Hielscher – Ultrasound Technology

Ultrasonic Solutions for Improved Vaccine Production

  • Sonication is used in various steps of vaccines preparation: for cell lysis, to homogenize cell suspensions, to stimulate cell growth, for encapsulation, for adjuvant protein binding etc.
  • Hielscher’s ultrasonicators are used in the antigen production, encapsulation and formulation as well as in the degassing step before filling the vaccine into vials or syringes.
  • Hielscher Ultrasonics is your long-experienced partner for reliable ultrasonic systems in the pharma industry. Find out during which process steps of vaccine manufacturing can improve your production!

Production of Vaccines

Ultrasonication can be beneficial during various stages of vaccine production. To produce a vaccine, the first step is to prepare the antigen itself. Depending on the typ of pathogen, the metod of antigen generation differs: Whilst viruses are grown either on primary cells such as chicken eggs (e.g. for influenza) or on continuous cell lines such as cultured human cells (e.g. for hepatitis A), bacteria are grown in bioreactors (e.g. Haemophilus influenzae type b). Recombinant proteins, which have been derived from viruses or bacteria, can be grown also in yeast, bacteria, or cell cultures. When the antigen is produced, it must be released from the cells in which it has been grown.
A virus may need to be inactivated, possibly with no further purification required. Recombinant proteins need many operations involving ultrafiltration and column chromatography. Depending on the vaccine formulation, an adjuvant, stabilizing agents, and preservatives are added. Adjuvants enhance the immune response of the antigen, stabilizers and preservatives increase the shelf life.
During vaccine manufacturing sonication can be applied at various stages. As a non-thermal processing method, heat degradation of valuable material is avoided. Find below the most common applications where ultrasound improves the production of vaccines:

Dispersion of Antigens

Ultrasonic shear disperses particles homogeneously.Antigens such as cell fragment or protein antigens must be homogeneously dispersed into a suspension, polymer, or liposomal encapsulation in order to obtain a stable vaccine formulation. Sonication is long-term proven to prepare fine dispersions in the manufacturing of pharmaceutical products and is therefore an established technique in the modern vaccine production.
Aluminum-based adjuvants, composed of very small primary particles, are a commonly used type of adjuvant, which can be easily aggregated into a functioning unit in vaccine formulations. In order to combine adjuvants with antigens, a uniform distribution of antigen throughout the aluminum-containing vaccine is required. Ultrasonic dispersion prepares homogeneous dispersions of antigens and adjuvants (e.g. Alhydrogel™).

Ultrasonic homogenizers such as the UIP1000hdT are used for various applications in the vaccine manufacturing.

UIP1000hdT with ultrasonic flow reactor

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Cell Lysis & Extraction

The antigens produced from microorganisms must be released from the microbial cell. Sonication is a proven technology of cell lysis and extraction. By adjustment of the sonication parameters, cells can be perforated or disrupted so that the targeted antigens become available and can be isolated.

Inactivation of Pathogens

Escherichia coli bacteria are reliably lysed using ultrasonic tissue homogenizers.Power ultrasound is applied to disrupt and kill microorganisms such as bacteria and viruses. For instance, ultrasonic deactivation of E. coli followed by irradiation has been shown to be the most potent technique for preparation of an effective colibacillosis vaccine. [Melamed et al. 1991]
Commonly used techniques for microbial inactivation are thermal pasteurization and sterilization, which are based on long exposure to high temperatures and often lead to thermally induced deterioration of functional properties. A combined treatment of sonication and heat (thermo-sonication) can accelerate the rate of sterilization; since the thermal intensity and duration is significantly reduced, thermal degradation of heat-sensitive compounds (e.g. proteins, antigens). Ultrasonic sterilisation and pasteurization is cost-efficient, energy-saving and environment-friendly.

Emulsions & Suspensions

Ultrasonically prepared double emulsion, e.g. for pharmaceuticals.Vaccine formulations may consist of water-lipid mixtures. Since water-lipid formulations are immiscible, a fine-size emulsion must be prepared by either overcoming the droplets’ surface tension or using a surfactant. Ultrasonic emulsification is a well-established technique to formulate nano-emulsions / mini-emulsions, double emulsions, and pickering emulsions. For instance, water-insoluble lipopeptides can be ultrasonically suspended with antigen at a 1:1 (w/w) ratio in an aqueous solution.
Further, sonication is applied in order to reduce cell aggregats and to distribute the single-dispersed cell evenly in the suspension.

