Milieux de chromatographie en agarose
Biovanix Agarose Media is a conventional biological separation medium crafted from highly crosslinked agarose microspheres. It utilizes high-performance agarose raw materials sourced from abroad to maintain a consistent level of product quality. This product preserves the remarkable hydrophilicity and expansive network structure inherent to natural polysaccharide compounds, demonstrating excellent affinity for bioactive macromolecules. It is characterized by high loading capacity, broad applicability, non-specific adsorption, and rapid flow dynamics. Consequently, it is extensively utilized in the laboratory-scale preparation of biological macromolecules, including proteins, nucleic acids, peptides, and polysaccharides, as well as in the large-scale industrial production of biopharmaceuticals and bioengineering applications.
Characteristics of Agarose Chromatography Media
Agarose-based media represent the gold standard matrix in the field of bioseparation. Derived from natural polysaccharides and optimised through modern cross-linking technologies, these matrices exhibit a unique combination of physicochemical properties ideal for the purification of proteins, antibodies, and other biomacromolecules.
Below are the key technical characteristics:
1. Exceptional Hydrophilicity and Bio-inertness
The fundamental advantage of agarose is its non-toxic, hydrophilic nature.
Low Non-Specific Binding (NSB): The agarose backbone is rich in hydroxyl groups, creating a surface that interacts favorably with water but exhibits minimal hydrophobic interaction with proteins. This significantly reduces non-specific adsorption, ensuring high recovery rates and preventing the denaturation of sensitive biological targets.
Biocompatibility: The inert environment preserves the tertiary structure and biological activity of enzymes, antibodies, and other complex proteins during the separation process.
2. Macroporous Reticular Structure
Agarose forms a three-dimensional hydrogel network characterised by large, open pores.
High Mass Transfer: The macroporous structure (typically adjusted via agarose concentration, e.g., 4% or 6%) allows for rapid diffusion of macromolecules into and out of the beads. This is critical for maintaining high resolution and dynamic binding capacity (DBC), even at higher flow rates.
Accessibility: Unlike silica or smaller-pore polymers, agarose is particularly suited for separating large biomolecules, such as immunoglobulins (IgG), virus-like particles (VLPs), and plasmids, as these molecules can easily access the interior surface area of the beads.
3. Versatile Surface Chemistry for Ligand Coupling
Agarose is chemically versatile and easily activated, making it the preferred backbone for affinity and ion exchange chromatography.
Ease of Derivatisation: The primary hydroxyl groups on the sugar residues serve as ready attachment points for various chemical activation methods (e.g., Cyanogen Bromide, NHS-ester, Epichlorohydrin).
Ligand Stability: It supports the multipoint attachment of ligands (such as Protein A, Protein G, or charged functional groups) without compromising the stability of the matrix or the activity of the ligand.
4. Enhanced Mechanical Stability (Cross-linking)
While native agarose is mechanically soft, industrial-grade agarose media undergoes chemical cross-linking to enhance its rigidity.
Pressure-Flow Characteristics: Highly cross-linked agarose (often designated as “Fast Flow” or “High Flow”) can withstand higher back-pressures. This rigidity prevents bed compression and allows for high linear flow rates, which are essential for shortening cycle times in industrial-scale manufacturing.
Évolutivité : The mechanical strength ensures that performance parameters remain consistent when scaling up from laboratory columns to industrial process columns.
5. Robust Chemical Stability
Cross-linked agarose exhibits significant resistance to chemical degradation, facilitating rigorous cleaning and sanitisation protocols.
CIP Resistance: It tolerates exposure to harsh cleaning-in-place (CIP) agents, most notably high concentrations of Sodium Hydroxide ( – ). This allows for the effective removal of precipitated proteins, lipids, and endotoxins, extending the lifespan of the resin.
Solvent Compatibility: It is stable in aqueous buffers across a wide pH range (typically pH 3–14) and compatible with many organic solvents and chaotropic agents (e.g., urea, guanidine hydrochloride) used for column regeneration.
