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CN-122012216-A - Oncolytic virus efficient intelligent packaging and purifying system

CN122012216ACN 122012216 ACN122012216 ACN 122012216ACN-122012216-A

Abstract

The invention provides an efficient and intelligent oncolytic virus packaging and purifying system, which comprises an intelligent virus packaging module, a totally-enclosed primary harvesting module, a self-adaptive chromatography purifying module and a central intelligent control system, wherein the totally-enclosed integrated flow path is designed, and AI image recognition and UV-Vis and DLS on-line detection are fused through a sterile pipeline in series connection with the packaging, harvesting and purifying modules, so that the intelligent judgment of harvesting time and purifying components is realized, and the pollution caused by manual intervention is avoided; the AI detection replaces manual judgment, greatly improves the accuracy of harvesting and purifying, accurately captures the target virus by self-adaptive chromatography, remarkably improves the purity and recovery rate, continuously optimizes parameters by machine learning, reduces batch difference and provides a reliable scheme for large-scale production of clinical oncolytic viruses.

Inventors

  • YANG JUXIANG

Assignees

  • 山东恒光生命科技有限公司

Dates

Publication Date
20260512
Application Date
20260130

Claims (6)

  1. 1. The efficient and intelligent oncolytic virus packaging and purifying system is characterized by comprising an intelligent virus packaging module, a totally-enclosed primary harvesting module, a self-adaptive chromatography purifying module and a central intelligent control system; the intelligent virus packaging module is used for realizing efficient transfection of plasmids into packaging cells, packaging and culturing the viruses, and judging the optimal harvesting time of the viruses by monitoring the cell states in real time; The fully-closed primary harvesting module is connected with the intelligent virus packaging module through a sterile pipeline and is used for receiving a virus culture solution and completing cell lysis, virus release and primary clarification of the culture solution in a closed environment; The self-adaptive chromatography purification module is connected with the totally-enclosed primary harvesting module through a sterile pipeline and is used for carrying out multi-step fine purification on the virus crude extract after preliminary clarification and accurately collecting target virus fractions based on real-time detection data; The central intelligent control system is respectively connected with the intelligent virus packaging module, the totally-enclosed primary harvesting module and the self-adaptive chromatography purification module in a wired or wireless communication mode and is used for receiving real-time operation data of each module and dynamically coordinating and optimizing process parameters of each module through a preset algorithm model; The intelligent virus packaging module, the totally-enclosed primary harvesting module and the self-adaptive chromatography purification module are connected in series with the sanitary-grade diaphragm valve through the sterile quick-mounting connector to form a continuous and enclosed integrated production flow path.
  2. 2. The efficient and intelligent oncolytic virus packaging and purifying system according to claim 1, wherein the intelligent virus packaging module comprises: the bioreactor is internally provided with an on-line sensor for temperature, pH and dissolved oxygen concentration; The online microscopic imaging system is integrated at the top of the bioreactor and comprises a positive microscope module with 20X-40X objective lens, a CMOS image sensor and an AI image recognition unit; The transfection unit is connected with the bioreactor through a sterile pipeline and is a microfluidic electric transfection unit or a high-flux nano material transfection unit; The AI image recognition unit is used for analyzing the cell density, the fusion degree and the pathological change effect in real time and transmitting the analysis result to the central intelligent control system.
  3. 3. The efficient and intelligent oncolytic virus packaging and purifying system according to claim 1, wherein the totally-enclosed primary harvesting module comprises: the sterile harvesting tank is connected with the bioreactor of the intelligent virus packaging module through a sterile pipeline; the processing unit is integrated inside the sterile harvesting tank and is an ultrasonic crushing unit or a tangential flow filtration unit; the power adjustable range of the ultrasonic crushing unit is 50-300W, the working frequency is 20-40kHz, the tangential flow filtration unit is provided with a hollow fiber membrane or a flat membrane with the molecular weight cut-off of 100-500kDa, and the membrane material is polyethersulfone or regenerated cellulose.
  4. 4. The efficient and intelligent oncolytic virus packaging and purifying system according to claim 1, wherein the adaptive chromatography and purifying module comprises: the chromatographic column group at least comprises an affinity chromatographic column and a molecular sieve chromatographic column which are connected in series in sequence, and an ion exchange chromatographic column can be optionally matched; The online detection unit is arranged at the sample injection end and the elution end of the chromatographic column group and comprises an ultraviolet-visible light spectrum detector and a dynamic light scattering detector; The electric diverter valve is arranged at the eluting end of the chromatographic column group and is electrically connected with the central intelligent control system; the detection wavelength range of the U ultraviolet-visible light spectrum detector is 220-300nm, the detection wavelength range is used for detecting protein absorption peaks of eluent, the detection range of the particle size of the DLS detector is 10-1000nm, and the detection wavelength range is used for detecting particle size distribution of particles in the eluent.
  5. 5. The efficient and intelligent oncolytic virus packaging and purifying system according to claim 1, wherein the central intelligent control system comprises: The data acquisition module is used for receiving temperature, pH, dissolved oxygen, cell state, eluent spectrum and particle size distribution data of each module; a process database pre-stores at least 50 batches of process parameter data sets for qualified oncolytic virus production, including transfection conditions, culture parameters and purification elution procedures; the algorithm processing module adopts a random forest and gradient lifting tree fusion algorithm, and dynamically adjusts transfection voltage, chromatographic elution flow rate and diverter valve switching time based on real-time data and a process database; the data recording module is used for storing the whole flow data of each batch of production, and the storage time is more than or equal to 5 years.
  6. 6. The efficient and intelligent oncolytic virus packaging and purifying system according to claim 2, wherein the microfluidic electrotransfection unit comprises: The microfluidic chip is provided with 3-5 parallel micro-channels, the width of each channel is 50-200 mu m, and the depth is 20-50 mu m; the pulse power supply outputs 100-800V of voltage range, the pulse width is 10-100 mu s, the pulse times are 1-5 times; the sample injection end of the microfluidic chip is connected with a plasmid solution storage tank and a packaging cell suspension storage tank, and the sample outlet end is connected with the bioreactor.

