Protein A Affinity Resin For mAb Purification
GALAK Chromatography Technology Co., Ltd
After nearly 30 years of development, therapeutic monoclonal antibodies have been playing an important role in the field of biomedicine. Like all biological drugs, they must be purified by liquid chromatography. The existing purification process can be divided into two categories: protein A affinity chromatography dominated and non-protein A affinity chromatography combined. The former is undoubtedly dominant.
This phenomenon is mainly attributed to the high multiple purification effect of protein A affinity chromatography. Usually, most of the high molecular weight antibody molecules aggregate and host cell protein and protein affinity chromatographic packing to produce A leakage of protein A, the host cell DNA all can in protein affinity chromatography to remove in this step, A single solution, 90% purity was obtained due to the affinity chromatography itself have enrichment effect, the processing of the sample volume is exponential decreases, and these are all important factors in favor of the refined chromatographic steps below.
The extensive use of affinity chromatography and its tremendous success is a pioneering effort in the biopharmaceutical industry. However, industry insiders also pointed out that the industrial production of therapeutic monoclonal antibodies has been developing faster in upstream technology than downstream technology, making downstream purification the bottleneck of the whole process. Therefore, more rapid and efficient purification requires more dynamic binding capacity (DBC) of protein A affinity chromatographic resins.
1. Protein A ligand of protein A affinity chromatography resin
The natural protein A from the cell wall of stapgylococcus aureus was discovered in the 1960s.
In 1972, scientists published further studies on natural protein A, revealing its structure and the specific recognition of IgG molecules. On this basis, the concept of the conjugation of natural protein A to chromatographic fillers to make affinity chromatographic fillers was proposed for the first time. The molecular weight of natural protein A is about 42 000Da. The literature in 1977 reported that it's n-terminal and c-terminal have different functions: at the n-terminal, there are 4 structural domains (D, A, B, and C from the n-terminal successively), each of which is about 7000Da. However, the part of c-terminal 15, 000Da has no Fc binding ability at all, whose function is to enable the natural protein A molecule to bind to the cell wall of staphylococcus aureus. In the 1980s, the amino acid sequence of protein A was fully revealed, and the cloning of A gene capable of achieving protein A expression in e. coli was first published. Meanwhile, the 5th Fc recognition structural domain E at the n-terminal was also discovered. These solid results laid the groundwork for large-scale production of protein A using genetic engineering. Repligen, an American firm, was the first to produce recombinant protein A.In the 1990s, U.S. FDA required that the protein A ligands used in the purification of chromatographic fillers for therapeutic monoclonal antibodies should not come into contact with any animal-derived molecules, which changed the production process and purification method of protein A. Now, natural protein A has been withdrawn from the therapeutic monoclonal antibody purified chromatographic filler market. Another result of high interest is the genetically engineered alkali-resistant protein A, which consists of four or five modified B domains (called Z domains). The aspartamine-alanine dipeptide was modified from an unstable aspartamine-lysine dipeptide in the B domain.
This achievement lays the foundation for the construction of relatively stable protein A under strong alkali conditions and is of great significance for the direct use of sodium hydroxide solution as in-situ cleaning (CIP) in industrial production. Protein A with two or more C domains is also the research object of several research institutions and companies, and the results have been published 7. Progress has also been made in the exploration of the specific recognition mechanism of protein A and antibody molecules 8.
Molecular studies have found that IgG Fc in the relatively stable structure, containing histidine and molecular recognition of A IgG protein structure domain the stability of the similar structure of histidine, both imidazole molecule can achieve face-to-face arrangement, under the condition of neutral pH, imidazole molecule didn't take charge of the two, the hydrophobic effect between the imidazole ring on the non-covalent bond combination of protein A and IgG have played an important role to strengthen, the formation of hydrogen bonds also promotes the combination of A and IgG protein molecules. When pH drops to acidic conditions, the imidazole rings of histidine are fully charged and repel each other, causing the non-covalent combination of protein A and IgG to separate 9. It has also been reported that there are differences in the affinity between protein A and different subtypes of human IgG. Generally speaking, the binding force of protein A to human IgG1 and IgG4 is stronger than IgG2, while the binding force of protein A to IgG3 is the worst. It was found that the degree of glycosylation of IgG had no effect on its binding to protein A. In theory, protein A should be able to bind to four or five IgG molecules simultaneously because it has four or five domains that recognize IgG molecules, but in practice, this phenomenon is impossible. According to literature reports, in solution, each protein A can only bind to an average of "2.0 ~ 3.3" IgG molecules, which indicates that not every domain can interact with IgG molecules at the same time. When protein A is coupled to the surface of the chromatographic filler, the number of IgG molecules that each protein A molecule may interact with simultaneously is smaller.
