Purification Techniques | The Secret of 99% Purity: BARONHAP® Hydroxyapatite Chromatography Resin

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In the field of biopharmaceuticals, how to efficiently separate complex biomolecules has always been a difficult problem for purification workers. As a unique chromatography resin, hydroxyapatite (HAP) has become a "secret weapon" for the purification of biological macromolecules such as antibodies and vaccines by virtue of its multi-modal interaction mechanism. This article will take you to have an in-depth understanding of the purification principle, product application and other aspects of HAP, and uncover the skills behind its "versatility".

Introduction to purification principle and product parameters

Different from conventional resin, HAP resin incorporate separation mechanisms such as anion exchange, cation exchange and metal chelation through their two-site structure Ca2+ and PO43-. The PO43- sites can bind to positively charged proteins through ionic bonds, showing the characteristics of cation exchange; The Ca2+ sites bind to negatively charged protein free carboxyl clusters through metal chelation or anion exchange.

Figure 1: Schematic diagram of bonding principle

Resin Name	BARONHAP® Type I	BARONHAP® Type II
Mean Particle Size	40 μm
Ligand	Ca2+, PO43-, -OH
Dynamic Binding Capacity (CHO cell-derived IgG1 )	≥45 mg/g G1/mL Chromatography resin	≥15mgIgG1/mL Chromatography resin
Density	0.60~0.66 g/mL	0.65~0.72 g/mL
Max. Back Pressure	3 MPa	4 MPa
Recommended Flow Rate	50-400 cm/h
pH stability (CIP)	6.5-14
Solvent Tolerance	Common phosphate solutions,1 M NaOH,8 M urea,pH should be ≥ 6.5 during use
Intolerance	EDTA, sodium citrate
Storage Conditions	0.1~0.5 M NaOH

Table 1. BARONHAP® Product Parameters

Binding capacity performance

The binding capacity test was performed using CHO cell-derived IgG1, and the main binding of the molecule was manifested as phosphate group binding, BARONHAP® Type I has more advantages in binding capacity.

Pressure-flow rate curve

Figure 2: BARONHAP® Type I

Pressure-flow rate curve (D: 300 mm * H: 20 cm)

Application Case 1

BARONHAP® Type I

Efficient removal of aggregate in antibody projects

Recommended process conditions

Sample information: Bispecific antibody anion flow through liquid
Chromatography column : 10 * 250 mm, 20 mL
Buffer A: 20 mM PB, 20 mM Tris，pH 7.2
Buffer A: 20 mM PB, 20 mM Tris，1 M NaCl, pH 7.2

Figure 3: BARONHAP® Type I chromatogram

#	TIME	Peak Area	Peak Height	Peak Width	Symmetry Factor	Peak Area%	Type
1	6.314	140.4	6.1	0.3511	1.611	0.478	BV E
2	7.152	29207.6	1134.3	0.3864	0.738	99.522	VB R

Summary: BARONHAP® Type I performed well in the biospecific antibody project, with a binding capacity of 36.9 g/L, purity increased from 91.8% to 99.5%, aggregate content decreased from 6% to 0.5%, and yield reached 86%.

Application Case 2

BARONHAP® Type II, 40 µm

Applications in recombinant factors

Figure 4: BARONHAP® Type II Elution Profile

Figure 5: Type II elution profile of company B

Resin Manufacturer	Conc (mg/mL)	Volume (mL)	Total (mg)	Yield (%)	HCP (ppm)	SEC-HPLC (%)
BARONHAP® Type II	0.422	25.81	10.9	50.9	929.1	94.9
Company B Type II	0.458	23.92	11.0	51.3	917.9	92.1

Summary: Compared with the synchronous comparison of imported competitors, the performance is comparable.

Frequently Asked Questions (FAQ)

Q1

What are the precautions in the column packing process?

A

Avoid using pure aqueous phase and require buffer with a certain ionic strength to pack the column.
Salt loading test is recommended for column efficiency test.

Q2

What buffers can be used with HAP?

A

A commonly used binding buffer is 5 mM phosphate, pH 6.8. Alkaline proteins can be eluted linear or stage with 1 M NaCl phosphate buffer pH 7.2. Acidic proteins can be eluted linear or stage with 500 mM phosphate buffer. It is critical that both binding and elution buffer pH be higher than pH 6.5.

Q3

How to extend HAP resin life?

A

It is recommended that all buffers, including storage solutions, contain at least 5 mM phosphate and have a pH equal to or higher than 6.5. Try to choose high pH buffers for all applications. Adding calcium ions at the ppm level can improve the firmness of the resin.
When NaCl elution is used for the first time, the pH will be lowered due to the shedding of hydrogen ions, so the addition of co-buffer, such as MES, will greatly improve the resin firmness.
Preservation of the column with NaOH prevents carbonate precipitation on the column surface.
Avoid sudden changes in the flow rate to reduce the pressure impact, which will destroy the column bed.
After alkali washing, it is recommended to add high-salt rinsing to fully cure.

Q4

HAP service life and shelf life?

A

If properly packed while maintaining pH above 6.5, HAP can be used for at least 40 cycles. If care is taken to avoid damage to the resin, the HAP can be repeatedly packed 20 times or more. When stored in closed containers at 4-30 ℃, the resin can be kept for 5 years.

Q5

How much scale-up experience does HAP currently have? What is the current maximum capacity of a single batch?

A

The maximum column size achieved is 450 mm, and the bed height of the column is about 20 cm. The maximum capacity of a single batch is 50 kg.

References

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