Description

Oncolytic virus efficient intelligent packaging and purifying system Technical Field The invention relates to the technical field of biological pharmacy, in particular to an efficient and intelligent packaging and purifying system for oncolytic viruses. Background Oncolytic viruses become one of the core research directions in the field of tumor immunotherapy by virtue of a unique mechanism of target lysis of tumor cells and activation of systemic anti-tumor immune response, and the clinical transformation of oncolytic viruses needs to rely on a stable large-scale production process. The typical oncolytic virus production process comprises the key links of packaging cell culture, virus plasmid transfection, virus packaging amplification, cell lysis and harvesting, crude extract clarification, fine purification and the like, and the clinical application has extremely high requirements on the sterility (no exogenous microorganism pollution), purity (impurity protein/nucleic acid residue is less than or equal to 0.1%) and batch consistency (titer variation coefficient is less than or equal to 5%) of products, the traditional general bioreactor and separation equipment are difficult to adapt to the special and fine production requirements, and the targeted integrated intelligent equipment is needed to break through the technical bottleneck; The existing oncolytic virus production technology has multi-dimensional defects, and the defect sources are directly related to process design and equipment configuration, namely, the pollution and inactivation risks are prominent due to process link splitting. In the existing production, virus packaging (bioreactor), harvesting (centrifugal equipment) and purification (chromatography system) are mostly independent equipment, material transfer depends on manual operation, even if a sterile butt joint pipeline is adopted, connection is still needed to be completed in an ultra clean bench, and environmental exposure risks are unavoidable. The method has the advantages that cytopathic effect (CPE) is observed by a microscope during harvesting, judging errors of different operators on the CPE grade can reach 15% -20%, the preset elution flow rate and buffer solution proportioning parameters are adopted in the purification stage, the strategy can not be adjusted according to the batch difference of virus concentration and impurity protein content in the crude extract, the parameters can be modified after the existing attempt is made to detect the quality of the crude extract in an off-line manner, the time is up to 2-4 hours, virus degradation is easy to cause, and the system lacks collaborative optimization capability. The device is independently provided with a controller, parameters such as transfection voltage, culture dissolved oxygen, chromatographic flow rate and the like are manually debugged one by one according to experience, the downstream process cannot be dynamically optimized based on the upstream cell state, the automatic accumulation and analysis functions of production data are avoided, the process improvement depends on manual summarization, and the efficiency is low. Disclosure of Invention (One) solving the technical problems Aiming at the defects of the prior art, the invention provides a high-efficiency intelligent oncolytic virus packaging and purifying system, which solves the problems of prominent pollution and inactivation risks caused by process link splitting, dependence on manual and fixed procedures for key node judgment and purification control and lack of collaborative optimization capability of the system in the prior art. (II) technical scheme In order to achieve the aim, the invention is realized by the following technical scheme that the high-efficiency intelligent oncolytic virus packaging and purifying system comprises an intelligent virus packaging module, a totally-enclosed primary harvesting module, a self-adaptive chromatography purifying module and a central intelligent control system; the intelligent virus packaging module is used for realizing efficient transfection of plasmids into packaging cells, packaging and culturing the viruses, and judging the optimal harvesting time of the viruses by monitoring the cell states in real time; The fully-closed primary harvesting module is connected with the intelligent virus packaging module through a sterile pipeline and is used for receiving a virus culture solution and completing cell lysis, virus release and primary clarification of the culture solution in a closed environment; The self-adaptive chromatography purification module is connected with the totally-enclosed primary harvesting module through a sterile pipeline and is used for carrying out multi-step fine purification on the virus crude extract after preliminary clarification and accurately collecting target virus fractions based on real-time detection data; The central intelligent control system is respectively connected with the i