Ultrapure alginate

PRONOVA Sodium Alginates

alginate_logo

PRONOVA™ sodium alginates product group belongs to a family of highly purified and well-characterized alginates developed for use in biomedical and pharmaceutical applications.
Alginate is one of the most versatile biopolymers. It is in commercial use or in advanced clinical evaluation in a range of pharmaceutical and biomedical applications, including bone regeneration and tissue bulking. Gels, pastes, fibers and solid structures of alginate are being evaluated for use in numerous tissue-engineering applications. The best known technology is immobilization of living cells in alginate gels. Such bioreactor systems are currently being developed for the treatment of a variety of diseases.

Chemistry

Alginate is a linear copolymer with homopolymeric blocks of (14)-linked b-D-mannuronate (M) and its C-5 epimer -L-guluronate (G) residues, respectively, covalently linked together in different sequences or blocks.

The monomers can appear in homopolymeric blocks of consecutive G-residues (G-blocks), consecutive M-residues (M-blocks), alternating M and G-residues (MG-blocks) or randomly organized blocks [1-4]. The relative amount of each block type varies with the origin of the alginate.

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Alternating blocks form the most flexible chains and are more soluble at lower pH than the other blocks. G-blocks form stiff chain elements, and two G-blocks of more than 6 residues each form stable cross-linked junctions with divalent cations (e.g. Ca2+, Ba2+, Sr2+ among others) leading to a three-dimensional gel network [5, 6].

At low pH, protonized alginates will form acidic gels. In these gels, it is mostly the homopolymeric blocks that form the junctions, where the stability of the gel is determined by the relative content of G-blocks [7].

Determination of primary structure is today possible by NMR techniques by analyzing the spectrum using appropriate statistical considerations [8, 9].

References

[1] Smidsrød, O. and Draget, K.I., Carbohydr. Eur., 1996, 14, 6
[2] Ertesvåg, H., Valla, S. and Skjåk-Bræk, G., Carbohydr. Eur., 1996, 14, 14
[3] Skjåk-Bræk, G. and Espevik, T., Carbohydr. Eur., 1996, 14, 19
[4] Onsøyen, E., Carbohydr. Eur., 1996, 14, 26
[5] Stokke, B.T., Smidsrød, O., Bruheim, P. and Skjåk-Bræk, G., Macromolecules, 1991, 24, 4645
[6] Grant, G.T., Morris, E.R., Rees, D.A., Smith, P.J.C. and Thom, D., FEBS Lett, 1973, 32, 195
[7] Draget, K.I., Skjåk-Bræk, G., Christensen, B.E., Gåserød, O. and Smidsrød, O., Carbohydr.
Polym., 1996, 29, 209
[8] Grasdalen, H., Larsen, B. and Smidsrød, O., Carbohydr. Res., 1981, 89, 179
[9] Grasdalen, H., Carbohydr. Res., 1983, 118, 255

Lab_Ultrapure_Alginate_2

Gelation

In contrast to most other polysaccharide gels, alginate gels can develop and set at constant temperature. This unique property is particularly useful in applications involving fragile materials like cells or tissue with low tolerance for higher temperatures.

img_alginate_1

An alginate gel will develop instantaneously in the prescence of divalent cations like Ca2+, Ba2+ or Sr2+ and acid gels may also develop at low pH. Gelling occurs when the divalent cations take part in the interchain ionic binding between guluronic acids blocks (G-blocks) in the polymer chain giving rise to a three dimensional network. Such binding zones between the G-blocks are often referred to as egg-boxes, and consequently alginates with a high content of G-blocks induce stronger gels. Gels made of M-rich alginate are softer and more fragile, and may also have a lower porosity. This is due to the lower binding strength between the polymer chains and to the higher flexibilities of the molecules.

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The gelling process is highly dependent on diffusion of gelling ions into the polymer network and there are essentially two main classical methods for the preparation of alginate gels: the dialysis/diffusion method, and the internal gelling method.  (NovaMatrix® self-gelling technology provides an exciting alternative.)

In the dialysis/diffusion method (diffusion setting) gelling ions are allowed to diffuse into the alginate solution. This method is most commonly used in biotechnology for immobilization of living cells in alginate gel.

An alginate solution can also be solidifed by internal gelation method/internal setting, i.e. in situ gelling. Here a calcium salt with limited solubility, or complexed Ca2+-ions are mixed with an alginate solution into which the calcium ions are released, usually by the generation of acidic pH with a slowly acting acid such as D-glucono-α-lactone (GDL). This method is chosen if the purpose is to create a homogenous, non-syneretic alginate macrogel to fill the space of a given container. The main difference between internal and diffusion setting is the gelling kinetics. Consequently, the gel network will also be different.

