The research and development and production of veterinary biological products

is a highly regulated field with extremely stringent compliance requirements.

According to the regulations of the Chinese Ministry of Agriculture, all veterinary

biological products must be produced in GMP-compliant facilities and require a

dual licensing system (production license and product approval number). CHO

cells (Chinese Hamster Ovary cells), as epithelial-like cells derived from the

ovarian tissue of Chinese hamsters, have significant advantages in veterinary

vaccine production: they possess complex post-translational modification

mechanisms similar to those of mammals, including humans, and can express

biologically active macromolecular proteins, which is crucial for vaccine

immunogenicity.

In the human pharmaceutical field, over 70% of recombinant therapeutic proteins

are produced using CHO cell expression systems; in the veterinary vaccine field,

CHO cell systems have also become one of the preferred platforms for

recombinant protein expression, including several important veterinary subunit

vaccines, such as classical swine fever E2 and porcine epidemic diarrhea (PEDV).

The high diversity of CHO cells also presents certain challenges. Due to genetic

and phenotypic instability, different culture conditions can lead to differences in

CHO cell proliferation capacity, glycosylation capacity, and recombinant protein

expression levels. This necessitates providing sufficient data to demonstrate the

stability of the cell line and the consistency of the product during the compliance

application process.

Regarding compliance applications, CHO cell-based vaccine projects must meet

stringent review requirements. The Ministry of Agriculture and Rural Affairs has

stipulated four different review methods for veterinary drug registration: general

review, priority review, emergency evaluation, and filing review. Enterprises

developing and using their own vaccines can apply for review using different

methods depending on the circumstances. In 2023, the Ministry of Agriculture

and Rural Affairs further stated that it would explore a fast-track approval system

for enterprises developing and using their own vaccines, to meet the needs of

animal disease prevention and control while ensuring safety.

The Horizon CHO K1 GS cell line represents a significant technological innovation in veterinary vaccine production in recent years. Derived from CHO-K1 cells at ECACC (European Cell Bank), this cell line features a gene-edited knockout of the glutamine synthase (GS) gene. This modification renders the cell line unable to survive in glutamine-free media unless successfully transfected with a plasmid expressing exogenous glutamine synthase. This provides a powerful selection system for screening cell clones that efficiently express recombinant proteins.

2.1 Technical features and advantages

The core advantages of the Horizon CHO K1 GS cell line lie in its highly efficient

screening mechanism and excellent adaptability to manufacturing processes.

The GS knockout system utilizes the necessity of glutamine synthase as a

selection marker, eliminating the need for toxic screening drugs such as

MSX (methionine thioxanil). This not only improves screening efficiency but also

reduces potential safety risks in later production. This characteristic is

particularly crucial for veterinary vaccine compliance applications, as regulatory

agencies impose strict restrictions on additives and potentially toxic substances

used in the production process.

Another important characteristic of this cell line is its ability to adapt to

suspension culture, which is essential for large-scale industrial production.

Suspension culture not only increases cell density and product titers but also

simplifies downstream purification processes and reduces production costs.

In practical applications, vaccine production processes using this cell line can

achieve recombinant protein yields of several grams per liter, fully meeting the

needs of commercial production.

2.2 Compliance advantages

From a compliance perspective, the Horizon CHO K1 GS cell line possesses a clear intellectual property status, avoiding potential patent disputes. Furthermore, this cell line has a clear genetic background and comprehensive testing records, including detection of exogenous pathogenic factors, GS function verification, and virus detection, all of which provide strong support for registration applications. In addition, this cell line has undergone independent evaluation by more than 15 institutions worldwide, and several drugs using this technology have received FDA IND approval in the United States. These successful precedents provide reference and confidence for the compliant application of veterinary vaccines.

Table 2: Key Advantages of Horizon CHO K1 GS Cell Line in Compliance Applications

In CHO cell culture, media optimization is a key factor affecting vaccine yield

and quality. Zhejiang Yishengke has developed Aplo CHOL2 medium and the

Aplo CHOF1 Feed-P1/P2 feeding system specifically designed for CHO cells to

meet the specific needs of veterinary vaccine production. These products are

highly compatible with the Horizon CHO K1 GS cell line, providing a complete

solution for veterinary vaccine production.

3.1 Characteristics of Aplo CHOL2 Medium

Aplo CHOL2 is a chemically defined, animal-free medium specifically designed

for suspension culture of CHO cells. This formulation avoids the potential viral

contamination risks from animal-derived components, improving product

safety. This is particularly critical for veterinary vaccine production, as

regulatory agencies strictly limit the use of animal-derived components to

avoid the risk of transmission of pathogens such as mad cow disease.

The unique feature of this medium is its optimized nutrient ratio, which meets

the needs of high-density CHO cell culture. By precisely controlling the

concentrations of key components such as glucose, amino acids, and trace

elements, Aplo CHOL2 medium can support a viable cell density of over

28 million per milliliter, a figure comparable to mainstream international

culture media products and fully meeting the needs of commercial production.