2 Main types of protein A affinity chromatography resin
Currently, GE Healthcare, Merck Millipore, and ThermoFisher are among the three top companies whose protein A affinity chromatography resins are widely used in the industrial production of therapeutic monoclonal antibodies in the United States. The different properties of these resins in base materials and protein A ligands result in significant differences in pressure resistance, dynamic loading and the ability to withstand CI conditions, which has an impact on production efficiency.
The company was Pharmacia of Sweden, where all protein A affinity chromatographic filler products were made from agarose cross-linked. The early product is rProtein ASepharose, which is made of porous agarose microspheres with low crosslinking degree and no more than two atmospheres of pressure resistance. The operating pressure must be strictly controlled when using it. It is a typical compressible filler. This product USES A recombinant protein A, which is coupled to CNBr-activated agarose porous microspheres via A thio-ether bond via cysteine at the c-terminal. This coupling mode has been continuously improved since then. Protein A connects to the activated arm through A single thioether bond, ensuring the n-terminal extension of protein A, thus increasing the chance of contact with antibody molecules. The use of CNBr activation, although effective, must use toxic chemicals, operation risk is higher.
Like rProtein A Sepharose FF product, MabSelect launched after the 20th century still USES recombinant protein A, but the activated arm is an epoxy compound containing ether structure, and the recombinant protein A is coupled with epoxy group activation arm by sulfur ether bond through cysteine located at the c-terminal. By increasing the crosslinking degree, MabSelect slightly improved the pressure resistance, reaching more than 3 atmospheres.
MabSelect Xtra is an improved version of MabSelect, which has a higher density of ligand, so it has a higher dynamic load, but it doesn't have any improvement in pressure resistance.
MabSelect SuRe, which follows MabSelect Xtra, uses an agarose microsphere that, like MabSelect Xtra, uses the previously mentioned alkali-resistant protein A, which combines four Z domains. As 0.1 ~ 0.5mol/L NaOH solution can be used as CIP, the production process is simplified. It is the first of its kind.
The company's products are branded as ProSep A. Although Merck-Millipore continues to develop new products, such as Eshmuno, PVA substrate. But for therapeutic monoclonal antibodies is protein A affinity chromatography resin from BioProcessing. BioProcessing was a British firm until Millipore bought it. There is something special about these products. It can be seen from the shape of the particles, which are based on the control pore glass (CPG) substrate.
This material is a special process of broken glass particles through screening to get a certain distribution of glass particles, completely irregular shape, and glass particles above the hole are treated by chemical methods. The pore diameter of ProSep-vA is 100nm, while that of ProSep-vA Ultra is 70nm. The latter has a higher specific surface area and can load more protein A13 due to the smaller pore diameter. The advantage of removing non-specific protein adsorbents from the glass surface using proprietary technology that has not been published is that it can be used at higher flow rates, increasing the productivity of the packing per column volume per unit time.
However, porous glass is unstable under alkaline conditions, so ProSepA series products cannot use in alkaline CIP. Due to the irregular shape of the packing glass particles, in addition to pressure during the column installation process, the use of oscillators to form a stable column bed.
The company's products use POROS for trademark, launched in the 1990s, as with other POROS products, polystyrene divinylbenzene(PS/DVB) was used the concept of tubular chromatography, characterized by a loss of microspheres in addition to the pore diameter of about 80 nm, also contains a through-hole, hole mass transfer conditions within the barriers reduced the microspheres. In addition, polystyrene-divinylbenzene non-specific protein adsorption was removed by surface coating with hydroxy-rich compounds.