Quality

NovaMatrix® manufacture PRONOVA™ sodium in our facilities in Norway. We operate according to GMP guidelines; ICH Q7, ICH Q1, ICH Q2 and ISO standards; ISO 9001:2008 and ISO 13485:2003. Furthermore NovaMatrix holds the Manufacturing License for API’s issued by the Norwegian Medicine Agency (NOMA).

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NovaMatrix® produces and supplies well-characterized and documented ultrapure, bio-compatible and bio-absorbable biopolymers for use in the pharmaceutical, biotechnology and biomedical industries. These may include, but not be limited to applications such as drug delivery, tissue engineering, cell encapsulation, and medical devices. For our ultrapure sodium alginates and sodium hyaluronate, we maintain Drug Master Files with the US FDA.

NovaMatrix® products are intended to be used in applications which require high and consistent quality provided by our manufacturing, Quality Control and Quality Assurance.

NovaMatrix® manufacture PRONOVA™ sodium alginate in our facilities in Norway. We operate according to GMP guidelines; ICH Q7, ICH Q1, ICH Q2 and ISO standards; ISO 9001:2008 and ISO 13485:2003. Furthermore NovaMatrix holds the Manufacturing License for API’s issued by the Norwegian Medicine Agency (NOMA).

Lab_Ultrapure_Alginate

Safety and toxicology

The safety and toxicology profile of ultrapure PRONOVA™ sodium alginate products is described in a Drug Master File submitted to the US FDA. The table below is meant to serve as a summary over some of the studies performed and the conclusions drawn from them.

Effect of sodium alginate on cell survival of V-79 and 3T3 cells cultured in vitro

Report number : ALG-95-001

  • Animal species : In vitro cell culture 3T3 mouse fibroblasts V79 Chinese hamster
  • Alginate products : PRONOVA™ UP LVG (132 mPas) – PRONOVA™ UP MVG (230 mPas)
  • Concentration : 0-1 mg/ml
  • Conclusion : Little or no effect on cell survival and colony-forming ability.

Single dose toxicity study by the intraperitoneal route

Report number : 658/521

  • Animal species : Mouse
  • Alginate products : PRONOVA™ UP MVG (230 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/522

  • Animal species : Rat
  • Alginate products : PRONOVA™ UP MVG (230 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/523

  • Animal species : Mouse
  • Alginate products : PRONOVA™ UP LVG (132 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/524

  • Animal species : Rat
  • Alginate products : PRONOVA™ UP LVG (132 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/529

  • Animal species : Rat
  • Alginate products : PRONOVA™ UP LVG (30 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/530

  • Animal species : Rat
  • Alginate products : PRONOVA™ UP LVG (3.5 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.

Single dose toxicity study by the intravenous route

Report number : 658/534

  • Animal species : Rat
  • Alginate products : Poly mannuronic acid – high molecular weight, intrinsic viscosity 16 dl/g
  • Concentration : 1, 10, 100 mg/kg
  • Conclusion : No mortality in 1 and 10 mg/kg groups, 50% mortality in 100 mg/kg group. No abnormal clinical signs in 1 of 10 mg/kg groups.
Report number : 658/535

  • Animal species : Rat
  • Alginate products : Poly mannuronic acid – low molecular weight, intrinsic viscosity 6.8 dl/g
  • Concentration : 1, 10, 100 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.

Pharmacokinetics of poly-M

Report number : Poly-M-94-1

  • Animal species : Mouse
  • Alginate products : Poly mannuronic acid (14C-labelled)
  • Concentration : 5 mg/kg
  • Conclusion : Oral: No absorption, IV: half-life approx. 4 hr, IP: half-life approx. 12.5 hr, Elimination via urine.
Report number : ALG-95-001

  • Animal species : In vitro cell culture 3T3 mouse fibroblasts V79 Chinese hamster
  • Alginate products : PRONOVA™ UP LVG (132 mPas) – PRONOVA™ UP MVG (230 mPas)
  • Concentration : 0-1 mg/ml
  • Conclusion : Little or no effect on cell survival and colony-forming ability.
Report number : 658/521

  • Animal species : Mouse
  • Alginate products : PRONOVA™ UP MVG (230 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/522

  • Animal species : Rat
  • Alginate products : PRONOVA™ UP MVG (230 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/523

