세포치료제 역가시험 자료집

2010. 12.
이 참고자료집은 미국, 유럽, 일본의 세포치료제 관련 최신 지견을 정보제공
하기 위하여 마련되었습니다. 세포치료제 연구개발 시, 미국 FDA, 유럽

EMEA, 일본 PMDA의 최신 가이드라인에 따라 제품의 특성에 맞는 역가

시험법을 설정하실 수 있도록 도움을 주기 위한 자료로서 작성되었으니,
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 세포치료제 역가시험 자료집

목 차

1. 역가시험 관련 가이드라인

1) 세포,유전자치료제 역가시험법 (Draft, 2008, FDA) _ 국문

2) 세포,유전자치료제 역가시험법 (Draft, 2008, FDA) _ 영문

3) 면역세포치료제 역가시험법 (2007, EMEA)

4) 세포치료제 가이드라인 중 역가시험법 (2008, EMEA)

5) 암치료를 위한 수지상세포치료제 평가가이드라인 (2005, 식약청)

2. 세포치료제 역가시험 관련 연구

1) 면역거부억제 줄기세포치료제 역가시험법 (07172응용연652(GVHD))

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1. 세포/유전자치료제 역가시험법 [FDA 가이드라인(안)]

산업계를 위한 가이드라인 : 세포유전자치료제의 역가시험법

[FDA 가이드라인(안)]

목 차

I. 서론

II. 배경
A. 허가된 생물학적제제의 역가에 대한 규제 요구사항
B. 임상시험용 세포유전자치료제의 역가 요구사항

C. 세포유전자치료제의 역가와 임상적 유효성과의 관계

III. 역가 측정을 위한 요구사항
A. 무엇으로 역가를 측정할 것인지 어떻게 결정하는가?

B. 역가측정에 사용되는 방법
C. 세포유전자치료제에서 역가와 임상적 유효성사이의 관계
D. 역가시험법 개발 시기
E. 진행성역가시험법 수행

IV. 분석법 설계과 검정

A. 분석법 설계시 고려사항
B. 참고물질과 대조품의 사용
C. 분석법 밸리데이션 계획시 고려사항
V. 참고문헌

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Ⅰ 서론

- 이 가이드라인은 세포유전자치료제(cellular and gene therapy, CGT) 제조자에게

역가2) 측정을 위한 검사법3) 개발에 대한 권고사항을 제시하고자 한다. FDA는
이 가이드라인에서 권고사항으로 임상시험계획승인신청(Investigational New
4)
Drug Application, IND) 이나 생물학적제제허가신청(Biologics License
5)
Application, BLA) 을 뒷받침 할 수 있는 역가에 대한 정보를 명확히 하고자 함
이며 특정 역가시험법에 대한 권고나 제품출하에 대한 기준을 제시하려는 것은
아니다. 이 가이드라인은 관련 가이드라인(참고문헌 1-11, 15)에 추가하려는 것
이며 기존의 가이드라인을 대체하거나 바꾸는 것은 아니다.
- 이 가이드라인은 공중보건법(Public Health Act, PHS Act)의 section 351하의
미국 FDA 생물의약품연구및평가센터 세포조직유전자치료제국
(FDA/CBER/OCTGT(Office of Cellular, Tissue and Gene Therapies)에서 검토
되는 세포/유전자치료제6)에만 적용된다. 이 가이드라인은 21 CFR 1271.10에서
언급한 것처럼 PHA act의 361(42 U.S.C. 264)에 의해서만 규제되는 사람 세포,
조직 그리고 세포조직 제품(HCT/Ps)이나 21 CFR Part 820에 의해 의료기기로
규제되는 제품에 대해서는 적용되지 않는다.
이 가이드라인은 또한 의약품 연구 및 평가센터(CDER)나 생물의약품 연구 및
평가 센터(CBER)의 백신 연구 및 평가국(OVRR, Office of Vaccine Research
and Review)나 혈액 연구 및 평가국(OBRR, Office of Blood Research and
Review)에 의해 검토되는 생물학적제제에는 적용되지 않는다.
- 이 가이드라인을 포함한 FDA 가이드라인은 법적 강제성이 있는 사항을 제시하지
않는다. 가이드라인 문서는 특정 주제에 대한 FDA의 방침을 기술하며, 일종의
권고 사항으로 보아야 한다.

II 배경

A. 허가된 생물학적 제제의 역가에 대한 규제 요구사항은 무엇인가?

- 모든 생물학적 제제는 생물학적제제 허가 신청 승인을 위한 안전성, 순도, 역가
의 규정된 요구사항을 충족시켜야 한다(42 U.S.C. 262, 연방 식품, 의약품, 화장
품법 (Federal Food, Drug and Cosmetic Act, FDC Act(21 U.S.C. 321 et
seq.); 21CFR 601.2). 세포/유전자치료제에 대해서는 제품 적합 시험(product
conformance testing, 21 CFR 601.20(a))과 제조 공정 관리(21CFR 601.20(c))
가 생물학적제제 규정(21 CFR Part 600 et seq.) 뿐만 아니라 완제 의약품(21
CFR Parts 210, 2117))의 FDA cGMP (현행 우수 제조기준, Current Good
Manufacturing Practice) 규정에 따라야 한다. 그러한 제제에 적용되는 기준에
적합함을 확인하는 시험을 완료하기 전에는 어떠한 로트도 출하될 수 없으며 여

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 기에는 역가, 무균, 순도 그리고 확인시험이 포함된다. 이러한 요구사항은 하나의
로트가 단일 투여 용량으로 정의되는 자가 그리고 단일 환자 동종 제제를 포함한
모든 생물학적제제에 적용된다.
- 역가(potency)는 적절한 실험실 시험이나 의도된 방법으로 제제의 투여를 통해
적절하게 관리된 임상시험에 의해 의도한 효과를 나타내는 제제의 특성이나 능력
8)
으로 정의된다(21 CFR 600.3(s)). 강도(strength) 는 역가, 즉 적절한 실험실 시
험이나 적절하게 개발되고 조절된 임상자료에 의해 나타내어지는 의약품의 치료
적 활성으로 정의된다(21 CFR 210.3(b)(16)(ii)).
규정에서 “역가 시험법은 각 제제에 대해 특이적으로 고안되어 600.3(S)의 규정
에 의한 역가의 설명을 충족시키기에 적절한 역가를 나타낼 수 있는 생체 외 또
는 생체 내 또는 두 방법 모두로 구성될 수 있다(21 cfr 610.10)”고 명기하고 있
다.
- FDA 규정은 각 제제에 대한 역가의 적절한 측정을 결정함에 있어 상당한 유연성
을 허용하고 있다. 역가는 각 제제의 특성에 기초해서 결정되므로 역가 시험법의
적절성은 각 제제별로 평가되어야 한다. 허가된 생물학적 제제의 출하시험을 위
한 모든 역가 시험법은 다음을 포함한 관련 생물학적제제와 cGMP규정에 부합하
여야 한다.

• 제제 특이적인 역가(생물학적 활성/활성들)를 제시(21 CFR 600.3(s), 610.10

그리고 21 CFR 210.3(b)(16)(ii))
• 제제 출하를 위한 시험결과를 제공(21 CFR 610.1; 21 CFR 211.165(a))
• 정량적인 자료를 제시(21 CFR 211.194; 또한 21 CFR 600.3(kk) 참조; 21
CFR 211.165(d); 211. 165(e))
• 미리 정한 수용(acceptance) 그리고/또는 기각(rejection) 기준 충족
• 적절한 참고물질(reference), 표준품, 그리고/또는 대조물질(control)을 포함(21
CFR 210.3(b)(16)(ii), 211.160)
• 사용한 시험방법들을 밸리데이션(validation)을 통해 정확성, 민감성, 특이성
그리고 재현성을 확립하고 문서화(21 CFR 211.165(e)와 211.194(a)(2))
• 모든 주성분(active ingredients)의 확인과 강도(활성)를 측정(21 CFR
211.165(a): 또한 21 CFR 210.3(b)(7) 참조)
• 유통기한(dating period) 확립을 위한 자료 제시(21 CFR 600.3(1)과
610.53(a) 참조)
• 라벨링 요구사항을 충족(21 CFR 610.61(g)(3)과 610.61(r))

B. 임상시험용 세포유전자치료제의 역가요구사항은 무엇인가?

- 초기단계의 임상시험에서는 위에서 언급된 허가된 생물학적제제에 대한 모든 요
구사항을 충족시키는 것이 가능하지 않을 수 있다(참고문헌. 3,4,8). 그렇지만 임

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 상연구의 모든 단계에서 사용하는 제제의 안정성(21 CFR 312.23(a)(7)(ii)) 뿐만
아니라 확인, 순도, 역가(21 CFR 312.23(a)(7)(i))를 보증하기 위한 자료를 제출
하여야 한다. 필요한 자료의 양은 임상시험 단계, 연구 기간, 다른 이용 가능한
정보의 양에 따라 다양하다.
- 역가 측정은 모든 임상시험단계에서 투여되는 제제가 일관성 있게 생산됨을 확립
9)
하기 위하여 사용되는 제제 특성 시험 , 비교 동등성시험, 안정성 시험계획서(프
로토콜)에 필요하다. 그러나 세포/유전자치료제의 복합성 때문에 역가 시험법을
확립하기 위한 많은 시도가 필요하다(표 1). 세포/유전자치료제의 개발을 촉진하
기 위하여 역가 시험법 개발을 포함한 제제 특성 시험을 위해 점증적 접근법
(incremental approach)을 권장한다. 진행식(progressive) 역가 측정 시험 수행
을 위한 일반적인 권장사항은 Section III.E에 나와 있다. 이 가이드라인의 III.A,
III.E 그리고 IV.C.4에서 기술한 바와 같이 역가 측정은 제제가 개발됨에 따라 개
선되고 현저하게 바뀔 것이다.

세포유전자치료제의 역가시험법 개발의 한계 예:

◦ 자가 그리고 동종의 공여자 변이성
초기물질(starting material) 고유의 변이성 ◦ 세포주의 이질성(heterogeneity)
◦ 에러 발생이 높은 복제 바이러스
◦ 적은 양의 액체에 부유되어 있는 자가 세포를
제한된 로트 크기 및 시험 물질 이용한 단회투여 치료제(single dose
therapy)
제한된 안정성 ◦ 세포제품에서 생존성
◦ 최종 제제에 포함된 여러 가지 세포주
◦ 펩타이드로 감작된(peptide pulsed) 종양세포
다수의 주성분
및/또는 면역조절세포의 이질적 혼합물
◦ 복합적으로 사용되는 다수의 벡터
◦ 같은 벡터에서 발현되는 여러 개의 유전자들
활성 성분사이의 간섭 또는 상승작용의 가능성
◦ 자가/동종 세포치료제에서 복합적 세포 유형
◦ 세포의 복잡하고 잠재적 효능
◦ 이식 유전자의 감염, 도입(integration),
복잡한 작용 기작
그리고 발현과 같이 기능에 필요한 여러 단계
◦ 여러 개의 유전자를 포함한 벡터

◦ 투여부위에서부터 이동
◦ 원하는 세포로 분화
제제의 생체 내 운명 ◦ 바이러스 또는 세포성 복제
◦ 바이러스 벡터 감염, 탈피(uncoating), 그리고
이식유전자의 발현

C. 세포/유전자치료제에서 역가와 임상적 유효성사이의 관계는 무엇인가?

- 단일 시험법으로는 세포/유전자치료제의 임상적 유효성을 적절하게 예측할 수 없
다. 임상적 유효성은 일관성 있게 제조된 제제로 행해진 적절하고 잘 통제된 임

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 상시험에 의해 입증된다. 임상적 유효성은 제제의 역가와 상관관계에 있지만 임
상시험 자료가 로트 출하를 위한 역가의 실질적인 정량방법은 아니다. 오히려 임
상시험결과는 제제의 임상적 유효성과 역가 시험법 사이의 상관관계10)를 확립할
때 사용되어 질 수 있으며, 이는 로트 출하, 안정성 그리고/또는 비교동등성 시
험에 사용될 수 있다(상관성연구와 관련해서 더 자세한 것은 III.C. 참조).