3.2 Advantages of the Aplo CHOF1 Feed-P1/P2 System

Regarding feeding strategy, the Aplo CHOF1 Feed-P1/P2 system employs a

fed-batch culture protocol, providing targeted nutritional support according to

different stages of cell growth and product expression. During the cell growth

stage, feeding primarily supports cell proliferation to achieve high cell density;

during the product expression stage, feeding optimizes the expression

conditions of recombinant proteins, increasing product titers.

This staged feeding strategy effectively controls the accumulation of metabolic

byproducts, such as lactate and ammonium salts. High concentrations of

metabolic byproducts are detrimental to cell growth and antibody expression.

By optimizing the feeding strategy, the accumulation of these byproducts can

be reduced, improving cell viability and product quality. In practical application

s, the culture process using this fed-batch system can achieve antibody titers

exceeding 10 g/L, far surpassing the levels of traditional batch cultures.

3.3 Process Optimization Strategies

To maximize the performance of the Horizon CHO K1 GS cell line and the

Zhejiang Yishengke culture medium system, systematic optimization of culture

parameters is necessary. Key parameters include temperature, pH, dissolved

oxygen, and osmotic pressure.

Temperature Control: Practice shows that switching the culture temperature

from 37℃ to a lower range of 29-35℃ during the exponential growth phase

has a positive impact on intact cell density, cell viability, and specific cell

productivity.

pH Optimization: The suitable pH range for CHO cells is 6.8-7.4. Optimizing the

pH of the cell culture is crucial for producing high-quality recombinant

proteins. Studies have found that when the pH is set to 7.10, the yield of a

certain recombinant protein even reached 4.1 g/L.

Dissolved Oxygen Control: Dissolved oxygen (DO) is one of the key

environmental parameters in large-scale processes. Most DO values in CHO

cells are typically in the range of 20% to 50%.

Osmotic pressure regulation: Appropriately increasing osmotic pressure can

increase mAb titers, reduce galactosylation, and appropriately increase the

proportion of high-mannose glycosylation, thereby improving production

efficiency.

Through these optimization strategies, Zhejiang Yishengke's culture medium

and feeding system have achieved a highly synergistic effect with the Horizon

CHO K1 GS cell line, providing veterinary vaccine manufacturers with a

complete solution from cell line to culture medium, greatly simplifying the

process development process and accelerating the progress of compliance

declarations.

Compliance application for veterinary vaccines based on CHO cells is a

systematic project requiring comprehensive consideration of technical,

regulatory, and quality requirements. In accordance with the requirements

of the *Veterinary Drug Registration Regulations* and the *Veterinary Drug

Manufacturing Good Manufacturing Practice*, and considering the

characteristics of the Horizon CHO K1 GS cell line and Zhejiang Yishengke's

culture medium system, we propose the following compliance application

solution:

4.1 Technical Material Preparation and Application Strategy

Sufficient technical materials are fundamental to success in the compliance

application process. According to the *Veterinary Drug Registration

Regulations*, the registration of new veterinary biological products requires

the provision of systematic identification data of starting materials, including

bacterial and viral strains, cell lines, and biological tissues, as well as data on

production processes, storage conditions, genetic stability, laboratory safety,

and efficacy tests. For vaccines expressing CHO cells, the following technical

documents require special attention:

Cell line characteristic analysis: This includes the origin, genetic background,

gene modification details, and stability data of the Horizon CHO K1 GS cells.

This section should describe in detail the scientific basis and technical

approach of the GS gene knockout, as well as the impact of this modification

on the safety and efficacy of the vaccine.

Production Process Validation: A detailed description of the culture process

parameters using Aplo CHOL2 medium and the Aplo CHOF1 Feed-P1/P2

feeding system is required, including inoculation density, temperature, pH,

dissolved oxygen, feeding time, and rate. Scale-up data should be provided

to demonstrate consistency from laboratory to pilot-scale and then to

production scale.

Product Quality Studies: This includes structural identification, glycosylation

analysis, purity testing, and biological activity of the target protein.

Special attention should be paid to the consistency of product quality

attributes, demonstrating the similarity between different batches.

Regarding the application strategy, the various review methods stipulated by

the Ministry of Agriculture and Rural Affairs can be fully utilized. For vaccines

urgently needed for the prevention and control of major animal diseases,

priority review can be applied for, and the Veterinary Drug Review Center of the

Ministry of Agriculture and Rural Affairs will review relevant vaccine

registration applications as soon as possible. For highly innovative CHO cell

expression vaccines, the review process can be accelerated through thorough

preliminary communication and data preparation.

4.2 Process Validation and Quality Control

Process validation is a core component of compliant application, requiring

proof of the consistency and reliability of the production process. Validation

of CHO cell-based vaccine manufacturing processes should include the

following aspects:

Cell Bank System Validation: A master cell bank (MCB) and working cell bank

(WCB) need to be established, and comprehensive cell bank characterization

data should be provided, including species identification, purity, viability,

genetic stability, and production stability. The clear genetic background of the

Horizon CHO K1 GS cell line facilitates this.

