharide vaccines and recombinant factors.

CASE 1 CHT for the further improvement of purity in double antibodies

The double-antibody project is used to remove aggregates to achieve higher purity. The SEC purity is increased from 95% to 99%, and the recovery rate is 86%.

• Chromatography column for sale: 4.7mL Baron CHTⅠ prepacked column

• Loading volume: 36.9mg/ml; linear flow rate 120cm/h

• Balance solution: 5mM PB PH6.8

• Eluent: 5mM PB+1M NaCl PH6.8

Figure 4: Chromatogram

Figure 5: HPLC detection chart

CASE 2 CHT for purification of recombinant factors

CHT is used for the purification of recombinant factors with a purity of up to 94%. Compare the performance of a certain reorganization factor project with imported competitors.

• Product：BaronCHT Type I,40μm

Figure 6: Comparison of performance between BioLink CHT I type and competitors from abroad

Through analysis, the yield, purity and quality are at the same level as competitors from abroad.

Table 1: Comparison of data between BioLink and competitors from abroad