III. 역가 측정을 위한 권고사항

A. 무엇으로 역가를 측정할 것인지 어떻게 결정하는가?

- 세포/유전자치료제의 복합성 때문에 적절하고 의미 있는 역가 시험법 개발을 위
해 제제의 생물학적 특성을 이해하는 것이 필요하다.
역가를 측정하는 방법에 대한 정보를 제공하고 다듬기 위하여 전임상과 임상 개
발동안 충분한 자료를 모아야 한다.
- 초기에 역가 시험법을 디자인하기 위한 생물학적 활성 또는 활성들을 결정할 때,
적절한 전임상 연구, 개념입증 연구, 초기임상연구, 이용 가능한 이전의 경험
(historical experience) 그리고 이용 가능한 참고물질이나 대조물질을 고려하여
야 한다(III.C 참조). 이러한 정보는 기능과 관련된 제제의 특성과 생물학적 활성
에 대한 기본적인 이해를 도울 수 있다. 제제 개발동안 얻어진 특성분석 자료는
처음에 선택한 역가 시험법을 뒷받침할 수도 있으며 또는 제제의 시판을 준비하
기 위해 역가시험법을 개선할 수도 있다(III.E와 IV.C.4 참조). 제제를 개발할 때,
일상적인 로트 출하를 위해 행해지는 것 외에 광범위한 범위의 제제 특성을 측정
하여야한다. 이것은 어떤 제제의 특성이 역가와 가장 상관관계에 있는지 평가하
는데 도움을 줄 수 있다. 평가한 몇몇 시험법이 로트 출하에 실제적이지 않을 수
있지만(예, 정량적인 결과를 일관성 있게 얻기 어렵거나, 시간이 많이 걸림) 가장
적절하게 디자인된 시험법(IV.A)은 생물학적 활성이나 임상적 유효성 또는 둘 다
와 관련해 제품의 특성에 대한 유용한 정보를 제공할 수 있다.
- 세포/유전자치료제는 정량적으로 역가를 나타내는 특이적인 생물학적 특성을 측
정하기 위한 시험법을 개발하는데 어려움이 있을 수 있다(표 1). 세포/유전자치
료제는 종종 작용기작이 복잡하거나 그리고/또는 잘 밝혀지지 않았기 때문에(적
절한 치료적 또는 임상적 기능), 어떤 특성이 역가를 측정하는 데 가장 적절한지
결정하기 어려울 수가 있다. 그럼에도 불구하고 역가 시험법은 적절한 생물학적
특성을 반영해야 한다. 예를 들면, 유전자 치료 벡터는 역가를 위한 적어도 두개
의 생물학적 특성(유전자를 세포로 전달하는 능력과 발현된 유전자의 생물학적
효과)을 반영해야 한다. 따라서 역가 분석법은 유전자 전달 효율과 전달된 유전
자의 생물학적 효과 둘 다를 포함해야 한다.
- 또한, 세포/유전자치료제의 작용기작이 하나 이상의 주성분11)(여러 종류의 세포,

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 여러 개의 벡터, 여러 개의 항원 결정기(epitope)를 가진 백신)에 의존할 수 있
다. 어떤 복합제품(예, 세포 종양 백신)의 경우, 어떤 성분이 역가를 나타내는지
모호할 수 있다. 알려진 하나 이상의 유효 성분을 포함하는 제제의 경우, 모든
유효 성분의 생물학적 활성(강도)을 결정할 수 있는 역가 시험법을 디자인해야
한다(21 CFR 211.165(a)) 참조). 따라서 제제가 하나 이상의 주성분을 포함하고
있다면, 하나의 시험법이 각 유효성분의 활성을 측정하는데 불충분할 수 있으므
로 제제의 역가를 측정하기 위해 하나 이상의 시험법이 필요하다(III.B.3 참조).
부가적으로, 시험법을 디자인 할 때, 주성분 간의 방해나 상승작용의 가능성을
고려하여야 한다.

B. 역가를 측정하기 위해 어떤 방법들이 사용될 수 있나?

1. 생물학적 시험법
- 생물학적 제제의 역가를 평가하는 전통적인 방법은 효과를 나타내는 특이적인 활
성과 관련된 제제의 활성을 측정하는 그리고 Section II.A.에 나타나 있는 기준을
충족하는 정량적인 생물학적 시험법(bioassay, 생물검정)을 개발하는 것이다. 생
물검정은 살아있는 생물학적 시스템 내에서 제제의 유효성분을 평가함으로써 역
가를 측정한다. 생물검정은 생체 내 동물실험, 생체 외 기관, 조직, 세포 배양 시
스템 또는 이들의 조합을 포함할 수 있다. 생체 외 또는 생체 내 시험법을 사용
할 수 있으나, 가능하다면 동물사용의 제한을 권장한다(참고문헌 12).

2. 비생물학적 분석시험법12)
- 어떤 세포/유전자치료제에 대한 정량적인 생물검정법의 개발은 제제의 특성 그리
고/또는 기술적인 한계 때문에 복잡할 수 있다(표 1). 생물검정법 개발이 가능하
지 않다면, 생물학적 활성의 대리지표를 확인하는 것이 필요하다. 예를 들어, 로
트 출하를 위해 실제적이며 신뢰할 수 있는 분석 시험법(analytical assays)을 사
용하는 것이 필요하다. 분석 시험법은 제제의 면역학적, 생화학적 그리고/또는
분자적인 특성을 평가함으로써 광범위한 제제의 특성 자료를 제공할 수 있다. 만
약 대리 지표측정법이 적절한 제제 특이적인 생물학적 활성과 상관성이 있음이
입증될 수 있다면 이러한 특성은 역가를 나타내는데 사용될 수 있다(Section
III.C, 참고문헌 13과 14 참조). 상관성을 확립하기 위해서는 이 가이드라인에서
권장하는 것과 같이 엄격한 제제의 특성 시험을 행해야 한다.

3. 복합시험법(assay matrix)
- 많은 경우, 하나의 생물학적 또는 분석 시험법으로는 역가를 측정하는데 적절하
지 않을 수 있다. 다음에 제시하는 가능성 때문이다.
• 제제가 복잡한 작용기작을 가지고 있음

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 • 제제가 여러 가지 유효 성분을 가지거나 그리고/또는 여러 가지 생물학적 활
성을 가지고 있음
• 제한된 제제 안정성
• 생물학적 분석법이 정량적이지 않고, 충분히 강건(robustness)하지 않으며 또
는 정확성이 없음
- 만약 하나의 분석법이 역가를 나타내는 제제의 특성을 측정하기에 충분하지 않다
면, 품질, 일관성, 안정성과 관련된 다른 제제의 특성을 측정하는 여러 가지 보
조 시험법을 개발할 수 있다. 같이 사용했을 때 그리고 결과가 적절한 생물학적
활성과 상관관계가 있다면 이러한 보조적인 시험법도 역가를 측정하는데 적절할
수 있다. 그러한 assay matrix(시험법의 모음)은 생물학적 시험법, 생물학적 및
분석 시험법 그리고 분석 시험법만으로 구성될 수 있다(참고문헌 13과 14).
Assay matrix는 정량적인 정보(예, 활성 단위) 또는 정성적인 정보(예, 적합/부적
합)를 주는 시험법을 포함할 수 있다.
만약 정성적인 시험법이 로트 출하, 안정성 또는 비교동등성 시험을 위한 역가를
결정하기 위한 assay matrix의 일부로 사용된다면, 하나 또는 그 이상의 정량적
인 시험법도 첨가되어야 한다(Section II.A 참조).

C. 분석시험과 분석생물학적 활성의 상관성 입증에는 무엇이 필요한가?

- 생물학적 활성의 대리지표 측정법의 분석시험법 사용의 유효성을 입증하기 위해
서는 유효성 관련 생물학적 활성과 대리지표 측정 사이의 상관성을 수립한 충분
한 자료가 제공되어야 한다.
- 생물학적 활성과 대리지표 측정법 간의 상관성은 전임상/개념입증연구의 결과 증
명, 동물실험 또는 임상시험 자료, 또는 in vitro 세포 및 생화학적 자료 비교 등
을 포함하는 다양한 접근법을 이용하여 확립될 수 있다.
- 만일 생물학적 제품의 허가를 목적으로 생물학적 활성에 대한 대리지표 측정으로
서 분석시험법을 선택하였다면, 아래의 section II, A에 기재된 기준을 만족하여
야 한다.
- 생물학적 활성 측정을 위한 대리지표 측정법에 이용되는 적절한 자료는 사례마다
다를 수 있으며 또는 다음 항목에 따라 영향을 받는다.
• 구축된 상관성의 유형과 적절성
• 축적한 제품 정보의 양
• 제품의 생물학적 활성이 얼마나 잘 이해되었는가
• 대리지표 측정법이 생물학적 활성을 얼마나 잘 반영할 수 있는가
- 만일 생물학적 활성과 대리지표 측정법의 상관성에 따른 역가를 산출하고자 한다
면, 초기 연구기간 동안 제품과 특성평가 시험 자료들을 모으기 시작하여야 한
다.

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D. 언제 역가측정법 개발을 시작해야 하는가
- 이 문서 전반에서 기술한 것처럼, 제품의 특성분석을 통해서 품질, 일관성 그리
고 안정성에 영향을 미치는 제품의 지표(들)에 대한 이해가 필요하다. 더욱이, 이
러한 지표들의 이해와 관리가 생산 로트 간의 일관성 증명, 다른 제조과정 그리
고/또는 다양한 측정법의 비교평가에 필요하며, 또한 어떤 제품이 유효한 제품인
지 결정하는 것이 필요할 수 있다. 따라서, 역가 측정 능력은 제품의 특성분석에
기초하기 때문에 가능한 많은 제품의 정보를 얻기 위하여 전임상과 임상시험 초
기단계 동안 역가시험법 개발을 시작하여야 한다.
- 제품 개발 초기 동안의 역가측정은 다음과 같은 많은 장점을 가진다.
• 제품개발 전기간 동안의 제품 활성, 품질 및 일관성을 증명할 수 있음
• 로트 출하를 위한 규격을 뒷받침하는 자료수집이 이루어짐
• 제조방법 변경을 평가하기 위한 기초자료를 제공
• •제품의 안정성 평가를 가능하게 함
• 별개의 시험법에서의 기술적 문제 또는 그 이유를 알 수있음
• 다중시험법(multiple assay)의 평가가 가능함
• 필요하다면, 상관성 연구를 뒷받침하는 충분한 양의 자료를 모을 수 있음

E. 진행성 역가시험법 수행이란?

1. 제품개발 초기
- 전임상, 임상 1상 및 2상 시험의 일부 제품에서는 생물활성의 제한된 정량 정보
로도 충분할 수 있다. 초기 임상연구에 이용된 제품 로트의 역가시험은 후기임상
연구에서 이용된 시험법보다 더 넓은 범위에서 수행할 수 있다. 임상시험의 진행
과 제품에 대한 정보지식이 증가한다 하더라도, 생물학적 제품의 적절한 정량적
특성평가를 적절히 평가할 수 있는 개선된 역가측정법을 개발하고 수행하여야
한다(21 CFR 312.23(a)(7) 참조).
2. 제품개발 후기
- 후기 임상시험의 주요목적(예: 3상, 주요임상시험13))은 제품이 유효성이 있는지에
대해 의미있는 자료를 수집하는 것이다. 유효성은 적절히 잘 관리된 임상연구에
의해 결정된다. 따라서 역가시험의 설계와 수용기준은 주요임상시험 동안 투여된
제품이 충분한 특성분석과 함께 일관성있게 제조되었음을 평가하는 것이 중요하
다. 확립된 역가 한계에 대한 확인은 향후 생산되는 제품의 로트가 환자에 주어
진 용량에서 작용하리라는 합리적인 신뢰성을 주어야 한다.
- 또한 인허가를 위한 유효기간(expiry dating) 설정에 이용된 적합한 로트의 안정
성시험 동안 확립된 한계를 가지는 잘 특성분석된 역가시험법을 이용해야 한다
(21 CFR 610.53; Ref. 7 참조).
3. 생물학적 제제 허가
- 생물학적 제제를 시장에 내놓기 위해서는 수용가능한 기준으로 정의된 승인된 역

- 11 -
 가시험법이 기술되어야 하며, 생물학적 제제허가신청 (21 CFR 601.2(a),
211.165(e) 및 Section II.A 참조)에서 정의되어 있어야 한다. 수용가능한 기준은
제조 경험을 통해 얻어진 지식정보, 임상조사 및 제품생산의 모든 기간 동안 이
루어진 시험으로부터 모아진 자료가 기초되어야 한다(참고문헌 5). 주요한 임상
시험에 사용하기 위해 제조된 로트 또는 적합한 로트의 제제를 평가할 때 수용
기준은 이러한 자료를 반영하여 개선되어야 한다.
- 역가시험법의 수용기준은 이후의 로트 출하시험을 위해 생물학적제제허가신청에
서 설명되어야 하며, 임상적 유효성을 증명하기 위한 주요 임상시험에서 사용되
는 제품 롯트에 대한 역가한계 설정이 상세히 기술되어야 한다(FDC Act,
Section 505(d), 21 U.S.C. 351 참조).