Culture Process Robustness: It needs to be demonstrated that the culture

process using Aplo CHOL2 medium and the Aplo CHOF1 Feed-P1/P2 feeding

system can stably produce products that meet quality requirements.

This includes determining the operating range of critical process parameters

(CPPs) through design space studies. Experimental data should be provided

demonstrating the adaptability of the culture medium system to different

culture scales (from shake flasks to bioreactors).

Purification Process Validation: It needs to be verified that downstream

purification processes can effectively remove product-related impurities

(such as host cell proteins, DNA, etc.) and process-related impurities, ensuring

the purity and safety of the final product. The characteristic of CHO cells,

which secrete almost no endogenous proteins, is advantageous in this regard.

Quality Control Strategy: This should cover the entire process from raw

materials to the final product. Quality control of key raw materials is

particularly important, including quality testing standards and methods for

Aplo CHOL2 culture medium and Aplo CHOF1 Feed-P1/P2 supplements.

Strict internal control standards should be established to ensure batch-to-batch

consistency of culture media, thereby guaranteeing product quality stability.

4.3 Strategies and Practices for Accelerated Approval

To accelerate the approval process for CHO cell-based veterinary vaccines,

the following strategies can be adopted:

Fully utilize expedited approval policies: The Ministry of Agriculture and Rural

Affairs prioritizes the review of vaccines urgently needed for the prevention

and control of major animal diseases; and adopts an emergency evaluation

approach for vaccines required for emergency response to major animal

diseases. Enterprises can apply for the corresponding fast-track review channel

based on product characteristics.

Strengthen communication with regulatory agencies: Communicating with the

Veterinary Drug Review Center of the Ministry of Agriculture and Rural Affairs

early in the R&D process to reach a consensus on technical routes, evaluation

standards, and application requirements can reduce uncertainty in later

applications.

Learn from mature experience in human biological products: CHO cell

technology is very mature in the field of human biological products, with

abundant technical data and regulatory experience available for reference.

By fully referencing the evaluation principles and technical requirements for

human products, the cognitive barriers to veterinary vaccine applications can

be reduced.

Providing sufficient comparative data: Providing comparative data with already

marketed products demonstrates that the new product's safety, efficacy, and

quality controllability are no less than those of already marketed products,

simplifying review requirements.

Through the above systematic solution, veterinary vaccine projects based on

the Horizon CHO K1 GS cell line and Zhejiang Yishengke culture medium

system can more efficiently complete compliance applications, accelerate the

product launch process, and meet the needs of animal disease prevention and

control.

With the continuous development of biotechnology and the gradual

improvement of regulatory policies, CHO cells have a very broad application

prospect in the field of veterinary vaccines. Based on current technological and

regulatory trends, we can foresee the following development directions:

Technological Innovation: CHO cell systems will continue to develop towards

higher expression, better quality, and stronger controllability. For example,

Horizon's newly launched CHO-K1 ADCC+ cell line, by eliminating the cell's

natural fucosylation activity, can express antibodies completely lacking the

fucosylate type, enhancing antibody-dependent cytotoxicity (ADCC) effects.

This type of technological innovation will also be applied to the field of

veterinary vaccines, improving vaccine immunization efficacy. At the same time,

culture media and feeding systems will become more refined and personalized,

such as Zhejiang Yishengke's products, which can provide customized

nutritional solutions for specific cell lines and product characteristics.

Regulatory Policies: The Ministry of Agriculture and Rural Affairs has stated that

it will further strengthen research and investigation to explore a fast-track

approval system for vaccines developed and used by enterprises themselves.

This means that in the future, more explicit and optimized review guidelines

may be introduced for innovative technology platforms such as CHO cell

expression systems, further accelerating the market launch process of

innovative vaccine products. Enterprises should closely monitor policy

developments and actively participate in the discussion and formulation of

relevant policies and regulations, providing practical references for industry

development and regulatory improvement.

In terms of market application: With the successful practices of companies such

as Zhejiang Hailong Biotechnology, the application of CHO cell platforms in the

field of veterinary vaccines will become more widespread. Currently, in addition

to the classical swine fever E2 subunit vaccine, several other important animal

disease CHO expression vaccines are in the research and development or

application stage, including swine pseudorabies vaccine, bovine viral diarrhea

virus (BVDV) vaccine, and bovine infectious rhinotracheitis (IBR) vaccine. These

innovative products will enrich the market supply of veterinary vaccines and

provide more and better tools for animal disease prevention and control.

In summary, by adopting the Horizon CHO K1 GS cell line and Zhejiang

Yishengke's Aplo CHOL2 medium and Aplo CHOF1 Feed-P1/P2 feeding system,

combined with a scientifically sound and compliant application strategy,

veterinary vaccine R&D companies can more efficiently promote product

development and registration, making significant contributions to animal

health and livestock development. With continuous technological

advancements and a constantly improving regulatory environment, the

application of CHO cell technology in the field of veterinary vaccines will

usher in a broader prospect for development.