Cell Lysis: Method Selection and Optimization for Various Types of Target Products

In host cell culture-based biopharmaceutical production, the target product can either remain within the cell as an intracellular product or be released into the surrounding culture medium as an extracellular product. The decision regarding whether the product is retained inside the cell or released into the external environment depends on the choice of host system. For instance, when using Escherichia coli (E. coli) as the host cell for production, the products are primarily intracellular. In the case of intracellular products, a cell lysis step is necessary to release them into the fluid surrounding the cells. Depending on the process design, this fluid can consist of either depleted media from the bioreactor or suspension media from the bioreactor. A suitable buffer solution is required for cells harvested from the reactor. Conversely, processes that focus on extracellular products do not necessitate a lysis step, as these products can be secreted from the cells and directly isolated and extracted using centrifugation and/or filtration methods.

Intracellular products are typically located in the cytoplasm or periplasm. Biopharmaceuticals produced by E. coli, such as insulin and human growth hormone, serve as prime examples. Recombinant intracellular products can be classified as soluble, insoluble, or a combination of both. Insoluble products are formed as inclusion bodies, which are micrometer-sized solid aggregates containing relatively "pure" but inactive protein. Since the proteins that make up the inclusion bodies are misfolded, these aggregates must be solubilized by unfolding (denaturing) the proteins using denaturants, followed by refolding (renaturing) the proteins to yield biologically active soluble biopharmaceutical products. The recovery of products during this step is often very low due to significant product precipitation. Consequently, engineering and optimizing cell line construction to promote soluble production is generally preferred, as it enhances overall process yields.

For intracellular products retained in the periplasm of the cell, yields tend to be lower, and the recovery of periplasmic proteins may be suboptimal. However, one advantage of periplasmic products is that a cell lysis step can be designed to selectively release products contained in the periplasm without releasing the contents of the cytoplasm. This selective release can result in higher product purity and allow the purification stage of the process flow to begin in a more favorable state. Nevertheless, designing methods for selectively releasing periplasmic products can be challenging.

Extracellular products are released into the medium used for cell growth. A common example is monoclonal antibodies produced by Chinese Hamster Ovary (CHO) cells, which can secrete proteins. The advantage of extracellular products is that they eliminate the need for a lysis step, which, while releasing the product, would also release unwanted cellular components into the process stream. Consequently, extracellular products typically enter the purification stage with only small amounts of soluble impurities.

Other processes yield specific ratios of intracellular and extracellular products. For instance, the production of adeno-associated virus (AAV) utilizing human embryonic kidney 293 (HEK293) cells serves as a prime example. AAV is widely employed as a vector for gene therapy. When HEK293 cells are used for upstream production, some serotypes, such as AAV5 and AAV9, are generated as both intracellular and extracellular capsids. In contrast, other serotypes, like AAV2, are predominantly intracellular due to AAV2's affinity for heparin present on the surface of HEK293 cells. Additionally, serotypes such as AAV1, AAV6, AAV7, and AAV8 are primarily found in the extracellular space.

Whether the product is intracellular, extracellular, or a combination of both, it will influence the design of the production process.

The figure below illustrates two distinct process route designs for intracellular products.

In the first process, cells are recovered from the bioreactor through centrifugation and then suspended in a buffer solution suitable for the product and the initial chromatography step of the purification stage. The resulting cell suspension is homogenized to create a lysate, also known as a homogenate, which is subsequently filtered to obtain a clarified lysate. In the second process design, the product is lysed directly in the bioreactor using a detergent. The resulting lysate is clarified through single or multi-stage depth filtration, followed by product concentration and buffer exchange using tangential flow ultrafiltration. This step ensures that the product is in the appropriate buffer for loading onto the first chromatography column of the purification stage. The second design is particularly advantageous when the product is both intracellular and extracellular, such as certain viral vectors used in gene therapy, because all contents of the bioreactor proceed to the next step in the process. In contrast, the first process design only allows cells from the bioreactor to advance to the next step, resulting in the loss of any product present in the culture medium. However, the advantage of the first process design is that no buffer exchange step is necessary before the purification stage, as the cells are already suspended in the buffer solution required for the initial chromatography step.

There are several lysis methods available for recovering intracellular products, which can be broadly categorized into two groups: non-mechanical and mechanical. The selection of a lysis method depends on various factors, particularly the ease of disrupting the cells to facilitate product release and achieve high recovery rates. Additionally, it is crucial to maintain the integrity of the target molecule to avoid denaturation, as well as to consider the scalability of the method. Other important factors include the ease of removing the resulting cell debris, the processing time, and, of course, the cost.

For non-mechanical methods, chemical treatments are employed to "permeabilize" the animal cell membrane, facilitating the release of intracellular products into the fluid surrounding the cell. Non-ionic surfactants, such as Triton X-100, are commonly utilized for this purpose, as well as organic solvents like toluene. However, the use of organic solvent treatments is not prevalent in biopharmaceutical production due to the associated risk of product denaturation.

Another method for cell lysis is alkaline lysis, which utilizes hydroxide ions (OH⁻) as the primary component to disrupt the cell membrane. The lysis buffer is composed of sodium hydroxide and sodium dodecyl sulfate (SDS). The OH- ions interact with the cell membrane, breaking the fatty acid-glycerol bonds, which subsequently renders the cell membrane permeable. Meanwhile, SDS solubilizes the proteins and the cell membrane. A pH range of 11.5–12.5 is optimal for effective cell lysis. Although this method is applicable to a variety of cell types, it is relatively slow, typically requiring 6 to 12 hours to complete. Alkaline lysis is primarily employed to isolate plasmid DNA from bacteria.

