4、病毒(du)清除工藝驗證方案

On January 21, 2020, the National Medical Products Administration (NMPA) issued the "Announcement on the Application of 11 International Technical Guidelines for Human Drug Registration, including Q2 (R1): Analytical Method Validation: Text and Methodology" (Announcement No. 7 of 2020). The announcement clarified that, starting 6 months after the release of this announcement (i.e., July 10, 2020), pharmaceutical studies (based on the time of experimental record) will apply ICH guidelines, including ICH Q5A (R1) and Q5B. Therefore, the virus clearance validation for human monoclonal antibodies, protein drugs, and other biopharmaceuticals now follows a unified set of standards and principles both domestically and internationally. The recommended validation plan (CHO expression system) is shown in the table below:

Table 2: Currently Recommended Overall Virus Clearance Process Validation Plan for IND and BLA Submissions

Process	臨床Ι期（IND） NMPA/EMA/UDFDA	臨床Ⅲ期后報產（BLA） NMPA/EMA/UDFDA
低 pH 孵育或 S/D 處理	1種病毒（MuLV），重復2次實(shi)驗(yan)	2 種病毒（MuLV，PRV），重復(fu)2次實驗
納米膜過濾	2種病毒（MuLV，MVM），重復(fu)2次實驗	4 種(zhong)病(bing)毒（MuLV，PRV，Reo3，MVM），重(zhong)復2次實驗
陰離子交換層析	2種病(bing)毒（MuLV，MVM），重復2次實驗	4 種病(bing)毒（MuLV，PRV，Reo3，MVM），重復2次實驗，需評估舊填(tian)料(liao)的病(bing)毒清除能力以及病(bing)毒殘留
另外一(yi)種(zhong)層析	可選	4 種病毒(du)(du)（MuLV，PRV，Reo3，MVM），重復2次(ci)實驗，需評估舊(jiu)填料的病毒(du)(du)清除能(neng)力(li)以及病毒(du)(du)殘留

Virus Clearance Process Validation Showcase

Canvest Bio has provided professional, comprehensive, and reasonable virus clearance process validation services to hundreds of domestic and international biopharmaceutical companies; the approval rate is 100%. We have professional project managers (PM) who respond to customer needs promptly, track project progress, and provide continuous service throughout the submission process, including offering rational suggestions for submission material writing.

Filing Stage: IND

 Agency: Dual filing (domestic and international)

Process and Results:

Process	去除/滅活指數(shu)（log10）
	X-MuLv	MVM	PRV
低(di)pH孵(fu)育	≥4.30±0.14	/	≥5.20±0.07
陰(yin)離子層析	≥5.05±0.00	≥5.9±0.35	/
納米膜過(guo)濾	≥4.85±0.07	≥5.15±0.07	/

Filing Stage:BLA

Agency: Domestic declaration

Process and Results:

Process		去除/滅活指數（log10）
		X-MuLv	MVM	PRV	Reo3
低pH孵育		≥5.80±0.28	/	≥5.15±0.07	/
納米膜過濾		≥4.95±0.00	≥4.55±0.00	≥4.30±0.14	≥4.15±0.07
親(qin)和層析	新填(tian)料	3.38±0.07	3.14±0.38	4.09±0.14	2.43±0.20
	舊填(tian)料	3.48±0.07	3.14±0.38	4.09±0.51	2.29±0.16
陰離子層(ceng)析(xi)	新填料	4.30±0.00	4.19±0.07	4.70±0.07	4.64±0.07
	舊填料(liao)	4.35±0.00	4.33±0.10	4.66±0.29	4.50±0.14

3、 指示病(bing)毒選擇

Indicator viruses can be classified into three types: "related" viruses (such as human viruses that may be carried by human blood products, including HAV, HBV, HIV, and B19), specific "model" viruses (such as MVM and x-MuLV viruses in CHO expression systems, and BV virus in baculovirus expression systems), and non-specific "model" viruses (such as PRV and Reo3 viruses in CHO expression systems). Priority should be given to selecting viruses that are closely related to potential contaminating viruses. If related viruses are unavailable or unsuitable for in vitro culture, specific "model" viruses can be used as substitutes.
When evaluating the overall virus clearance capability, non-specific model viruses with different characteristics should be selected. These include DNA/RNA, enveloped/non-enveloped, particle size, and viruses that are particularly resistant to physical/chemical treatments. The indicator virus should have a sufficiently high titer, and the selected indicator virus should not pose a biological hazard to operators or the environment.
The validation platform has a rich collection of virus strains, with clear backgrounds, high purity, and titers reaching 8-9 Log10/ml. Table 1 lists the four most commonly used indicator viruses for CHO expression product process validation.