IV. 시험법 설계 및 검정
A. 시험법 설계 동안 무엇이 고려되어야 하는가?
- cGMP 규정을 따르면, 시험법 설계는 반드시 시험법 평가를 위한 자료수집이 이
루어져야 한다. 여기에는 통계분석을 고려한 충분한 반복시험, 편향성(bias)(예:
96-well plate의 위치에 따른 편향성)을 줄이기 위한 무작위 샘플의 사용 및 적
절한 관리 등이 포함된다. 또한 시험법 설계는 시험법 가변성에 영향을 주는 인
자에 대한 지식을 반영해야 한다. 따라서, 시험법의 가변성에 대한 원인들을 고
려하고, 시험법 설계에서 이 원인들을 제한시키기 위한 조치를 취해야 한다. 가
변성을 감소시키기 위한 일반적인 원칙은 잘 정제된 시약, 잘 조정된 실험기기의
이용 그리고 충분히 훈련된 실험자 등이다. 또한 시험법의 가변성은 자세하게 기
술된 표준작업수순서(SOP)와 적절한 장소 및 적절한 관리를 통해 충분히 감소시
킬 수 있다. 시험법 특이적 관리(assay-specific controls)는 이용하는 시험법 뿐
만 아니라 분석되는 제제에 따라 달라질 수 있다. 또한 기준물질(reference
materials)과 대조물질(controls)을 포함하는 중요한 시약의 장기간의 유효성도
고려하여야 한다. 제조자는 시험법 설계 전략의 상세한 검토를 위해 각기 다른
공급원을 구할 수 있다(예. 참고문헌 13-20).

B. 표준물질 및 대조물질은 어떻게 이용되는가?

- 모든 실험 설계가 잘 이루어졌다면, 역가시험법의 개발은 적절한 기준물질과, 적
절한 대조물질과의 비교가 포함되어야 한다. 표준물질 그리고/또는 대조물질을
제제에 동시에 적용(running)하는 것은 시험법이 기대한 대로 수행될 수 있음을
보증하는데 일조할 수 있다. 또한 대조물질은 시험기기와 시약들이 확립된 범위
안에서 제대로 작용함을 확인할 수 있게 도와준다. 하나의 잘 설계된 대조시료
세트는 의미있고 재현성있는 결과의 신뢰성을 충분히 높여준다.
- 표준물질과 대조물질은 시험법 개발에 도움을 주며, 더 적절한 자체개발 표준물
질 그리고/또는 대조물질의 개발 및 검정에 이용할 수 있다. 다수의 표준물질,

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 표준품 및 대조물질이 이용가능하거나, 생물학 특성평가를 위해 계속 개발되고
있다. 예를 들면, 시험기기를 보정하거나 정량적 FACS 분석(참고문헌. 18)을 위
해 수용가능한 parameter를 정의하는데 도움을 주는 형광성의 비드(bead)/항체
14)
및 입자크기의 표준품 그리고가이드라인서15)가 있다. 기준물질은 또한 현재
adenovirus type 5(Ref. 19)16) 그리고 retrovirus17) vector에 이용할 수 있다.
adeno-associated virus type 2 vectors18)에 대한 기준물질은 개발중에 있다.
lentivirus vectors를 위한 표준물질과 대조물질 역시 기술되어 있다(참고자료.
20).
- 만일 일반적인 표준 또는 기준물질이 유효하지 않다면, 당신은 독자적인 회사내
(“in house") 표준물질을 개발하여야 한다(참고문서. 9 - 11). 이를 위해서는 당
신 또는 다른 곳으로 부터의 잘 특성분화된 임상적 로트들 또는 다른 잘 특성 분
석된 물질들(예를 들어 당신의 제품과 유사한 특성을 가진, 특성분석이 잘 되어
있는 세포주)이 준비되어야 한다. 이때에는 표준물질(자체개발 표준물질/대조물질
을 포함)을 어떻게 그리고 왜 선정했는지에 대한 분명한 이론적 근거가 있어야
한다. 우리는 표준물질을 개발 또는 획득할 때 생물의약품 평가 연구센터의 심사
부서(review team)에게 상의할 것을 권장한다.
- 표준물질은 제품 개발 및 특성분석의 다양한 단계에서 사용되므로, 이 물질에 대
한 안정성 연구를 제품의 안정성 연구와 동시에 수행하여야 한다(참고자료. 7).
또한 표준물질의 각 새로운 배치에 대하여 적절하게 특성분석을 실시하고 원형
과의 비교를 통해 적절한 절차를 수립하여, 새로운 표준물질의 유효성을 증명해
야 한다. 가능하다면 새로 제조된 표준물질과의 비교를 위해 각 로트의 표준물질
시료를 보존(참고자료. 6-8)하고 표준물질의 소진 또는 유효기간 만기에 대비하
여 미리 준비해야 한다.

C. 시험법 밸리데이션 계획을 위해 무엇이 고려되어야 하는가?

1. 규정
- 생물학적제제의 인허가를 취득하기 위해서는 생물학적제제허가신청(BLA)과 관련
된 시험 자료를 제출해야 한다. 무엇보다 개발된 제품이 규정된 역가의 필요조건
을 만족시켜야 한다(21 CFR 601.2). 이 역가의 필요조건은 미리 결정된 수용기
준을 이용한 역가시험의 밸리데이션이 요구된다(21 CFR211.165(e) 참조). 밸리
데이션 과정을 수행하는 데에는 가능한 많은 공급원에서의 분석방법이 밸리데이
션에 유효하다(참고자료. 9-11). 아래를 포함하는 모든 적절한 시험법의
parameter(참고자료. 9-11)에 대해 분석과 밸리데이션이 수행되어야 한다.
• 정밀성
• 정확성(반복, 재현성)
• 민감도(측정한계/정량한계)
• 특이성

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 • 직선성과 한계범위
• 조직적합성
• 강건성/강직성

2. 통계학적 설계 및 분석
- 역가측정에서 실험의 분석 및 설계에 대한 적절한 통계학적 방법은 아주 중요하
다. 그렇지 않다면, 그러한 실험 자료로부터의 추론은 유효하지 않을 수 있다.
반복실험에서 측정법의 변경성 및 변경의 잠재적인 근원은 결과를 보고할 때 고
려되어야 한다. 당신은 정당성과 이론적 근거를 포함한 분석방법을 완전히 기술
하여야 한다. 이러한 설명은 연구보고서상에서 독립적인 통계학적 분석과 평가가
이루어졌음을 명확히 해야 한다. 역가시험법 밸리데이션 연구에서 수집된 자료가
전자파일로 되어 있다면 CBER 검토위원회에서 이루어지는 통계학적 평가를 용
이하게 할 수 있다. 밸리데이션 연구 결과는 표적된 검정 parameter를 기술하여
야 하며, 이는 수용기준과 일치하여야 한다. 우리는 역가시험의 분석과 설계에서
피드백을 받을 수 있도록 심사부서와 초기부터 논의하기를 권장한다(반드시 보관
해야 하는 실험실 기록의 필요요건: 21 CFR211.194 참고)

3. 품질시험의 밸리데이션
- Section III.B.3에서 기술한 바와 같이, 품질시험법은 역가를 평가하기 위해서 시
험법 매트릭스(assay matrix)의 한 부분으로 사용될 수 있으며, 적절한 상관성
연구를 수행할 수 있도록 한다. 반드시 품질시험을 반영할 수 있는 모든
parameter에 대하여 밸리데이션하여야 하며, 품질과 연관성이 없다고 결정한
parameter 들에 대한 이론적 근거를 제시하여야 한다. 예를 들어 어떤 시험법의
검정 parameter가 품질평가시험에 잘못된 정보 판독으로 적용가능하지 않더라도
(예를 들어 직선성), 적절한 대조시료가 특이성, 민감도 시험 뿐 아니라 다른 수
용 가능한 연구특징(강건성 , 시험법 체계의 적절성 등)을 규명하기 위해 사용되
어야 한다
- 정량적 자료가 없어도, 정확성과 정밀성의 결과로 고려할 수 있다: 어쨌든 적당
한 시험법 설계(예: 충분한 반복시험)를 가지고, 재현성을 기술할 수 있다. 부분
적 정량시험(semi-quantitative assays)(아주 변하기 쉬운 정량적 판독정보, 예:
동물모델에서의 반응)에서는 강직성과 재현성 시험에서 더 넓은 수용범위가 고려
될 수 있다. 또한 검출한계 그리고/또는 정량이 적절한 기준 시험설계에서 세워
질 수 있다. 예를 들어 만일 합리적인 양의 대조 또는 기준물질이 충분한 통계적
정의에서 기대했던 활성을 나타내지 않는다면, 시험법은 일반적으로 받아들이기
어려울 수 있다. CGT제품의 복합성 때문에 특정 시험법의 적절성을 결정하는 상
황은 시험법 마다 다를 수 있다.

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4. 시험법의 평가 및 변경
- 제품개발 동안 또는 인허가 후, 또는 둘 모두에서 제조 및 시험과정은 역가시험
법 재평가에 필수적이며/또는 유익할 수 있다. 만일 승인된 시험법을 변경하거나
새로운 시험법을 제안할 계획이라면, 반드시 적절한 역가측정을 통해 변경된 또
는 새로운 시험법을 증명하는 검정연구를 수행하여야 한다(21 CFR 211.165(e)).
승인된 적용에 대한 이러한 변경들은 반드시 보충자료를 제출하여야 한다(21
CFR 601.12(b)(3)(vi)).
- 역가측정법 변경을 뒷받침하는데 필요한 자료의 양은 인자(factor)의 수에 따라
결정된다.
• 제품개발 단계
• 현행 시험법 내의 변화 유형
• 다른 제품의 특성 측정에 이용되는 어떤 시험법이 이용되는지
• 제안된 시험법이 상단에 기술된 시험법 기준을 충족하는지(상단 또는 Section
ILA 참조)
- 만일 조사연구기간 동안 역가측정법을 변경한다면 시험법 자체가 수정되어야 하
며 제안된 변경(들)이 정당하다는 증명이 제시되어야 한다(예를 들어 더 적절하
고 더 유용하고 더 정량적인 방법임을 입증)
- 권고사항으로 어떠한 경우라도 시료(예: 제품, 표준물질들, 주요시약들)의 보존유
지가 중요함을 더욱 강조하게 된다. 만일 새로운 시험법이 적절한 시료보관 분석
이 없이 적절히 수행된다면 시험법들의 비교 또는 결정은 어려울 것이다.
- 이 가이드라인서에서 지적하듯이, 제품의 적절한 역가측정법 개발에 상당량의 자
료가 필요할 수도 있으며(참고자료. 14 참조), 당신의 시험법(들)은 당신의 제품
개발 시기에 따라 또는 새로운 정보와 방법 습득여부에 따라 변할 수도 있다. 우
리는 당신이 역가측정법의 설계, 평가 및 밸리데이션에 따라 적시에 심사부서와
논의하기를 권장한다.