Milieux de chromatographie en agarose
Affinity Chromatography Media
Produit | Capacité de liaison dynamique | Application |
40 mg His/mL | Capacité de charge élevée Isolement et purification de protéines recombinantes marquées à l'histidine (His-Tag) | |
40 mg His/mL | ||
50 mg His/mL | Faible Ni2+ fuite Isolement et purification de protéines recombinantes marquées à l'histidine (His-Tag) | |
50 mg His/mL | ||
25 mg His/mL | Principalement utilisé pour la séparation et la purification des protéines du génie génétique marquées à l'histidine (His-Tag) contenant de l'EDTA ou du DTT et d'autres composants. | |
25 mg His/mL | ||
35 mg d'IgG/mL | Purification par affinité de divers anticorps polyclonaux et monoclonaux | |
50 mg d'IgG/mL | Résistance alcaline, élution facile Purification par affinité de divers anticorps polyclonaux et monoclonaux | |
10 mg GST/mL | Isolement et purification de la protéine marquée par la glutathion transférase (protéine de fusion GST), de la glutathion transférase et de la protéine dépendante du glutathion | |
1,5 mg AT Ⅲ/mL | Isolement et purification de l'AT Ⅲ, du facteur de coagulation, de la lipoprotéine, de la lipase et du polysaccharide | |
1,5 mg AT Ⅲ/mL | ||
20 mg Trypsine/mL (High Sub) 10 mg Trypsine/mL (Low Sub) | Isolement et purification de la trypsine, de la thrombine, de l'urokinase, de la kallikréine, de la prékallikréine et d'autres protéases à sérine | |
25 mg BSA/mL | Largement utilisé pour la séparation et la purification des protéines, en particulier pour l'élimination de la protéine A des anticorps monoclonaux qui ont été éliminés par le milieu d'affinité de la protéine A, ainsi que des dimères d'anticorps, des protéines de l'hôte, des acides nucléiques et des virus. | |
60 mg BSA/mL | Largement utilisé dans la séparation et la purification des protéines |
Produit | Capacité de liaison dynamique | Application |
Prosep MMA | 20 mg BSA/mL | Grande rigidité Débit élevé Haute résolution Chargement rapide |
35 mg BSA/mL | ||
60 mg lgG/mL | ||
45 mg BSA/mL | ||
| 35 mg BSA/mL | ||
| 50 mg BSA/mL |
Milieux de chromatographie par échange d'ions
| Produit | Capacité de liaison dynamique | Application |
| DEAE 6FF | 60 mg BSA/mL | Milieu d'échange d'anions faible : Haute applicabilité (FF) Haute résolution (HP) Grande capacité (XL) |
| DEAE 6HP | 60 mg BSA/mL | |
| DEAE 6XL | 120 mg BSA/mL | |
| Q 6FF | 50 mg BSA/mL | Milieux d'échange d'anions forts : Haute applicabilité (FF) Haute résolution (HP) Grande capacité (XL) |
| Q 6HP | 70 mg BSA/mL | |
| Q 6XL | 140 mg BSA/mL | |
| CM 6FF | 100 mg LZM /mL | Milieu d'échange cationique faible : Haute applicabilité (FF) Haute résolution (HP) Grande capacité (XL) |
| CM 6HP | 120 mg LZM /mL | |
| CM 6XL | 120 mg LZM /mL | |
| SP 6FF | 130 mg LZM /mL | Milieu d'échange cationique fort : Haute applicabilité (FF) Haute résolution (HP) Grande capacité (XL) |
| SP 6HP | 160 mg LZM /mL | |
| SP 6XL | 160 mg LZM /mL |
Produit | Capacité de liaison dynamique | Application |
90 mg BSA/mL | Grande rigidité Débit élevé Haute résolution Chargement rapide | |
120 mg BSA/mL | ||
120 mg de lysozyme/mL | ||
35 mg BSA/mL | ||
45 mg BSA/mL | ||
75 mg de lysozyme/mL | ||
70 mg de lysozyme/mL |
Hydrophobic Chromatography Media
| Produit | Capacité de liaison dynamique | Application |
| Butyl 4FF | 20 mg BSA/mL | Weakly hydrophobic Suitable for the separation and purification of aliphatic Protein |
| Butyl 6HP | 25 mg BSA/mL | |
| Phenyl 6FF(HS) | 30 mg BSA/mL | Strong hydrophobicity Suitable for the separation and purification of aromatic Protein (such as monoclonal antibody) |
| Phenyl 6FF (LS) | 15 mg BSA/mL | |
| Phényle 6HP | 20 mg BSA/mL | |
| Octyl 4FF | 8 mg BSA/mL | Medium hydrophobicity Suitable for the separation and purification of strong lipophilic Protein |
| Octyl 6HP | 8 mg BSA/mL |
Frequently Asked Questions (FAQ): Agarose Chromatography Media
1. Technical Characteristics & Media Selection
Q: What distinguishes Agarose media from Polymer or Silica-based matrices?