Follow the technologies of POROS, GALAK Chromatography creates its own independent technologies on Protein A Affinity Chromatographic Filler. GALAK products use Absolut A for trademark. Absolut A is for titer analysis of antibodies and fusion proteins using HPLC systems
Protein A binds to the Fc region of immunoglobulins through interactions with the heavy chain. The binding of protein A has been well documented for IgG from a variety of mammalian species and for some IgM and IgA as well. Compare with protein A, recombinant protein A (rProtein A) has the advantage of good alkali-tolerant that is resistant to harsh cleaning agents (0.1 to 0.5 M NaOH).
The spherical PS/DVB (Poly(styrene/divinylbenzene)) particles are highly cross-linked for enhanced mechanical stability, and bigger hole diameter. After GLK® hydrophilic treatment process, PS/DVB surface removes non-specific binding that increases the column efficiency. Compared with agarose substrate, hydrophilic PS/DVB has a higher dynamic binding capacity (DBC), longer lifetime and less shedding of rProtein A ligand.
3 Dynamic Binding Capacity (DBC) Of Protein A Affinity Resin
The affinity purification of antibody molecules takes place at a certain velocity, so dynamic binding capacity (DBC) is used.
To characterize how many antibody molecules can be "captured" per unit volume of A protein A affinity resin under certain flow rate or residence time (RT) conditions has direct practical significance. In order to improve the efficiency of affinity chromatography, the researchers determined that the best scenario was to achieve a high dynamic load of 14 for shorter contact time. However, between the two has a trade-off between contradiction, higher dynamic loads often need to obtain a longer contact time, the main problem is the antibody molecules in the filler hole mass transfer barriers, this is liquid chromatography, especially large-scale preparation chromatography, one of the biggest challenges facing and scientists are trying to solve the problem for many years.
The C phase in the van Deemter formula and the later theories based on the velocity and band broadening of liquid chromatography are described in detail.
van Deemter formula:
R is a measure of band broadening; U is the velocity; A and B are constants, independent of flow rate, which can be ignored in large-scale preparation. C phase is the coefficient related to mass transfer barrier.
As the velocity increases, the Cu term also increases, and the contribution to spectral band broadening increases.
GALAK Chromatography Technology Co., LTD. (GALAK) has made a new attempt in this field. The main parameters of Sepromax® A40 Plus protein A affinity chromatographic filler introduced by the company are shown below.
|Support Matrix||Poly(styrene/divinylbenzene) (PS/DVB)|
|Ligand||Recombinant Protein A|
|Dynamic Binding Capacity (DBC)||Approx. 40 mg human IgG/ml resin (Determined at 10% breakthrough by frontal analysis at a mobile phase velocity of 500 cm/h in a column with a bed height of 5 cm, Residence time 0.6 min)|
|Shrinkage/Swelling||< 1% from 1-100% organic solvent|
|pH Range||pH 2-10 (Working), pH 2-13 (Cleaning)|
|Maximum Operating Pressure||1500 psi (100 bar / 10 MPa)|
|Cleaning In Place (CIP)||0.1-0.5M NaOH|
|Temperature Stability||4-25 °C|
|Delivery Conditions||20% ethanol (2-8℃)|
Details about Sepromax® A40 Plus, click here >>
GALAK resin uses surface hydrophilic treatment similar to POROS series. Since most protein A molecules are coupled into the resin's pores, antibody molecules must be diffused into the pores to interact with protein A. The molecular weight of the antibody molecule is higher than 150kDa and the diffusion rate is lower.
Yeung published the simulation results of similar protein molecules' diffusion in chromatography packing hole in 2012, and the diffusion rate of surface protein molecules in chromatography packing hole was 20 times slower than that in solution. Integrated the findings and the predecessors' work, and lake researchers use highly uniform microspheres particle size as the backing material, can ensure the pore of microsphere between highly consistent, this reasoning, using such microspheres, the guarantee of back-pressure, the microspheres particle size will be the smallest, namely the hole diffusion path length minimum and is almost unanimously, spread in the hole is synchronized. Using a resin with a wide particle size distribution, such as MabSelect SuRe, the diffusion of antibody molecules in the pore is asynchronous.
GALAK's results confirm this. Under the same experimental conditions, the dynamic loading of 40mg/mL was obtained. MabSelect SuRe required 2.4min of contact time, while Sepromax A40 Plus with uniform microspheres only required 1.3min of contact time.