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2. 세포,유전자치료제 역가시험법 (Draft, 2008, FDA) _ 영문

Potency Tests for Cellular and Gene Therapy Products (Draft Guidance)

Table of Contents

I. INTRODUCTION ................................................................................................................. 1
II. BACKGROUND .................................................................................................................. 2
A. What Are the Regulatory Requirements for Potency of Licensed Biological Products? .2
B. What are the Potency Requirements for Investigational CGT Products? ................. 3
C. What Is the Relationship Between Potency and Clinical Effectiveness for CGT Products? ... 4

III. RECOMMENDATIONS FOR POTENCY MEASUREMENTS ................................... 5

A. How to Determine What to Measure for Potency? ................................................... 5
B. What Methods May be Used to Measure Potency? ................................................... 6
1. Biological assays ........................................................................................................ 6
2. Non-biological analytical assays ............................................................................... 6
3. Multiple assays (assay matrix) ................................................................................. 7
C. What is Necessary to Correlate an Analytical Assay with Biological Activity? .... 7
D. When Should Potency Assay Development Initiate? .................................................. 8
E. What is Progressive Potency Assay Implementation? ................................................. 8
1. Early product development: ...................................................................................... 8
2. Later phase product development: ........................................................................... 9
3. Biological License ...................................................................................................... 9
IV. ASSAY DESIGN AND VALIDATION ......................................................................... 9
A. What Should be Considered During Assay Design? .................................................. 9
B. How Should Reference Materials and Controls be Utilized? .................................. 10
C. What Should be Considered for an Assay Validation Plan? .................................. 11
1. Regulations ................................................................................................................ 11
2. Statistical design and analysis ................................................................................ 11
3. Validation of qualitative assays .............................................................................. 12
4. Assay evaluation and modification ........................................................................ 12

V. REFERENCES ................................................................................................................... 14

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 Guidance for Industry
Potency Tests for Cellular and Gene Therapy Products

This draft guidance, when finalized, will represent the Food and Drug Administration’s
(FDA’s) current thinking on this topic. It does not create or confer any rights for or
on any person and does not operate to bind FDA or the public. You can use an
alternative approach if the approach satisfies the requirements of the applicable statutes
and regulations. If you want to discuss an alternative approach, contact the appropriate
FDA staff. If you cannot identify the appropriate FDA staff, call the appropriate
number listed on the title page of this guidance.

I. INTRODUCTION
We, FDA, are issuing this guidance to provide you, manufacturers of cellular and gene
therapy (CGT) products, with recommendations for developing tests1 to measure
potency.2 These recommendations are intended to clarify the potency information that
could support an Investigational New Drug Application3 (IND) or a Biologics License
Application4 (BLA). Because potency measurements are designed specifically for a
particular product, this guidance does not make recommendations regarding specific types
of potency assays, nor does it propose criteria for product release. This guidance is
intended to supplement related documents (Refs. 1 through 11, and 15) and does not
replace or supersede any existing documents.
This guidance applies only to CGT products5 reviewed by FDA’s Office of Cellular,
Tissue and Gene Therapies (OCTGT), CBER under Section 351 of the Public Health
Service Act (PHS Act) (42 U.S.C. 262) (Refs. 1 and 2). This guidance does not apply
to human cells, tissues, and cellular and tissue products (HCT/Ps), which are regulated
solely under section 361 of the PHS Act (42 U.S.C. 264) as described under 21 CFR
1271.10 or to products regulated as medical devices under 21 CFR Part 820. This
guidance also does not apply to biological products reviewed by CDER or by CBER’s
Office of Vaccine Research and Review (OVRR) or Office of Blood Research and
Review (OBRR).
FDA’s guidance documents, including this guidance, do not establish legally enforceable
responsibilities. Instead, guidances describe the FDA’s current thinking on a topic and

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should be viewed only as recommendations, unless specific regulatory or statutory
requirements are cited. The use of the word should in FDA’s guidances means that
something is suggested or recommended, but not required.

BACKGROUND

What Are the Regulatory Requirements for Potency of Licensed Biological Products?

All biological products must meet prescribed requirements of safety, purity and potency
for BLA approval (42 U.S.C. 262, Federal Food, Drug and Cosmetic Act, (FDC Act)
(21 U.S.C. 321 et seq.); 21 CFR 601.2). For CGTs, product conformance testing (21
CFR 601.20(a)) and control of the manufacturing process (21 CFR 601.20(c)) are
required to comply with FDA’s Current Good Manufacturing Practice (CGMP) For
Finished Pharmaceuticals regulations (21 CFR Parts 210 and 2116) as well as the
biologics regulations (21 CFR Part 600 et seq.). No lot of any licensed product may be
released by the manufacturer prior to the completion of tests for conformity with
standards applicable to such product, (21 CFR 610.1), which include tests for potency,
sterility, purity, and identity (21 CFR Part 610, Subpart B). These requirements apply to
all biological products, including autologous and single patient allogeneic products,
where a lot may be defined as a single dose.
Potency is defined as “the specific ability or capacity of the product, as indicated by
appropriate laboratory tests or by adequately controlled clinical data obtained through the
administration of the product in the manner intended, to effect a given result.” (21 CFR
600.3(s)). Strength7 is defined as “potency, that is, the therapeutic activity of the drug
product as indicated by appropriate laboratory tests or by adequately developed and
controlled clinical data. . . .” (21 CFR 210.3(b)(16)(ii)). Regulations stipulate that
“[t]ests for potency shall consist of either in vitro or in vivo tests, or both, which have
been specifically designed for each product so as to indicate its potency in a manner
adequate to satisfy the interpretation of potency given by definition in § 600.3(s) of this
chapter.” (21 CFR 610.10).
FDA regulations allow for considerable flexibility in determining the appropriate
measurement(s) of potency for each product. Potency is determined based on individual
product characteristics; therefore, the adequacy of potency assays is evaluated on a

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case-by-case basis. All potency assays used for release testing of licensed biological
drug products must comply with applicable biologics and CGMP regulations including:

• Indicate potency (biological activity/activities) specific to the product (21 CFR 600.3(s)
and 610.10; and 21 CFR 210.3(b)(16)(ii));
• Provide test results for release of the product (21 CFR 610.1; 21 CFR 211.165(a))
• Provide quantitative data (21 CFR 211.194; see also 21 CFR 600.3(kk); 21 CFR
211.165(d); 211.165(e););
• Meet pre-defined acceptance and/or rejection criteria (21 CFR 211.165(d); see also 21
CFR 600.3(kk); and 21 CFR 210.3(b)(20));
• Include appropriate reference materials, standards, and/or controls (see; 21 CFR
210.3(b)(16)(ii) and 211.160);
• Establish and document the accuracy, sensitivity, specificity and reproducibility of the
test methods employed through validation (21 CFR 211.165(e) and 211.194(a)(2));
• Measure identity and strength (activity) of all active ingredients (21 CFR 211.165(a);
see also 21 CFR 210.3(b)(7));
• Provide data to establish dating periods (see 21 CFR 600.3(l) and 610.53(a))
• Meet labeling requirements (21 CFR 610.61(g)(3) and 610.61(r))

What are the Potency Requirements for Investigational CGT Products?

In early clinical phase investigations, it may not be possible to meet all of the
requirements described above for licensed biological products (Refs. 3, 4, 8).
Nonetheless, you must submit data to assure the identity, quality, purity and strength
(21 CFR 312.23(a)(7)(i)) as well as stability (21 CFR 312.23(a)(7)(ii)) of products used
during all phases of clinical study. “[T]he amount of information needed to make that
assurance will vary with the phase of the investigation, the proposed duration of the
investigation, the dosage form, and the amount of information otherwise available.” (21
CFR 312.23(a)(7)(i)).
Potency measurements are necessary for product characterization testing,8 comparability
studies (Ref. 6), and stability protocols (Ref. 7), which are used to establish that a
consistently manufactured product is administered during all phases of clinical
investigation. However, the complexity of CGT products can present significant
challenge(s) to establishing potency assays (see Table 1). To facilitate the development

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of CGT products, we recommend an incremental approach to product characterization
testing, including the development of potency assays. General recommendations for
progressive potency assay implementation are outlined in Section III.E. As described in
Sections III.A, III.E, and IV.C.4 of this document, your potency measurement will
evolve and may change significantly as you develop your product.
Table 1:

C. What Is the Relationship Between Potency and Clinical Effectiveness for CGT
Products?
There is no single test that can measure adequately those product attributes that predict
clinical efficacy. Clinical effectiveness is demonstrated by adequate and well-controlled
clinical investigations conducted with a consistently manufactured quality product.
Clinical effectiveness may be correlated to product potency, but clinical study data is
not a practicable quantitative measure of potency to release a lot. Rather, clinical study
results may be used to establish a correlation(s) 9 between the product’s clinical efficacy
and a potency measurement(s), which can be used for lot release, stability, and/or
comparability studies (see Section III.C for more details related to correlation studies).

III. RECOMMENDATIONS FOR POTENCY MEASUREMENTS

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A. How to Determine What to Measure for Potency?
Because of the complexity of CGT products, you need to acquire an appropriate
understanding of the biological properties of your product in order to develop relevant
and meaningful potency measurements. You should collect sufficient data throughout
preclinical and clinical development to inform and refine your approach to measuring
potency.
When initially determining the biological activity or activities that will guide your
potency assay design, you should consider relevant pre-clinical investigations, proof of
concept studies, early clinical studies, available historical experience, and available
reference materials and controls (see Section III.C). This information may provide you
with a basic understanding about product characteristics and biological activities that
contribute to function. Characterization data obtained during product development may
provide support for the potency assay that you choose initially, or it may lead to an
improved potency measurement as you prepare to market your product (see Sections
III.E and IV.C.4). As you develop your product(s), you should measure a wide range of
product properties in addition to those performed for routine lot release. This may help
you to assess which product attribute(s) best correlate(s) with potency. Although some
of the assays you evaluate may not be practical for lot release (e.g., difficult to
consistently obtain quantitative results, time-consuming), most properly designed assays
(see Section IV.A) have the potential to provide valuable information about product
attributes related to biological activity or clinical effectiveness, or both.
CGT products may present challenges for developing assays to measure specific
biological attributes that quantitatively demonstrate potency (see Table 1). CGT products
often have complex and/or poorly defined mechanism(s) of action (i.e., relevant
therapeutic or clinical functional activity), making it difficult to determine which product
attribute is most relevant to measuring potency. Nonetheless, potency measurements
should reflect the relevant biological attributes. For example, a gene therapy vector
should rely on at least two biological activities for its potency: the ability to transfer a
genetic sequence to a cell and the biological effect of the expressed genetic sequence.
Therefore, the potency assay should incorporate both a measure of the gene transfer
frequency and the biological effect of the transferred gene.
In addition, the proposed mechanism(s) of action for CGT products may be dependent
on more than one active ingredient10 (e.g., multiple cell types, multiple vectors,
multi-epitope vaccines). For some complex products (e.g., cellular tumor vaccine) there

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could be ambiguity about which ingredients contribute to potency. For products that
contain more than one known active ingredient, you should design potency
measurement(s) to determine the biological activity (strength) of all active ingredients
(see 21 CFR 211.165(a)). Thus, if your product contains more than one active
ingredient you might need more than one assay to measure potency of the product
because one assay might be insufficient to measure the activity of each of the active
ingredients (Section III.B.3). Additionally, when designing your assay(s), you should also
consider the potential for interference or synergy between active ingredients.