Table 1: Properties of Commonly Used Indicator Viruses in Virus Clearance Process Validation

Virus Name	Virus Characteristics	Testing Method	Viral Titer Log10/ml
Reo3（呼腸孤病毒）	雙鏈 RNA 病毒，無包膜(mo)，抗性中50-70nm	TCID50 Q-PCR	8-9
MVM（鼠細小病(bing)毒 ）	單鏈(lian) DNA 病(bing)毒無包(bao)膜，抗性極高 18-26nm	TCID50 Q-PCR	8.5-9.5
PRV(偽狂病(bing)毒)	雙鏈 DNA 病毒有包(bao)膜，抗性中120-200nm	TCID50 Q-PCR	8-9
MuLv （鼠白血病病毒）	單鏈 RNA 病毒有(you)包(bao)膜，抗性低(di) 80-120nm	TCID50 Q-PCR	7.5-8.5
BV (桿狀病毒)	雙鏈 DNA病(bing)毒(du)有(you)包膜(mo) 45×275nm	TCID50 Q-PCR	8-9
POL-1 (人脊髓灰質炎(yan)I型病毒)	單鏈 RNA 病毒無包膜，抗性中 25-30nm	TCID50	8-9
VSV (水皰性口炎病毒)	單鏈 RNA 病毒有包(bao)膜，抗性低 70×150nm	TCID50 Q-PCR	8-9
PPV （豬細小病毒(du)）	單鏈 DNA 病毒無包(bao)膜(mo)，抗性高 18-24nm	TCID50 Q-PCR	7.5-8.5
HAdV-5 (人腺病毒(du)5型)	雙鏈 DNA 病(bing)毒無包膜，抗性(xing)低 70-90nm	TCID50	8.5-9.5
BPIV-3 （牛副(fu)流感病毒(du)3型）	單鏈 RNA 病毒有包膜(mo)，抗性低(di) 150-200nm	TCID50	8-9

2、法(fa)規(gui)依據

1、服務信息（簡介）

Canvest Bio provides customized virus clearance process validation services, offering virus detection and titer analysis on samples before and after processing using Q-PCR or infectivity assays to validate the effectiveness of virus removal/inactivation by downstream processes.

Canvest Bio operates a BSL-2 laboratory certified by national authorities and holds both CMA and CNAS certifications for virus clearance process validation. The laboratory is equipped with multiple biosafety cabinets, AKTA purification systems, nanofiltration equipment, ultracentrifuges, and other instruments. These enable virus clearance process validation for various processes, including low pH incubation, heat treatment/pasteurization, S/D treatment, chromatography (affinity chromatography, anion exchange chromatography, hydrophobic chromatography, etc.), and nanomembrane filtration. The company has rich industry experience, having completed hundreds of virus clearance process validation projects.

The director of Canvest Bio’s virus clearance process validation platform holds a Ph.D. in Microbiology from Wuhan University and possesses extensive experience in virology, cell biology, and microbiology, with a focus on cell testing and virus clearance process validation. The professional team has over ten years of industry experience, constantly optimizing technical systems to enhance virus titer and purity to meet regulatory and customer requirements. Core team members have solid backgrounds in biomedical and pharmaceutical fields, with over five years of experience in virus clearance process validation.

In recent years, Canvest Bio has provided virus clearance process validation services to hundreds of domestic and international biopharmaceutical companies. The company considers both domestic and international regulatory requirements, offering comprehensive and reasonable validation plans based on customer needs, along with authoritative testing reports. This has supported companies in process development, new drug clinical trial applications (IND), and biologics license applications (BLA), achieving both positive social and economic impacts.