A: Agarose is a naturally derived, hydrophilic polysaccharide matrix. Its primary advantage over synthetic options is its intrinsic hydrophilicity and low non-specific binding (NSB). This characteristic minimizes the adsorption of non-target biomolecules, ensuring high protein recovery and preserving the biological activity of sensitive targets.
Vs. Silica: Agarose offers superior chemical stability under alkaline conditions, allowing the use of Sodium Hydroxide (NaOH) for Cleaning-in-Place (CIP), which can degrade silica.
Vs. Synthetic Polymers: While rigid polymers (e.g., polystyrene-divinylbenzene) offer higher pressure tolerance, they often require surface hydrophilization. Agarose is naturally hydrophilic, reducing the risk of protein denaturation, though it generally operates at lower pressure limits than rigid polymer beads.
Q: What is the difference between “Native” and “Cross-linked” Agarose?
A: Native” vs “Cross-linked
Native Agarose: The gel structure is stabilized solely by hydrogen bonds. It lacks mechanical rigidity and compresses easily under pressure, making it unsuitable for industrial flow rates. Its primary application is analytical gel electrophoresis.
Cross-linked Agarose: This variant undergoes chemical modification (typically using epichlorohydrin) to form covalent bonds between the polysaccharide chains. This cross-linking significantly enhances the mechanical rigidity of the bead, enabling the high flow rates and pressure tolerance required for industrial-scale manufacturing (comparable to the “Fast Flow” or “Capto” standards).
Q: How should I choose between 4% and 6% Agarose concentrations?
A: The agarose concentration is inversely related to the pore size of the resin:
4% Agarose: Features a larger pore structure, making it ideal for the purification of very large biomolecules such as viruses, plasmids, and large protein complexes. However, it exhibits lower mechanical strength compared to higher concentrations.
6% Agarose: Features slightly smaller pores but offers enhanced mechanical rigidity. It balances capacity and flow performance, serving as the industry standard for general protein purification, including monoclonal antibodies (mAbs) and recombinant proteins.
2. Comparative Performance & Validation
Q: How does your media perform compared to industry benchmarks like Cytiva (GE)?
A: Through significant advancements in manufacturing technology over the past decade, our media has achieved high parity with established benchmarks.
Performance: Our “Fast Flow” equivalent resins demonstrate comparable Dynamic Binding Capacity (DBC), resolution, and pressure-flow characteristics to Sepharose™ Fast Flow.
Commercial Advantage: The primary differentiators are cost-efficiency and supply chain resilience, offering significantly shorter lead times compared to imported brands.
Q: Can I utilise your media as a direct “drop-in” replacement for Sepharose FF?
A: While the base matrix (cross-linked agarose) is chemically analogous, we recommend a formal Comparability Study before full-scale implementation. Key Critical Quality Attributes (CQAs) to validate include:
Particle Size Distribution: Verification that the mesh size aligns (e.g., ~90 μm) to ensure consistent back-pressure.
Densité du ligand : Confirmation that functional group density supports the required binding capacity and elution profile.
Non-specific Binding: Evaluation of background binding levels to ensure impurity clearance meets specifications.
Q: What regulatory support is provided for GMP production?
A: We provide comprehensive support for GMP-compliant manufacturing. Our quality system is ISO 9001 certified.
3. Operational Guidelines & Maintenance
Q: What is the recommended Cleaning-in-Place (CIP) protocol?
A: Cross-linked agarose exhibits excellent alkaline stability. The standard CIP protocol utilizes 0.1 M to 1.0 M NaOH.
Parameters: A contact time of 30 to 60 minutes is standard.
Mechanism: This effectively hydrolyzes precipitated proteins, saponifies lipids, and inactivates endotoxins and viruses without compromising the integrity of the agarose backbone.
Q: What is the typical lifecycle of the resin?
A: In a well-controlled GMP process, cross-linked agarose media typically sustains 100 to 200 purification cycles. Resin lifetime is generally limited by irreversible fouling (gradual loss of DBC) or increased column back-pressure due to particulate accumulation, rather than chemical degradation of the bead itself.
Q: What are the storage requirements for the media?
A: Agarose media should be stored in a bacteriostatic solution, typically 20% Ethanol, at temperatures between 4°C and 30°C.
Critical Warning: Do not freeze agarose beads. Freezing causes the formation of ice crystals within the pore network, which fractures the gel structure and irreversibly destroys the media’s chromatographic performance.
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