B. What Methods May be Used to Measure Potency?

1. Biological assays
The traditional approach for assessing the potency of biological products is to develop a
quantitative biological assay (bioassay) that measures the activity of the product related
to its specific ability to effect a given result, and that also meets the criteria listed in
Section II.A. Bioassays measure potency by evaluating a product’s active ingredients
within a living biological system. Bioassays can include in vivo animal studies, in vitro
organ, tissue or cell culture systems, or any combination of these. You may use in vitro
or in vivo assays; however, we encourage the responsible limitation of animal use
whenever possible (Ref. 12).
2. Non-biological analytical assays11
Development of a quantitative bioassay for some CGT products may be complicated by
properties of the product and/or technical limitations (see Table 1). In cases where
bioassay development is not feasible, it may be necessary to identify a surrogate of
biological activity. For example, you may need to use an analytical assay(s) that is
practical and reliable for lot release. Analytical assays can provide extensive product
characterization data by evaluating immunochemical, biochemical, and/or molecular
attributes of the product. These attributes may be used to demonstrate potency if the
surrogate measurement(s) can be substantiated by correlation to a relevant
product-specific biological activity(s) (see Section III.C, Refs. 13 and 14). To establish
meaningful correlations, you should conduct rigorous product characterization testing, as
recommended throughout this document.
3. Multiple assays (assay matrix)
In many cases, a single biological or analytical assay may not provide an adequate
measure of potency. The following are some potential reasons:

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• Product has complex mechanism of action
• Product has multiple active ingredients and/or multiple biological activities
• Limited product stability
• Biological assay is not quantitative, not sufficiently robust, or lacks precision

If one assay is not sufficient to measure the product attribute(s) that indicates potency,
then an alternative approach could be used to develop multiple complementary assays
that measure different product characteristics associated with quality, consistency and
stability. When used together and when results are correlated with a relevant biological
activity, these complementary assays should provide an adequate measure of potency.
Such a collection of assays (referred to as an assay matrix) might consist of a
combination of biological assays, biological and analytical assays, or analytical assays
alone (Refs. 13 and 14). The assay matrix may include assays that give a quantitative
readout (e.g., units of activity) or qualitative readout (e.g., pass/fail). If qualitative assays
are used as part of an assay matrix to determine potency for lot release, stability or
comparability studies, they should be accompanied by one or more quantitative assays
(see SectionC. What is Necessary to Correlate an Analytical Assay with Biological
Activity?
To demonstrate potency using an analytical assay as a surrogate measurement of
biological activity, you should provide sufficient data to establish a correlation between
the surrogate measurement(s) and the biological activity(ies) that is related to potency.
The relationship between the surrogate measurement and biological activity may be
established using various approaches, including comparison to preclinical/proof of concept
data, in vivo animal or clinical data, or in vitro cellular or biochemical data. If you
choose to use an analytical assay as a surrogate measurement of biological activity to
meet the potency requirements for licensed biological products, you should meet criteria
listed above in Section II.A. This could necessitate that you stress the product (i.e.,
show that the assay can detect an inactive or degraded product) and perform sufficiently
controlled studies (see Section IV.).
The suitability of data used to support surrogate assays for biological activity is
evaluated on a case-by-case basis and depends on or is influenced by the following:

• Type and relevance of the correlation(s) being made;

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• The amount of product information you have accumulated;
• How well the biological activity of the product is understood; and
• How well the surrogate measurement(s) reflects biological activity.

If you intend to demonstrate potency by correlating a surrogate assay(s) to a relevant

biological activity, you should start collecting product and assay characterization data
during early investigational phases.

D. When Should Potency Assay Development Initiate?

As discussed throughout this document, thorough product characterization is necessary to
understand the product parameter(s) that affect quality, consistency, and stability.
Moreover, understanding and controlling these parameters will be necessary to
demonstrate consistency between production lots, to assess comparability of different
manufacturing processes and/or various assays, and may also be necessary to allow you
to determine which product attributes are related to an effective product. Thus, because
the ability to measure potency is essential to product characterization, you should initiate
potency assay development during preclinical and early clinical investigations to obtain
as much product information as possible.
In addition, measuring potency during early product development has a number of
advantages, such as allowing you to:

• Demonstrate product activity, quality and consistency throughout product development;

• Generate a collection of data to support specifications for lot release;
• Provide a basis for assessing manufacturing changes;
• Evaluate product stability;
• Recognize technical problems or reasons a different assay might be preferable;
• Evaluate multiple assays; and
• Collect sufficient data to support correlation studies, if necessary.

E. What is Progressive Potency Assay Implementation

1. Early product development:
For some products in pre-clinical, Phase 1 and early Phase 2 studies, limited
quantitative information on bioactivity may be sufficient. Potency assays performed on
product lots used for early clinical studies are likely to have wider acceptance ranges

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than assays used in later phase investigations. Nevertheless, as clinical studies progress
and product knowledge increases, you should develop and implement improved potency
measurement(s) that quantitatively assesses relevant biological product attribute(s) (see 21
CFR 312.23(a)(7)).
2. Later phase product development:
The primary objective of later phase investigational studies (i.e., Phase 3, pivotal12) is to
gather meaningful data about product efficacy. Efficacy is determined by adequate and
well-controlled clinical study(ies). Therefore, your potency assay design and acceptance
criteria should be sufficient to assure that a well-characterized, consistently manufactured
product was administered during your pivotal study(ies). Conformance to established
limits for potency should thus provide reasonable confidence that future product lots will
perform as expected at a given dose in patients.
In addition, you should use a well-characterized potency assay with established limits
during stability testing of conformance lots used to establish expiry dating for licensure
(see 21 CFR 610.53; Ref. 7).
3. Biological License
To market a biological product, a validated potency assay with defined acceptance
criteria must be described and justified in the BLA (21 CFR 601.2(a) and 211.165(e),
see also Section II.A). The acceptance criteria should be based on knowledge gained
through manufacturing experience and data collected from assays performed during all
phases of product development and clinical investigation (Ref. 5). As you evaluate
product conformance lots or lots manufactured explicitly for use in your pivotal clinical
studies, acceptance criteria should be refined to reflect these data.
The potency assay acceptance criteria defined in your BLA, which are intended for
subsequent lot release testing, should depict the potency limits established for product
lots used in the pivotal clinical studies demonstrating clinical effectiveness (see FDC
Act, Section 505(d), 21 U.S.C. 351).

IV. ASSAY DESIGN AND VALIDATION

A. What Should be Considered During Assay Design?
In accordance with CGMP regulations, assay design should allow you to collect data
that will permit you to evaluate your assay(s). This includes incorporating a sufficient
number of replicates to allow for statistical analysis, using sample randomization to
reduce biases (e.g., sources of bias associated with placement in a 96-well plate), and

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including appropriate controls. Assay design should also reflect knowledge of the factors
that influence assay variability. Therefore, you should consider sources of variability in
the assay method and take steps to limit them in your assay design. General principles
for reducing variability include using well-defined reagents, well-calibrated equipment,
and adequately trained operators. Assay variability can also be substantially reduced by
following detailed standard operating procedures (SOPs) and having appropriate controls
in place. Assay-specific controls will depend on the product being analyzed as well as
the assay used. You should also consider the long-term availability of critical reagents,
including reference materials and controls. Manufacturers may refer to several resources
for a more detailed discussion of assay design strategies (e.g., Refs. 13 through 20).
B. How Should Reference Materials and Controls be Utilized?
As with all well designed experiments, developing a potency assay should include
appropriate controls and a comparison to an appropriate reference material, when
available. Running a reference material and/or control samples in parallel with the
product helps ensure that the assay is performing as expected. In addition, controls help
establish that the equipment and reagents are working within established limits. A well
designed set of control samples can substantially increase confidence that results are
meaningful and reproducible.
Reference materials and standards can help with assay development and can be used to
develop and qualify more relevant “in house” reference materials and/or controls. A
number of reference materials, standards, and controls are available or are being
developed for characterizing biologics. For instance, there are fluorescent bead/antibodies
and particle size standards13 and guidelines14 available to help calibrate equipment and
help define acceptable parameters for quantitative flow cytometry analysis (Ref. 18).
Reference materials are also currently available for adenovirus type 5 (Ref. 19)15 and
retrovirus16 vectors. A reference material for adeno-associated virus type 2 vectors17 is
under development. Standard materials and controls for lentivirus vectors have also been
described (Ref. 20).
In the event that a universal standard or reference material is not available, you should
develop your own “in house” reference material(s) (Refs. 9 through 11). These may
include well characterized clinical lots or other well characterized materials prepared by
you or another resource (e.g., a well characterized cell line with a profile similar to
your product). There should be a clear rationale for how and why the reference material
(including “in house” reference material/control) was developed. We encourage you to

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consult with your CBER review team when developing or obtaining reference materials.
Because you will use reference materials at various stages of product development and
characterization, you should subject them to stability studies in parallel with your
product stability studies (Ref. 7). Moreover, you should appropriately characterize each
new batch of reference material, compare it with the original, and establish appropriate
procedures to qualify and eventually validate new reference materials. When possible,
you should retain samples (Refs. 6 through 8) of each lot of reference material for
comparison with newly manufactured reference material and prepare in advance for
depletion or expiration of reference materials.
C. What Should be Considered for an Assay Validation Plan?
1. Regulations
To obtain a biologics license, you must submit data in your BLA demonstrating, among
other things, that your product meets prescribed requirements of potency (21 CFR
601.2), which requires that you validate your potency assay with predefined acceptance
criteria (see 21 CFR 211.165(e)). The validation process identifies potential sources of
errors and quantifies them within the assay method. Numerous resources are available
for analytical methods validation (Refs. 9 through 11). You should perform analysis and
validation of all relevant assay parameters (Refs. 9 through 11), including:

• Accuracy
• Precision (Repeatability, Reproducibility)
• Sensitivity (Limit Of Detection/Quantitation)
• Specificity
• Linearity and Range
• System Suitability
• Robustness/Ruggedness

2. Statistical design and analysis

It is critically important to apply sound and appropriate statistical methods to the design
and analysis of laboratory experiments for potency measurements. Otherwise, inferences
drawn from such experimental data might not be valid. Potential sources of assay
variability and variations from replicates should be taken into account when reporting
results. You should fully describe your methods of analysis, including your justification
and rationale. These descriptions should be sufficiently clear to permit independent

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statistical analysis and evaluation of the results presented in the study reports. Data
collected from potency assay validation studies, when provided in electronic format, can
facilitate statistical evaluations by the CBER review committee. The results of
validation studies should address the targeted validation parameters and their
conformance to acceptance criteria. We encourage you to initiate early discussions with
the review team to receive feedback on the design and analysis of potency experiments.
(See 21 CFR 211.194 for requirements pertaining to the laboratory records you must
keep.)
3. Validation of qualitative assays
As discussed in Section III.B.3, qualitative assays may be used as part of an assay
matrix to assess potency, provided that you conduct suitable correlation studies. You
should validate all parameters relevant to your qualitative assay and provide a rationale
for those parameters that you determine are not relevant. For example, although certain
assay validation parameters (e.g., linearity) may not be applicable to a qualitative assay
with a pass or fail readout, appropriate control samples should be used to characterize
the assay for specificity and sensitivity as well as for other features of acceptable
performance (e.g., robustness, system suitability).
Without quantitative data, demonstrating accuracy and precision could be challenging;
however, with proper assay design (e.g., sufficient replicates), you might be able to
demonstrate reproducibility. For semi-quantitative assays (assays with highly variable
quantitative readout, e.g., response in an animal model), broader acceptance ranges may
be considered for determining assay robustness and reproducibility. Also, limits of
detection and/or quantitation may be built into the assay design suitability criteria. For
example, if a reasonable amount of the control or reference material does not exhibit
the desired activity with sufficient statistical justification, the assay would not generally
be considered acceptable. Importantly, because of the complex nature of CGT products,
specific circumstances for determining assay suitability will vary from assay to assay.
Therefore, we encourage you to discuss planned experiments with your CBER review
team before you initiate specific assay designs and/or detailed experimental analyses of
potency measurements.