Enzyme treatments can be employed to digest the cell walls of bacteria, such as Escherichia coli, and yeast, such as Saccharomyces cerevisiae, leading to cell rupture. For instance, lysozyme disrupts the cell wall of Gram-negative bacteria by catalyzing the cleavage of polysaccharide chains in the peptidoglycan layer. However, in Gram-negative bacteria, access to the peptidoglycan is restricted by the outer membrane, which may hinder the effectiveness of enzymatic treatments. Consequently, it may be necessary to expose the cells to Triton X-100 or other surfactants to dissolve the outer cell membrane. Generally, the disadvantages of using enzymatic treatments to lyse bacteria or yeast include high costs and the potential unavailability of the required enzymes.

Osmotic shock can lyse animal cells by creating an osmotic pressure gradient between the fluid surrounding the cell and the cell's interior. Osmotic pressure is the hydrostatic pressure generated by the difference in solute concentration across a semipermeable membrane. When the extracellular solute concentration decreases, the osmotic gradient causes water to flow into the cell, ultimately leading to cell rupture. Another non-mechanical method involves the use of freeze-thaw cycles, particularly for animal cells, during which ice crystals form and compromise the integrity of the cell membrane. This method is typically employed in laboratory settings and is not commonly used in production environments due to its lack of scalability.

Many mechanical methods can be employed for cell lysis. In the ultrasonic method, an electronic generator emits high-frequency energy waves through a metal tip. The vibrations in the cell slurry induce cavitation, resulting in cell destruction; however, this method is not commonly utilized in biopharmaceutical production and may not be suitable for lysing resilient cells such as yeast. Another method is bead milling, which involves shaking cells with glass or stainless steel beads to effectively lyse yeast.

High-pressure homogenization is the most widely used method for cell lysis in biopharmaceutical processing. This technique achieves cell disruption by shearing a cell jet at high speeds against a solid surface. The cell suspension is pumped to high pressure within a compression chamber. The compressed cell suspension is then forced into a low-pressure chamber at high velocity through a small nozzle or a narrow orifice of a valve. The accelerated cell jet collides violently with the valve surface, resulting in the rapid transfer of the cell suspension from the high-pressure area to the low-pressure area. During this process, cell rupture occurs due to shear stress from collisions with the hard surface and the pressure drop. The mechanisms of cell rupture associated with high-pressure homogenization may vary according to different theories, which consider factors such as collision, turbulence, high-pressure shear, pressure drop, flow rate reduction, and cavitation. However, it is generally accepted that the primary causes of cell rupture are impact force, shear force, and cavitation. The fundamental principle of cell rupture is attributed to the non-specific tearing of the cell wall. High-pressure homogenization can be scaled up to process large volumes of feed while maintaining a high recovery rate of biological products. Nevertheless, this technology can generate elevated temperatures, which may be detrimental to heat-sensitive products such as enzymes, lipids, and proteins. To mitigate these challenges, a cooling system can be implemented.

Mechanical and non-mechanical cell lysis technologies each have their own advantages and disadvantages. Mechanical methods are generally effective at lysing resilient microbial cells; however, they tend to completely disrupt the cells, resulting in the release of all intracellular material. This can lead to increased challenges with impurities during the purification stage. In contrast, non-mechanical methods may produce milder lysis, thereby releasing relatively fewer unwanted intracellular impurities.

Mechanical methods generate more heat than non-mechanical methods, which can adversely affect the activity of the product. Therefore, it is essential to implement strategies to minimize temperature increases. Most equipment utilizing mechanical methods is equipped with heat exchangers to cool the lysed product. Additionally, it is considered best practice to cool the feed to these units to a temperature range of 2-8°C. This ensures that, despite the heat generated, the lysate temperature remains sufficiently low to prevent denaturation of the product. Furthermore, chemical methods necessitate the use of chemical additives, which must be removed in subsequent downstream processing steps. In contrast, mechanical methods do not require additives, eliminating concerns about the removal of additional process-related impurities.

Antuos Nanotechnology (Suzhou) Co., Ltd., a subsidiary of Duoning Biotech, provides a full range of high-pressure homogenizer products that can support applications of different scales from laboratory to production, with a maximum processing speed of 3,000 - 4,000 L/H.

Ÿ   High disruption rate, which can reach more than 95% after one pass;

Ÿ   Special feed valve design, no need for exhaust, direct feeding;

Ÿ   Variable frequency control system, flow rate can be adjusted according to requirements;

Ÿ   Built-in cooling system, directly absorbs heat to ensure the activity of product of interest;

Ÿ   Compliant with GMP requirements, with EU CE certification;

Ÿ   Optional: pneumatic pressurization, PLC control.

Duoning Biotech DuoMix/Mini DuoMix mixing systems can be used as containers for non-mechanical lysis operations, such as chemical lysis methods, enzyme lysis and alkaline lysis. This series of products can support applications ranging from 2-20 L benchtop applications to 3,000 L production scale applications. The products have powerful mixing functions and can quickly reach homogenization conditions. Also, the sophisticated stirring impeller design can reduce the shear stress effect on the target product. In addition to basic functions such as stirring and weighing, pH, temperature, conductivity monitoring elements and auxiliary peristaltic pumps can be optionally configured to better monitor and control the lysis process.

In addition, we provides hollow fiber tangential flow microfiltration modules suitable for harvesting cells such as E.Coli and yeast, which can achieve rapid and continuous harvesting of  fermentation broths, and simultaneously perform buffer exchange, avoiding the tedious operation of using centrifuges. Combined with the optimized selection of different pore size specifications, hollow fiber tangential flow filtration technology can also be well used for inclusion body washing, lysate clarification, target protein concentration, and buffer exchange. For applications that require maintaining the integrity of the whole cell, such as separating extracellular products, the mild laminar flow state within the hollow fiber can avoid cell breakage and release of unwanted impurities, reducing the burden of further purification in downstream.