  • Animal species : Mouse
  • Alginate products : PRONOVA™ UP LVG (132 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/524

  • Animal species : Rat
  • Alginate products : PRONOVA™ UP LVG (132 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/529

  • Animal species : Rat
  • Alginate products : PRONOVA™ UP LVG (30 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/530

  • Animal species : Rat
  • Alginate products : PRONOVA™ UP LVG (3.5 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/534

  • Animal species : Rat
  • Alginate products : Poly mannuronic acid – high molecular weight, intrinsic viscosity 16 dl/g
  • Concentration : 1, 10, 100 mg/kg
  • Conclusion : No mortality in 1 and 10 mg/kg groups, 50% mortality in 100 mg/kg group. No abnormal clinical signs in 1 of 10 mg/kg groups.
Report number : 658/535

  • Animal species : Rat
  • Alginate products : Poly mannuronic acid – low molecular weight, intrinsic viscosity 6.8 dl/g
  • Concentration : 1, 10, 100 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : Poly-M-94-1

  • Animal species : Mouse
  • Alginate products : Poly mannuronic acid (14C-labelled)
  • Concentration : 5 mg/kg
  • Conclusion : Oral: No absorption, IV: half-life approx. 4 hr, IP: half-life approx. 12.5 hr, Elimination via urine.

Alginate Products Shelf -Life

NovaMatrix® conducts stability studies according to ICH guidelines for all our product ranges. For sodium alginates, the parameters monitored are appearance of powder, dry matter content, intrinsic viscosity, apparent viscosity, pH, molar mass, chemical composition, ash content, endotoxins, and microbial purity.

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For most applications of alginates, the molar mass (molecular weight) is an important parameter determining the functionality of the alginate for that specific application. Aqueous solutions of alginates are viscous, the observed solution viscosity being dependent on molar mass, concentration of alginate, temperature, and potentially also presence of salts and other compounds in the formulation. Viscosity measurements are often used as a simple measurable parameter of changes in the molar mass of a polymer, and thereby can be used as a stability indicating parameter.

img_alginate_2

Figure 1: Apparent viscosity values observed during 60 months of storage of PRONOVA™ UP LVG.

Depolymerization of alginates will occur in solution and in the solid state due to a variety of mechanisms. Smidsrød et al (1963) observed that the presence of oxygen affected the stability of non-purified alginates in solution due to the presence of phenolic reducing substances that gave rise to oxidative-reductive free radical depolymerization (ORD). On the other hand, Holme et al. (2003; 2008) have shown that purified alginates in the solid state and in solution are depolymerized primarily by acid catalyzed hydrolysis and alkaline catalyzed mechanisms. The highest stability was observed in a pH interval of 5 to 8. Increased temperature will increase the rate of depolymerization for all mechanisms mentioned above (Holme et al. 2003; 2008). In vivo data on the kinetics and mechanism of depolymerization of alginates are scarce. There are no mammalian enzymes that are active towards alginates, however, even in mammals, one must expect random depolymerization mechanisms like the ones described above to occur. This document reports on the stability, shelf-life and storage conditions for PRONOVA™ UP sodium alginate powders.

NovaMatrix® conducts stability studies according to ICH guidelines for all our product ranges. For sodium alginates, the parameters monitored are appearance of powder, dry matter content, intrinsic viscosity, apparent viscosity, pH, molar mass, chemical composition, ash content, endotoxins, and microbial purity.

Apparent viscosity, intrinsic viscosity, and molar mass, which are all indicative measurements of depolymerization, have been observed to change significantly upon time under certain storage conditions. Figure 1 shows example data for the apparent viscosity (measured in 1% (w/w) sodium alginate solution at 20°C using Brookfield viscosimeter at 20 rpm) from a stability study conducted on PRONOVA™ UP LVG (low viscosity, high-G sodium alginate). No significant change in apparent viscosity is apparent after 5 years of storage at 2-8°C/ambient relative humidity (RH), or at -18°C. For storage at 25°C/60% RH, apparent viscosity displayed approximately 20% decrease after 5 years, while at 40°C/75% RH, apparent viscosity was reduced by approx. 40% after 12 months.

Based upon stability studies, NovaMatrix recommends refrigerated storage (2-8°C) of all PRONOVA™ UP sodium alginates. We label PRONOVA™ UP VLVG/ LVG/LVM/MVG with a shelf life of 5 years under refrigerated storage. PRONOVA™ UP VLVM and MVM have a shelf life of 3 years. All PRONOVA™ UP sodium alginates are delivered in a closed polyethylene container. During storage, the lid of the container should be kept closed.