4. Assay evaluation and modification

Manufacturing and testing practices evolve during product development or post-licensure,
or both, making it necessary and/or beneficial to re-evaluate your potency assay. If you

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plan to modify an assay that is used in an approved application or propose a new
assay, you must perform validation studies to demonstrate that the modified/new assay
continues to be an appropriate measure of potency (21 CFR 211.165(e)). These changes
must be submitted as a supplement to an approved application (21 CFR
601.12(b)(3)(vi)).
The quantity of data needed to support changes to potency measurements(s) will depend
upon a number of factors, including:

• Stage of product development

• Type of change within an existing assay
• Whether the assay is being used to measure a different product attribute(s)
• Whether the proposed assay meets assay criteria outlined above (see above and
Section II.A)

If you modify the potency measurement used during an investigational study, you
should qualify the assay and provide justification for the proposed change(s) (e.g., more
relevant, more practical, more quantitative).
These recommendations further emphasize the importance of maintaining retention
samples (e.g., product, reference materials, critical reagents) whenever possible. It will
be difficult to compare assays or determine if new assays are performing appropriately
without analyzing appropriate retention samples.
As this guidance indicates, a considerable amount of data might be necessary to develop
a suitable measurement of potency for your product (see also Ref. 14), and your
assay(s) might change over time as you develop your product and learn new
information and methods. We recommend that you have timely discussions with your
review team as you design, evaluate and validate your potency measurement.

V. REFERENCES

1. Application of Current Statutory Authorities to Human Somatic Cell Therapy Products

and Gene Therapy Products. October 14, 1993; 58 FR 53248. Available at
http://www.fda.gov/cber/genadmin/fr101493.pdf.

2. Guidance for Industry: Source Animal, Preclinical, and Clinical Issues Concerning the

- 29 -
Use of Xenotransplantation Products in Humans. (April 2003). Available at
http://www.fda.gov/cber/gdlns/clinxeno.htm.

3. Guidance for FDA Review Staff and Sponsors: Content and Review of Chemistry,
Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational
New Drug Applications (INDs). (April 2008). Available at
http://www.fda.gov/cber/gdlns/gtindcmc.htm.

4. Guidance for Reviewers: Instructions and Template for Chemistry, Manufacturing, and
Control (CMC) Reviewers of Human Somatic Cell Therapy Investigational New Drug
Applications (INDs). (April 2008). Available at
http://www.fda.gov/cber/gdlns/cmcsomcell.htm.

5. International Conference on Harmonisation: Guidance on Specifications: Test

Procedures and Acceptance Criteria for Biotechnological/Biological Products (ICH Q6B).
64 FR 44928, August 18, 1999. Available at http://www.fda.gov/cber/gdlns/ichtest.pdf.

6. International Conference on Harmonisation: Guidance for Industry: Q5E Comparability

of Biotechnological/Biological Products Subject to Changes in Their Manufacturing
Process. (June 2005). Available at http://www.fda.gov/cder/guidance/6677fnl.pdf.

7. International Conference on Harmonisation: Final Guidelines on Stability Testing of

Biotechnological/Biological Products (ICH Q5C). 61 FR 36466, July 10, 1996. Available
at http://www.fda.gov/cber/gdlns/ichq5c071096.pdf.

8. Guidance for Industry: CGMP for Phase 1 Investigational Drugs. (July 2008).
Available at http://www.fda.gov/cber/gdlns/indcgmp.htm.

9. Draft Guidance for Industry: Analytical Procedures and Methods Validation

Chemistry, Manufacturing, and Controls Documentation. (August 2000). Available at
http://www.fda.gov/cber/gdlns/methval.htm.∗

10. International Conference on Harmonisation Guideline Validation of Analytical

Procedures: Text and Methodology Q2(R1). (November 2005). Available at
http://www.ich.org/LOB/media/MEDIA417.pdf.

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11. Chapter <1225> Validation of Compendial Methods. US Pharmacopeia 28, United
States Pharmacopeia Convention, Inc., Rockville, MD: 2005.

12. The Interagency Coordinating Committee on the Validation of Alternative Methods

(ICCVAM) Mission, Vision and Strategic Priorities. (February 2004). Available at
http://iccvam.niehs.nih.gov/about/ni_Mission.htm.

13. Kawakami K, Puri, RK. Regulatory Expectations During Product Development for
Tumor Vaccines. Brown F, Petricciani J editors. Development of therapeutic cancer
vaccines. Dev. Biol, Basel, Karger, 2004, vol 116, pp. 53-9.

14. Potency Measurements for Cellular and Gene Therapy Products, Cellular, Tissue and
Gene Therapies Advisory Committee (CTGTAC) Meeting. Gaithersburg Hilton, February
9, 2006. Available at
http://www.fda.gov/ohrms/dockets/ac/cber06.html#CellularTissueGeneTherapies.

15. Chapter <111> Design and Analysis of Biological Assays. US Pharmacopeia 28,
United States Pharmacopeia Convention, Inc., Rockville, MD: 2005.

16. Brown, F., Mire-Sluis A. (Eds.), The Design and Analysis of Potency Assays for
Biotechnology Products. Brown, F. (ed.), Developments in Biologicals, Vol. 107, Basel:
Karger (2002).

17. Montgomery, D. C. Design and Analysis of Experiments. John Wiley & Sons; 6th
edition (2005).

18. Stelzer, GT., et al., U.S.-Canadian Consensus Recommendations on the

Immunophenotypic Analysis of Hematologic Neoplasia by Flow Cytometry:
Standardization and Validation of Laboratory Procedures. Cytometry (Comm Clin
Cytometry) 30:214-230 (1997).

19. Hutchins, B., et al., Working Toward an Adenovirus Vector Testing Standard.
Molecular Therapy Vol. 2, No. 6, (December 2000).

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20. Kiermer, V., et al., Report from the Lentivirus Vector Working Group: Issues for
Developing Assays and Reference Materials for Detecting Replication-Competent
Lentivirus in Production Lots of Lentivirus Vectors. BioProcessing Journal, March/April
2005: 39-42.

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GUIDELINE ON POTENCY TESTING OF CELL BASED IMMUNOTHERAPY
MEDICINAL PRODUCTS FOR THE TREATMENT OF CANCER
(EMEA/CHMP/BWP/271475/2006)

TABLE OF CONTENTS

1. EXECUTIVE SUMMARY .................................................................................................. 3

2. INTRODUCTION (BACKGROUND) ............................................................................. 3

3. SCOPE .................................................................................................................................... 3

4. LEGAL BASIS ...................................................................................................................... 4

5. ASPECTS TO POTENCY TESTING OF CELL BASED IMMUNOTHERAPY

PRODUCTS ................................................................................................................................ 4

5.1 IN VIVO (ANIMAL) POTENCY TESTING .............................................................................. 5

5.2 IN VITRO POTENCY TESTING .............................................................................................. 5

5.3 VIABLE CELL COUNT .......................................................................................................... 6

5.4 AUTOLOGOUS CELL BASED PRODUCTS .............................................................................. 6

5.5 REFERENCE PREPARATION .................................................................................................. 6

5.6 ADJUVANT CONTAINING IMMUNOTHERAPY PRODUCTS. ................................................... 6

DEFINITIONS ........................................................................................................................... 7

REFERENCES (SCIENTIFIC AND / OR LEGAL) ................................................... 8

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1. EXECUTIVE SUMMARY
Licensed biological medicinal products must meet specifications for appearance, identity,
purity, biological activity and/or quantity of the drug substance. Determining the
biological activity of cell based immunotherapy products is not easy since the active
ingredient is usually composed of whole cells and the activity of these products can
generally not be attributed to one specific cell characteristic. The potency (i.e., the
quantitative measure of biological activity) of cell based immunotherapy products can be
measured using in vivo or in vitro tests. An appropriately validated potency assay
should be based on a defined biological effect as close as possible to the mechanism(s)
of action/clinical response. Surrogates for potency may be developed to demonstrate
biological activity of the test sample. Development and validation of such assays for
cell based immunotherapy products need special considerations. This document represents
CHMP’ current thinking on these issues.

2. INTRODUCTION (background)
Cell based immunotherapy aims at treating patients by stimulating their immune system
using autologous or allogeneic cells. Immunotherapy of cancer is based on an immune
response targeted against tumour-specific/tumour associated antigen(s), leading to
destruction of malignant cells. The targeting of interactions between the immune system
and the tumour constitute a complex approach of which the precise mechanisms of
action are often not fully understood.

In the scientific literature, cell based immunotherapy products for the treatment of
cancer are sometimes called cell based tumour vaccines or cancer vaccines.

Assessment of the biological properties constitutes an essential step in establishing a

complete characterisation profile of a biological medicinal product. Due to their
complexity, cell based immunotherapy products cannot be fully characterised like
products derived by recombinant DNA techniques. Nevertheless, as for any biological
medicinal product, the biological activity is an important characteristic and needs to be
determined for cell based immunotherapy products.

According to the ICH guideline1 the biological activity describes the specific ability or
capacity of a product to achieve a defined biological effect. Potency is the quantitative

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measure of biological activity based on the attribute of the product, which is linked to
the relevant biological properties.

Current guidance on cell therapy based medicinal products is found in the Guideline for
Human Cell based Medicinal Products (CHMP/410869/2006) replacing the CPMP Points
to consider (PtC) on the manufacture and quality control of human somatic cell therapy
medicinal products (CPMP/BWP/41450/98). According to these guidelines the final cell
therapy product should be subjected to quality control and lot release testing as well as
to tests to evaluate the shelf-life of the product. This should include a potency assay,
which should be properly validated. However, specific guidance related to the
development and validation of such assays is not available.

This document intends to provide further guidance on specific requirements related to

the development and validation of potency assays for cell based immunotherapy
products. Other existing guidelines related to testing may be relevant and should be
consulted1,2.

3. SCOPE
This guidance document covers viable cell products for cancer-immunotherapy from
autologous or allogeneic origin, consisting of e.g. whole tumour cells or autologous
dendritic cells loaded with tumour antigens, all intended to induce tumour-specific
cytotoxity although the immunological pathway may differ between products.
Tumour-specific cells intended for adoptive transfer (i.e. passive immunisation strategies)
are also included, for example ex-vivo primed T-cells. Some principles outlined in this
document may also be applicable to tumour cell lysates.

The cells may be chemically treated or genetically modified in vitro to immortalize

them or to express certain gene products like growth factors or tumour antigens. If the
medicinal product is to be considered as a gene therapy medicinal product3, further
guidance can be found in the Note for Guidance on the Quality, Preclinical and Clinical
Aspects of Gene Transfer Medicinal Products.

4. LEGAL BASIS

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This guideline has to be read in conjunction with the introduction and general principles
(4) and Part I: Standardised marketing authorisation dossier requirements as well as Part
IV: Advanced therapy medicinal products of the Annex I to Directive 2001/83/CE as
amended.

5. ASPECTS TO POTENCY TESTING OF CELL BASED IMMUNOTHERAPY

PRODUCTS

Appropriately designed potency assays provide an accurate, reliable and consistent

demonstration of the biological activity of the active ingredient either at the level of
drug substance and/or drug product. In principle the results of a potency assay should
provide assurance that the amount of the active ingredient is sufficient to induce a
meaningful response and that the amount is consistent from batch to batch. As such, the
potency assay should be able to detect clinically meaningful changes in the amount
of active ingredient in a human dose of a product.

Determining the biological activity of cell based immunotherapy products is not easy
since the active ingredient is usually composed of whole cells and the activity of these
products can generally not be attributed to one specific cell characteristic. Potency
assays for immunotherapy products will be based on complex immune mechanisms
which are often poorly or incompletely understood and which may be complicated by
multi-antigen formulations and inherent variability of the starting material.