Ultrasonication is used to produce vaccines (Click to enlarge!)

Ultrasonic glass reactor

Adjuvants and Preservatives

Vaccines typically contain one or more adjuvants, used to boost the immune response. By ultrasonication, adjuvant microfibres are detangled and homogeneously dispersed so that the protein binding on the surface is improved. Emulsion-based adjuvant systems are widely used in vaccine development and formulation. Such emulsion-based adjuvant systems can be formulated using various emulsion types such as oil-in-water (o/w), water-in-oil (w/o), water-in-oil-in-water (w/o/w), or protein-stabilized emulsions.
Furthermore, preservatives are added to prevent the vaccine’s contamination with bacteria or fungi. Preservatives may be used at various stages of production of vaccines.
The use of ultrasonic homogenizers promote a more even and fine mixing and dispersing and is thereby a reliable tool for a more efficient vaccine production.

Formulation & Liposomal Encapsulation

Liposome-encapsulated vaccines can be administered orally, intranasally, intramuscularly, subcutanously and are an advantageous vaccine delivery method and adjuvant, which could improve targeted delivery and reduce toxicity of the entrapped antigens. Sonication is a reliable technique to encapsulate active compounds into liposomal formulations. Read here more about the ultrasonic formulation of liposomes!
Dor instance, in order to formulate a veterinary vaccine against the Newcastle disease, Zhao et al. (2011) prepared a phosphatidylcholine/cholesterol small unilammelar vesicles (SUV) under sonication. The ultrasonically encapsulated vaccine showed an enhanced immune response, higher IgG and IgM antibody titers as well as T-cell and B-cell proliferation.

Degassing

During vaccine and pharmaceutical production and before packaging, vaccines and liquids such as suspensions, solutions, emulsions and final formulations must be degassed. During the deagassing / de-aeration step gas bubbles (e.g. oxygen, carbon dioxide, which are entrapped in the liquid, are removed. Ultrasonic waves promote the coalescence of gas bubbles entrapped in liquids. The coalesced bubbles have a higher buoyancy and rise to the liquid surface. The removal of gas bubbles can be enhanced when a slight vacuum is applied to the sonication vessel. Ultrasonically assisted degassing is an easy and rapid deaeration technique of aqueous suspensions.

Cell Growth

Ultrasonic agitation during inoculation (the process of introducing microorganisms into a culture medium) can increase the growth of cell cultures. The intensity of sonication, temperature and retention time in Hielscher’s ultrasonic bioreactors can be exactly regulated regarding the cell type and its requirements.
For instance, mild sonication can be applied to increse the glucose uptake of cells and promote thereby the growth of cell cultures and suspensions. Ultrasound is known to increase cell permeability, which, in turn, can enhance nutrient / waste exchange thus leading to enhanced vaccine production. Thereby, the vaccine production time can be shortened and/or the yield of proteins used as vaccines can be increased.

Hielscher Ultrasonics’ Pharma-Reactors

Hielscher Ultrasonics is specialised in the production of high-power ultrasonic systems and sono-bioreactors for the implementation in R&D and industrial production of pharmaceuticals (e.g. vaccines, APIs)
Closed pharma-batch reactorSonication can be applied to open vessels, closed reactors and continuous flow-through reactors. All parts of the ultrasonic systems, which get in contact with the liquid medium, are made from stainless steel, titanium, or glass. Autoclavable parts and sanitary fittings ensure the production of pharma-grade products.
Intelligent software records the parameters of the sonication process automatically on the integrated SD memory card. The precise control of all process parameters ensure the reproducibility and standardisation of production.
Hielscher Ultrasonics’ industrial ultrasonic processors are highly reliable and can be precisely controlled. All industrial ultrasonicators can adjusted to deliver the full range from lower to very high amplitudes. Amplitudes of up to 200µm can be easily continuously run in 24/7 operation. For even higher amplitudes, customized ultrasonic sonotrodes are available. The robustness of Hielscher’s ultrasonic systems allows for 24/7 operation under heavy duty and in demanding environments.

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Hielscher Ultrasonics manufactures high-performance ultrasonicators for full-spectrum extracts.

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Literature/References



Facts Worth Knowing

Working Principle of Power Ultrasound: Accoustic Cavitation

Ultrasonic / acoustic cavitation creates highly intense forces which opens the cell walls known as lysis (Click to enlarge!)

Ultrasonic extraction is based on acoustic cavitation and its hydrodynamic shear forces