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Cell Disruption (Lysis) for Recombinant Protein Production

Industrial Sonomechanics (ISM) offers laboratory, bench and industrial-scale high-amplitude ultrasonic processors, which can be used for efficient cell disruption. The processors are based on our patented Barbell Horn Ultrasonic Technology (BHUT), which makes it possible to directly implement laboratory accomplishments in a production environment, guaranteeing reproducible and predictable results at any scale.

Yeast CellsBACKGROUND

Cell disruption (lysis) is an important step in recombinant protein production procedures. Ultrasonic cell disruption (lysing with ultrasound) has been the laboratory-scale method of choice for this application for several decades. The process requires high ultrasonic amplitudes to be applied to cell suspensions, producing extreme shear forces. The shear forces are the result of intense ultrasonic cavitation, which creates violently and asymmetrically imploding vacuum bubbles and causes micro-jets that rupture cell walls. However, due to limitations of conventional ultrasonic technolgy, this method's industrial implementation has not been possible without reducing ultrasonic amplitudes, diminishing the intensity of cavitation-generated shear forces and, therefore, compromising the efficiency of the lysis process. These limitations have been overcome with BHUT, which, as explained below, permits constructing industrial-scale ultrasonic cell disruptors with the same lysis efficiency as the laboratory devices, while offering much higher productivities.

Ultrasonic Processor with Water-Cooled TransducerWhy ISM's Ultrasonic Technology?

Conventional ultrasonic liquid processing systems comprise acoustic horns that reduce their diameters in the output direction and can only provide high ultrasonic amplitudes when their output tips are small. Process scale-up requires switching to horns with larger output tip diameters, able to output the ultrasonic energy into greater volumes of processed liquids, while still maintaining high amplitudes. If, however, the output tip diameter of a conventional horn is increased to an industrialy acceptable size, its maximum vibration amplitude becomes significantly lower and insufficient for cell disruption. The use of conventional high-amplitude ultrasonic processors is, therefore, limited to laboratory investigations that cannot be directly scaled-up. ISM has successfully overcome this limitation by developing BHUT, which permits constructing pilot and industrial-scale ultrasonic processors able to generate extremely high amplitudes and operate continuously. Our industrial cell disruption equipment is compact, requires very little technical support and includes only two wetted parts (HBH-type Barbell horn and Reactor chamber) with easy access for cleaning.

Cell Disruption (Lysis) by High-Intensity UltrasoundCell Disruption of S. Cerevisiae Yeast

In this section, we demonstrate the use of high-amplitude ultrasound for the disruption of a notoriously difficult-to-break yeast cell system: S. Cerevisiae. The experiments were first conducted on a laboratory scale utilizing ISM's LSP-500 ultrasonic processor, equipped with a conventional horn (CH) with the 12.7 mm tip diameter. The procedure was then transferred to a pilot scale using ISM's BHUT-based BSP-1200 processor, equipped with a Half-wave Barbell Horn (HBH) with the 32 mm tip diameter. LSP-500 was used in the batch mode: 45 ml samples were processed in a beaker with a temperature control jacket. BSP-1200 was utilized in the recirculating flow-through mode to process 1,500 ml samples. The temperature in all experiments was 18 oC and the yeast cell suspension concentration was 20 %. Lab-scale experiments were conducted at the ultrasonic amplitudes of 75 and 20 microns (μpp), whereas in pilot-scale experiments the amplitudes were 75 and 95 μpp. Standard chemical cell disruption was also performed using CelLyticTM-Y Yeast Cell Lysis reagent (Sigma, St. Louis, MO).

The results presented above for the extraction of Alkaline Phosphatase protein (AP) show that 1) ultrasound is far superior to chemical cell lysis, 2) high ultrasonic amplitudes are needed for efficient protein extraction, and 3) the sonication process can be directly scaled up with BHUT. For the resulting AP activity of 200 mIU/ml and the ultrasonic amplitude of 75 μpp, the productivity rates of the LSP-500 and BSP-1200 processors were ~ 4.5 ml/min and ~ 30 ml/min (scale-up factor of ~ 7, as expected from the ratio of the horns' radiating areas). When the amplitude was raised to 95 μpp, the productivity rate of the BSP-1200 processor increased to ~ 90 ml/min. A further scale up factor of about 5 can be achieved by transfering this process to the commercial scale utilizing ISM's industrial ultrasonic processor, ISP-3000.

The data presented above was collected in collaboration with Allied Innovative Systems, LLC (ALLIS).