Nevertheless, to assure a consistent functional activity of the medicinal product in the

recipient, the potency of the product within justified limits should be demonstrated by a
bioassay based on a defined biological effect as close as possible to the mechanism(s)
of action/clinical response. To define the biological effect, a proper understanding of the
biology of these cells is necessitated. Therefore, phenotypic and functional properties of
the cells should be extensively characterised. Based on these characteristics and the
mode(s) of actions established in non-clinical studies the concept of the analytical assay
should be deduced. One or more antigens may be selected that are linked to the
defined mechanisms of action. It is generally acknowledged that cellular immunity plays
a key role in the immunological destruction of tumours. Therefore, several assays under
development have been based on this principle. The mechanisms of action may be more

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complex involving both a cellular and humoral immune response. Assays based on
antibody formation against selected antigens or assay based on quantitative antigen
expression could thus be considered as well. However, the results of the pivotal studies
should ultimately support the chosen assay. Induction of a non-relevant immune
response (e.g. an antibody response that is not relevant as regards to the defined
biological effect) in animals following administration of the medicinal product is
generally not accepted as a measurement of potency.

Ideally, one single properly developed and validated assay is sufficient to cover both
characterisation issues and batch release testing. However, different kinds of assays may
be needed depending on the purpose of the assay, e.g. to characterise the active
substance, to validate the production process, to show batch-to-batch consistency, and to
determine the stability during shelf life. A potency assay is an extremely valuable tool
to provide assurance of unaltered biological characteristics of the product throughout the
development of the product. This is especially important when changes to the
manufacturing process are introduced after production of material for non-clinical studies
or pivotal clinical studies.

It may be prudent to develop in parallel different potency assay most suitable for their
intended use. These may comprise for example functional bioassays or, where justified,
assays based on quantitative antigen expression.

Preferably, a suitable potency assay should be in place already when material for the
first clinical trial is produced and it should be validated prior to phase III clinical trials
unless otherwise justified. Lot release and shelf life specifications for potency should be
determined and amended during product development, as appropriate. It is strongly
recommended that the development of a suitable potency assay be started as soon as
possible.

Potency of cell based immunotherapy products can be measured in a number of

different assays including in vivo and in vitro test systems.

5.1 In vivo (animal) potency testing

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An in vivo potency assay is a useful tool to verify the biological activity of the active
ingredient. However, the development of a relevant biological in vivo potency assays for
cell based immunotherapy products may be hampered by the lack of a relevant animal
model due to the inherent immunological differences between man and animals. In
addition to the lack of suitable animal models, it is acknowledged that such assays very
often suffer from wide inherent biological viability.
In vivo potency testing may also be particularly lengthy to perform and as such may
not be practical for lot release. However, the use of relevant animal models should be
fully explored for their applicability for routinely performed assays. Moreover, they
might be useful as a product characterization tool, e.g., after the introduction of a
process change or any other change that may impact the quality of the medicinal
product. For example, animals which are transgenic for human major histocompatibility
antigens can be used to present human antigens to the immune system of these animals.
Also, immuno-compromised animals (e.g., athymic mice) might be used to determine
the functional response of adoptively transferred human T-cells as the measurement of
potency.

As for any animal based potency assay, suitable conditions for conducting in vivo
animal testing should be set after appropriate validation. Some principles outlined in
current available guidance for biological assays of prophylactic vaccines and their
statistical analysis may be useful (e.g. Ph.Eur. 2.7 & 5.3.6).

5.2 In vitro potency testing

With in vitro assays, a biochemical or physiological response can be measured at the
cellular level. Such assays may be suitable as a direct measure of the biological activity
on a routine basis, i.e. for monitoring product consistency in batch release testing.
Measurable parameters are, for example, in vitro lysis of target cells by tumour-specific
(CD8) T-cells, in-vitro cytokine production by specific cells, e.g. lymphocytes in
response to the product, and co-stimulatory capacity of dendritic cells (DCs).

Where a direct measure of potency is not possible, surrogates for potency may be
developed to verify biological activity of the test sample provided that a correlation
between the surrogate and the defined biological activity has been demonstrated.
Surrogate analysis may comprise different kind of tests including determination of cell

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surface markers, activation markers, secretion of factors, expression of a single gene
product or protein expression pattern. Surrogate for potency may be developed for both
in vitro and in vivo potency tests.

If the mechanism of action of the medicinal product can be clearly related to specific
antigens (i.e., tumour-specific antigens, tumour-associated antigens), the potency assay
could be based on quantification of these antigens by suitable methods (e.g. flow
cytometry analysis). However, special consideration should be given to the validation of
non-standard methods if used for batch release testing. The possibilities of using
combinations of certain parameters (e.g. viability, cell marker expression, antigen
expression) could be envisaged.

5.3 Viable cell count

One of the requirements included in Directive 2003/63/EC (Annex I, part IV) is that
human somatic cell therapy medicinal products are made of a defined number (pool) of
viable cells. Cell viability is an important parameter of product integrity and may be
used as an in-process control after manipulation of certain cell characteristics e.g.
up-regulation of cell surface expression of specific antigens after cytokine treatment. Cell
viability may also be an important element of the potency of cell based products.
However, it should be linked with other measures of potency that demonstrate the
potential for biological activity of the product, such as quantitative antigen expression or
biological activity as measured in the bioassay.

5.4 Autologous cell based products

For cell based immunotherapy products comprised of autologous cells, sample and time
constraints may hamper complete batch control testing at release. In addition, there may
be an inherent variability within the sourced autologous cell population, which cannot be
fully rectified by the manufacturing process. In this case the use of variable cell
populations may be clinically justified. This variability in cell characteristics could pose
difficulties in validation of the potency assay and in assigning acceptance limits for
potency.

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Nevertheless, whenever a manipulation generates a more homogeneous subpopulation, the
development of an appropriate potency assay should be fully explored, which could
effectively be applied either as a characterisation tool or batch release test, or both. In
this situation, the absence of a suitable potency assay is not accepted without proper
justification, as this will pose difficulties in demonstrating production consistency of
autologous cell preparations after changes in manufacture or product composition have
been implemented.

5.5 Reference preparation

In general, potency assays on biological medicinal products rely heavily on the use of
reference preparations with an established potency. Most likely, no international reference
preparation will be available for highly specific cell based immunotherapy products and
it may be difficult to generate such preparations for autologous products.
‘n-house’reference materials should be characterised in terms of their composition, purity
and biological activity as thoroughly as possible by physicalchemical- biological
methods. The in-house reference material should preferably be clinically qualified
or shown to be comparable to materials shown to be efficacious in clinical trials.

5.6 Adjuvant containing immunotherapy products

There may be cases, where immunotherapy products will require an adjuvant to raise
their low immunogenicity. However, it should be kept in mind that these adjuvants may
exert activities that may interfere with the intended potency assay. For example,
Mycobacterium bovis (bacillus Calmette- Guerin - BCG)5 has been used as an adjuvant
but one of the BCG activities is associated with activation of monocytes/macrophages6.
Where the adjuvant is combined with the active cellular moiety prior to performing the
potency assay and the adjuvant may interfere with the specific biological activity,
special considerations should be given to this issue during assay development.
Compounds that are given separately and/or at a different time point in order to
pre-condition the immune system and that may be needed for biological activity, are not
considered to be adjuvants. As such, those compounds are outside the scope of this
specific section.

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DEFINITIONS

Biological activity:
The specific ability or capacity of the product to achieve a defined biological effect.

Potency:
The measure of the biological activity using a suitably quantitative biological assay (also
called potency assay or bioassay), based on the attribute of the product, which is linked
to the relevant biological properties.

REFERENCES (scientific and / or legal)

1. ICH Topic Q6B, Step 4 Note for Guidance on Specifications: Test Procedures

and Acceptance Criteria for Biotechnological/Biological Products.

CPMP/ICH/365/96 - Adopted March 99.

2. ICH Topic Q5C, Step 4 Note for Guidance on Quality of Biotechnological

Products: Stability Testing of Biotechnological/Biological Products.

CHMP/ICH/138/95 – Adopted Dec. 95.

3 EU Commission Directive 2003/63/EC, Annex I, Part IV: Advanced Therapy

Medicinal Products

4 EMEA/CHMP Note for Guidance on the Quality, Preclinical and Clinical

Aspects of Gene Transfer Medicinal Products. CPMP/BWP/3088/99

5 Mesa C., Fernandez L. Challenges facing adjuvants for cancer immunotherapy.

Immunology and Cell Biology 82 (2004): 644-650

6 Suttmann H., Jacobsen M., Reiss K., Jocham D., Bohle A,. Brandau S.

Mechanisms of bacillus Calmette-Guerin mediated natural killer cell activation. J

Urol. Oct 174 (2004): 1490-1495

7 CHMP Explanatory note on immunomodulators for the guideline on adjuvants

in vaccines for human use. EMEA/CHMP/VWP/244894/2006 – Adopted 27

July 2006,

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 GUIDELINE ON HUMAN CELL-BASED MEDICINAL PRODUCTS

TABLE OF CONTENTS

EXECUTIVE SUMMARY ....................................................................................................... 3

1. INTRODUCTION (BACKGROUND) ................................................................................ 3
2. SCOPE .................................................................................................................................. 3
3. LEGAL BASIS .................................................................................................................... 4
4. MAIN GUIDELINE TEXT ................................................................................................ 4
4.1 RISK ANALYSIS ................................................................................................................... 4
4.2 QUALITY AND MANUFACTURING ASPECTS ........................................................................ 5
4.2.1 Starting and raw materials ............................................................................................ 5
4.2.2 Manufacturing process ................................................................................................... 8
4.2.3 Characterisation ............................................................................................................ 10
4.2.4 Quality control .............................................................................................................. 13
4.2.5 Validation of the manufacturing process ................................................................... 14
4.2.6 Development Pharmaceutics ......................................................................................... 15
4.2.7 Traceability ................................................................................................................... 17
4.2.8 Comparability ................................................................................................................ 17
4.3 NON-CLINICAL DEVELOPMENT ......................................................................................... 18
4.3.1. Pharmacolog y............................................................................................................... 18
4.3.2. Toxicology ..................................................................................................................... 19
4.4 CLINICAL DEVELOPMENT .................................................................................................. 21
4.4.1 General aspects ............................................................................................................. 21
4.4.2 Pharmacodynamics ....................................................................................................... 21
4.4.3 Pharmacokinetics .......................................................................................................... 21
4.4.4 Dose finding studies ..................................................................................................... 21
4.4.5 Clinical Efficacy ............................................................................................................ 22
4.4.6 Clinical Safety ............................................................................................................... 22
4.4.7 Pharmacovigilance and Risk Management Plan ....................................................... 23

REFERENCES (SCIENTIFIC AND / OR LEGAL) .......................................................... 23

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EXECUTIVE SUMMARY
This guideline replaces the Points to Consider on the Manufacture and Quality Control
of Human Somatic Cell Therapy Medicinal Products (CPMP/BWP/41450/98). It takes
into account the current legislation and the heterogeneity of human cell-based products,
including combination products. A risk analysis approach can be used by the applicants
to justify the development and evaluation plans and can be a basis for the preparation
of a risk management plan.
In the quality and manufacturing section, guidance is provided on the criteria and
testing of all starting materials, on the design and validation of the manufacturing
process, on characterisation of the human cell-based medicinal products, on quality
control aspects, on the development programme, traceability and vigilance* and on
comparability issues. Guidance specific to the matrix/device/scaffold component in
combination products is
The guideline acknowledges that conventional non-clinical pharmacology and toxicology
studies may not be appropriate for cell-based medicinal products. Therefore the guideline
addresses which non-clinical studies are necessary to demonstrate proof-of-principle and
to define the pharmacological and toxicological effects predictive of the human response.
Special problems might be associated with the clinical development of human cell-based
medicinal products. Guidance is therefore provided on the conduct of
pharmacodynamic/pharmacokinetic studies, dose finding and clinical efficacy and safety
studies. The guideline describes the special consideration that should be given to
pharmacovigilance aspects and the risk management plan for these products.

1. INTRODUCTION (background)

Rapid development in the fields of biology, biotechnology and medicine has led to the
development of new treatments and highly innovative medicinal products, including
medicinal products containing viable cells. These new cell-based medicinal products have
a high potential in the treatment of various diseases where there is a previously unmet
medical need.
Human cell-based medicinal products are heterogeneous with regard to the origin and
type of the cells and to the complexity of the product. Cells may be self-renewing stem
cells, more committed progenitor cells or terminally differentiated cells exerting a
specific defined physiological function. Cells may be of autologous or allogeneic origin.