4 The ability to remove proteins and DNA from host cells
After 20 years of technical progress and process development, the industrial purification process of the therapeutic antibody has basically achieved "modularization", that is, by affinity chromatography, ion-exchange chromatography, and hydrophobic chromatography. The first step in most purification processes is the protein A Affinity chromatography, which traps only antibody molecules and thus has an excellent ability to remove host cell proteins HCP and DNA. The content of HCP in supernatant and eluate after purification by protein A affinity chromatography could be accurately determined by ELISA. Sepromax® A40 Plus and competitive products in the HCP purification efficiency and DNA purification efficiency of the comparative data are as follows.
|The ability of remove host cell proteins of Sepromax® A40 plus|
|HCP||Sepromax® A40 Plus||Competitive Product|
|Protein A Eluant||1.9ng/mg||4.5ng/mg|
|The ability of remove host cell DNA of Sepromax® A40 plus|
|rDNA||Sepromax® A40 Plus||Competitive Product|
|Protein A Eluant||154.4pg/mg||431.3pg/mg|
The protein, DNA, protein A molecule, antibody fragment, virus and endotoxin of the residual host cell must be purified by protein A affinity CHROMATOGRAPHY to reach the final purity requirement.
5. Abscission of protein A ligand
The abscission of protein A ligands occurs in the elution process of all protein A affinity chromatography resins after contact with antibody molecules. Exfoliated protein A is usually eluted with the target antibody, which is one of the problems encountered in the purification of antibody by protein A affinity chromatography. The shedding molecule can be either the whole protein a or the fragment of protein a, the molecular weight is 6k ~ 40kDa, but the degree of shedding is different.
It was reported that the abscission amount of ProSep-vA Ultra was higher than that of Mabselect SuRe, and the abscission amount of ProSep-vA Ultra was higher at the beginning, lower at the middle and higher at the end of the purification process. In terms of stability, the method using single covalently-coupled protein A is no worse than that using multiple covalently-coupled Protein A, then the argument that the latter is superior to the former is self-defeating. The ProSep-vA of porous glass substrate has a high drop-off rate in acid-eluted antibody, and the porous glass substrate itself is stable in an acid condition, so the drop-off of protein a should not be related to the decomposition of the substrate, it may be related to the protein A structure. The mechanism of protein a shedding is unclear and further studies are needed.
A brief description of the various protein A affinity chromatographic resins mentioned above are given in the table below.
|Name||Ligand/Source||Substrate, Particle Size||Supplier||Time To Market|
|Protein A Sepharose CL-4B||nProtein A/S aureus||4% cross-linked (CL) agarose， 90um||GE Healthcare||1975|
|nProtein A Sepharose 4 Fast flow||nProtein A/S aureus||4% cross-linked agarose (FF)， 90um||GE Healthcare||1982|
|rProtein A Sepharose Fast flow||rProtein A/E.coli||4% cross-linked agarose (FF)， 90um||GE Healthcare||1994|
|Rmp Protein A Sepharose Fast flow||rProtein A/E.coli||4% cross-linked agarose (FF)， 90um||GE Healthcare||1996|
|MabSelectTM||rProtein A/E.coli||high-flow agarose， 85um||GE Healthcare||2001|
|MabSelect XtraTM (High Capacity)||rProtein A/E.coli||high-flow agarose， 75um||GE Healthcare||2005|
|Mabselect suReTM |
|rProtein A/E.coli||high-flow agarose， 85um||GE Healthcare||2005|
|ProSep-vA High Capacity||nProtein A/S aureus||1000-angstrom controlled pore glass CPG, 100um||Merck-Millipore||1990|
|ProSep-vA High Capacity||nProtein A/S aureus||1000-angstrom controlled pore glass CPG, 100um||Merck-Millipore||2003|
|ProSep-vA Ultra||nProtein A/S aureus||7000-angstrom controlled pore glass CPG, 100um||Merck-Millipore||2004|
|POROS A||rProtein A/E.coli||polystyrene-divinylbenzene, 50um||ThermoFisher||1991, 1993|
|POROS MabcaptureTM A||rProtein A/E.coli||polystyrene-divinylbenzene, 45um||ThermoFisher||2007|
|Sepromax A40 plus||rProtein A/E.coli||coated mono dispersed cross-linked poly(styrene divinylbenzene)，40um||GALAK Chromatography||2013|
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