Our stability programs show that PRONOVA™ UP sodium alginates can be stored at room temperature (25°C/ 60% RH) for 6-9 months without significant reduction of apparent viscosity. Therefore, we do not consider cool/cold shipment of products to customers a necessity, as transportation and transit times are normally a matter of a few days.

PRONOVA™ UP specifications with respect to apparent viscosity are intended to be broad and suitable for a range of applications.

References:

  • Smidsrød, O., Haug, A., and Larsen, B. “Degradation of alginates in the presence of reducing compounds”, Acta. Chem. Scand. 17 (1963), p2628-2637.
  • Holme, H.K., Lindmo, K., Kristiansen, A., and Smidsrød, O. ”Thermal depolymerization of alginate in the solid state”, Carbohydr. Polym. 54 (2003), p431-438.
  • Holme, H.K., Davidsen, L., Kristiansen, A. and Smidsrød, O. ”Kinetics and mechanisms of depolymerization of alginate and chitosan in aqueous solution”, Carbohydr. Polym., Manuscript in press (2008).
  • (e.g. http://www.novamatrix.biz/Portals/novamatrix/Content/Docs/Technology/PIB-Alginate%20Stability%20093009.pdf)

Application examples

  • Anti-adhesion
  • Additives for cryopreservation of cells/tissues
  • Sealants
  • Drug delivery
  • 3D printing
  • 3D cell culture
  • Hydrogel, extracellular matrix scaffolds
  • Internal wound healing
Please select the product to obtain more informations or order

PRONOVA UP VLVM

  • Viscosity  [mPa*s] :  < 20
  • Appr. Mw [kDa] : < 75
  • G/M Ratio : ≤ 1
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

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PRONOVA UP LVM

  • Viscosity  [mPa*s] :  20-200
  • Appr. Mw [kDa] : 75-200
  • G/M Ratio : ≤ 1
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

Purchase the product

PRONOVA UP MVM

  • Viscosity  [mPa*s] > 200
  • Appr. Mw [kDa] : > 200
  • G/M Ratio : ≤ 1
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

Purchase the product

PRONOVA UP VLVG

  • Viscosity  [mPa*s] :  < 20
  • Appr. Mw [kDa] : < 75
  • G/M Ratio : ≥ 1.5
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

Purchase the product

PRONOVA UP LVG

  • Viscosity  [mPa*s] :  20-200
  • Appr. Mw [kDa] :  75-200
  • G/M Ratio : ≥ 1.5
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

Purchase the product

PRONOVA UP MVG

  • Viscosity  [mPa*s] :  > 200
  • Appr. Mw [kDa] : > 200
  • G/M Ratio : ≥ 1.5
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

Purchase the product

Product

Viscosity [mPa*s]

Appr. Mw [kDa]

G/M Ratio

Endotoxins [EU/g]

Total viable count [cfu/g]

< 20

< 75

≤ 1

≤ 100

≤ 100

20-200

75-200

≤ 1

≤ 100

≤ 100

> 200

> 200

≤ 1

≤ 100

≤ 100

< 20

< 75

≥ 1.5

≤ 100

≤ 100

20-200

75-200

≥ 1.5

≤ 100

≤ 100

> 200

> 200

≥ 1.5

≤ 100

≤ 100

PRONOVA SLM20

  • Viscosity  [mPa*s] :  20-99
  • Appr. Mw [kDa] : 75-150
  • G/M Ratio : ≤ 1
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : Sterile

Purchase the product

PRONOVA SLM100

  • Viscosity  [mPa*s] :  100-300
  • Appr. Mw [kDa] : 150-250
  • G/M Ratio : ≤ 1
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : Sterile

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PRONOVA SLG20

  • Viscosity  [mPa*s] :  20-99
  • Appr. Mw [kDa] : 75-150
  • G/M Ratio : ≥ 1.5
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : Sterile

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PRONOVA SLG100

  • Viscosity  [mPa*s] :  100-300
  • Appr. Mw [kDa] : 150-250
  • G/M Ratio : ≥ 1
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : Sterile

Purchase the product

Product

Viscosity [mPa*s]

Appr. Mw [kDa]

G/M Ratio

Endotoxins [EU/g]

Total viable count [cfu/g]

20-99

75-150

≤ 1

≤ 100

Sterile

100-300

150-250

≤ 1

≤ 100

Sterile

20-99

75-150

≥ 1.5

≤ 100

Sterile

100-300

150-250

≥ 1.5

≤ 100

Sterile