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In addition, the cells may also be genetically modified. The cells may be used alone,
associated with biomolecules or other chemical substances or combined with structural
materials that alone might be classified as medical devices (combined advanced therapy
medicinal products).

2. SCOPE

This multidisciplinary guideline will address development, manufacturing and quality

control as well as non-clinical and clinical development of cell-based medicinal products
(CBMP) including somatic cell therapy medicinal products as defined in Directive
2001/83/EC, Part IV, Annex I1 and tissue engineered products as defined in Regulation
1394/2007/EC2. This guideline is intended for products entering the Marketing
Authorisation (MA) procedure. However, the principles laid down in the guideline
should be considered by applicants entering into clinical trials.
Cell-based medicinal products discussed in this document have the following
characteristics:
- They contain viable human cells of allogeneic or autologous origin undergoing a
manufacturing process;
- They may be combined with non-cellular components;
- The cells may be genetically modified. The present document applies only to the
cellular component of the cell based medicinal products containing genetically
modified cells.

Although this document does not cover non-viable cells and cellular fragments
originating from human cells, the underlying scientific principles may be applicable.

This guideline does not cover xenogeneic cell-based medicinal products.

3. LEGAL BASIS

This guideline should be read in conjunction with the introduction and general principles
(4) and part 4 of the Annex I to Directive 2001/83/EC1 as amended and the Regulation
on Advanced Therapy Medicinal Products 1394/2007/EC2.
Also, donation, procurement and testing of cells from human origin must comply with

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overarching Directive 2004/23/EC4 and technical directives drawn from it, Directives
2006/17/EC5 and 2006/86/EC6.

4. MAIN GUIDELINE TEXT

4.1 Risk analysis

The risk posed by the administration of a cell-based medicinal product is highly

dependent on the origin of the cells, the manufacturing process, the non-cellular
components and on the specific therapeutic use. The variety of cell-based medicinal
products can lead to very different levels of risks for the patients, the medical personnel
or the general population. Therefore the development plans and evaluation requirements
need to be adjusted on a case by case basis according to a multifactorial risk based
approach (see Annex I to Directive 2001/83/EC2).

At the beginning of the product development, an initial risk analysis may be performed
based on existing knowledge of the type of product and its intended use. This should
be updated by the applicant throughout the product life cycle as data are collected to
further characterise the risk.

The comprehensive risk analysis should be used to justify the product development. It
should also serve as a basis for the preparation of a risk management plan in
accordance with the guideline on risk management systems for medicinal products for
human use (EMEA/CHMP/96268/2005)7. In particular, the results of the comprehensive
risk analysis should be used:

• to identify risk factors associated with the quality and safety of the product;
• to determine the extent and focus of the data required during non-clinical and
clinical development;
• when establishing the need for risk minimisation activities;
• when determining the post market risk management activities specified

in the pharmacovigilance plan.

The following general risk criteria (non-exhaustive) can be used in the estimation of the

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overall risk of the product:
• origin (autologous-allogeneic);
• ability to proliferate and/or differentiate;
• ability to initiate an immune response (as target or effector);
• level of cell manipulation (in vitro/ex vivo expansion/activation/ differentiation
/genetic manipulation/ cryo-conservation);
• mode of administration (e.g. ex vivo perfusion, local or systemic surgery);
• duration of exposure or culture (short to permanent) or life span of cell;
• combination product (cells and bioactive molecules or structural materials);
• availability of clinical data on or experience with similar products.

4.2 Quality and manufacturing aspects

This part of the guideline describes activities by manufacturers following procurement of

the cells and tissues. The manufacture of cell-based medicinal products should be in
compliance with the principles of good manufacturing practices, as set out in Directive
2003/94/EC8 and its Annex 29.

The active substance of a cell-based medicinal product (CBMP) is composed of the

engineered (manipulated) cells and/or tissues. Additional substances (e.g. scaffolds,
matrices, devices, biomaterials, biomolecules and/or other components) when combined as
an integral part with the manipulated cells are considered part of the active substance
and are therefore considered as starting materials, even if not of biological origin.

CBMP often contain, or consist of cell samples of limited size and many are intended
to be used in a patient-specific manner. This can raise specific issues pertaining to
quality control testing designs for each product under examination. Since this document
covers a variety of CBMPs, processes involved can vary from very simple to highly
complex. For certain CBMPs, the starting material, the active substance and the finished
product can be closely related or nearly identical. For such products, some requirements
listed below could be inadequate and in those cases only relevant sections and items
should be addressed.

4.2.1 Starting and raw materials

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The manufacturing process of CBMP usually does not include terminal sterilisation,
purification steps, viral removal and/or inactivation steps. Therefore, stringent sourcing
requirements and acceptance criteria for all materials derived from human or animal
origin should be adequately defined according to their intended use.

4.2.1.1. Cells

Donated cellular material from single or pooled donors, once processed (see 4.2.2.1)
may be:
• A single primary cell isolate used directly for the CBMP;
• Primary cells cultured for a few passages before being used for the CBMP;
• Cells based on a well-defined cell bank system consisting of a master cell
bank and a working cell bank.

An adequately controlled cell storage system should be established to allow proper

maintenance and retrieval of cells without any alteration of their intended final
characteristics. Storage conditions should be optimised to ensure cell viability, density,
purity, sterility and function. Identity should be verified by relevant genotypic and/or
phenotypic markers and the proportion of cells bearing these identity markers evaluated
as an indicator of the intended cell population.

A. Cells of primary origin

The specific requirements for donation, procurement and testing laid down in Directive
2006/17/EC5 shall be met.

Procedures and standards employed for the selection of appropriate donors and the
exclusion of high-risk or otherwise unsuitable candidate donors should be clearly
delineated and justified. If it is necessary to pool cells from different donors, the risk
analysis should address the possibility that pooling of allogeneic cell populations may
increase the risk of undesired immunological responses in the recipient and compromise
its therapeutic activity. In addition, pooling of cells may increase the risk of disease
transmission. Depending on the nature of the source of the cells and tissues, other risk

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factors, e.g. previous radiation exposure, should be also considered and addressed.

On receipt of the cells for use in a medicinal product, a specific microbiological

screening programme should be in place, adapted to the type of cells, with validated
assays capable of detecting human infectious agents with appropriate sensitivity and
taking into consideration the medium components that might interfere with the assays
(e.g. antibiotics). When cells originate from non-healthy tissues, the product specific
acceptance criteria should be defined according to the intended use.

Quality parameters aimed at the definition of acceptance criteria for a given organ or
tissues should be specified, taking into consideration general aspects such as shipment
and storage conditions.

In the case of autologous donation, the testing regimen of the starting material should
be justified, taking into account the autologous use.

Where allogeneic primary cells are collected and expanded for use in multiple patients,
the cell lot should be appropriately characterised. The same characterisation programme
shall be applied to each new cell lot.

B. Banking system for established cell lines

Where cell lines are used, an appropriately characterised Master Cell Bank (MCB) and
Working Cell Bank (WCB) should be established, whenever possible. Cell banking and
characterisation and testing of the established cell banks should comply with the ICH
guideline Q5D10.

4.2.1.2. Other materials, reagents and excipients

Various materials are needed for collection, selection, culture or even genetic or
phenotypic modification of cells, such as other cells, enzymes, antibodies, cytokines, sera
and antibiotics. Exposure to such materials can also impact on the quality, safety and
efficacy of the final therapeutic product. As a consequence, each substance used in the
procedure should be clearly specified and evaluated as to its suitability for the intended

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use. The microbial purity and low endotoxin level of these materials should be ensured.

Materials, including cells that function as support for growth and adhesion e.g. feeder
cells should be evaluated and/or validated as to their suitability for the intended use.

The quality of biologically active additives in culture media such as growth factors,
cytokines and antibodies, should be documented with respect to identity, purity, sterility
and biological activity and absence of adventitious agents. It is recommended to keep
the use of such materials to a minimal and to avoid the use of reagents with
sensitisation potential e.g. β-lactam antibiotics.

For viral safety aspects, the guidelines on viral safety11, 12

and Eudralex vol. 2B13 should
be taken into consideration. The principles laid down in the general text of the
European Pharmacopoeia on viral safety14 should be followed for every substance of
animal and human origin that is used during the production.

Measures should be taken to reduce the risk of transmissible spongiform encephalopathy

according to the relevant European legislation and guidelines15.

Where appropriate, the Note for Guidance on the “Production and quality control of
medicinal products derived by recombinant DNA technology”16 and the Note for
Guidance on the “Production and quality control of Monoclonal Antibodies”17 should be
taken into account.

When the raw materials, reagents and/or excipients have a marketing authorisation or
mentioned in a Pharmacopoeia, appropriate references may be given.

The following information must be added for materials of human or animal origin:

A. Human derived materials

Reagents of human origin (e.g. albumin, immunoglobulins) should be evaluated for their
suitability in a manner identical to that employed for plasma-derived products as
recommended in the CPMP Note for guidance on plasma-derived medicinal products18.
The use of synthetic alternatives should be investigated. If serum is required in the

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culture media, the use of serum isolated from the same individual who donated the cells
is preferred, where possible, to alternate allogeneic serum.

B. Animal derived material

Where cells or tissues of animal origin are used e.g. as supportive cells, the guidance
given in “Points to consider on Xenogeneic Cell Therapy Medicinal Products”19 should
be followed.

Animal derived reagents may harbour infectious agents and may increase undesirable
immunological responses in the recipient. When applicable, the use of animal reagents
should be avoided and replaced by non animal derived reagents of defined composition.

When bovine serum is used, the recommendations of the Note for Guidance on the
“Use of Bovine Serum in the Manufacture of Human Biological Medicinal Product”20
should be followed. The use of irradiated sera and/or alternative synthetic media is
encouraged and should be considered.

For viral safety testing of materials of other animal species, the table of extraneous
agents to be tested for in relation to the general and species-specific guidelines on
production and control of mammalian veterinary vaccines21 and Note for Guidance on
Production and Quality Control of Animal Immunoglobulins and Immunosera for Human
use22 should be consulted.

C. Special considerations

Special recommendations for the starting materials of cell-based Gene Therapy Medicinal
Products

When the cells in the active substance are genetically modified, the “Note for Guidance
on the quality, preclinical and clinical aspects of gene transfer medicinal products”23
should be followed, which gives details on the quality control, characterisation and
preclinical testing of gene transfer vectors. Cell populations which are transformed
should be assayed for appropriate and reproducible expression of the newly acquired

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characteristics. Special attention should be paid to the level and length of expression
and quality of the gene product(s) produced by the cells. As far as applicable and
practicable, the new characteristics of the cells should be quantified and controlled.

Special recommendations for matrix/device/scaffold components of combined products

Cell-based medicinal products may incorporate structural components which

independently are medical devices or active implantable medical devices. Those devices
should meet the essential requirements laid down in Directive 93/42/EEC24 concerning
medical devices and Directive 90/385/EEC25 on the approximation of the laws of the
Member States relating to active implantable medical devices, respectively, and this
information shall be provided in the marketing authorization application. In the case
where a Notified Body has evaluated the device part, the result of this assessment shall
be included in the dossier. Cell-based medicinal products may also incorporate structural
components which are not identical to, or used in the same way as in a medical
device. All structural components should be appropriately characterised and evaluated for
their suitability for the intended use (See sections on Characterisation and Development
Pharmaceutics).

Any matrices, fibers, beads, or other materials that are used in addition to or in
combination with the cells should be described and their function underpinned by means
of chemical, biological, physical (e.g. structure and degradation) and mechanical
properties. Inclusion of additional bioactive molecules should also be described and their
impact should be evaluated.

4.2.2 Manufacturing process

The manufacturing process of cell-based medicinal products should be carefully designed

and validated to ensure product consistency. The requirements should be defined and
justified.

A detailed description of the manufacture of the active substance and of the finished
product should be provided. The type of manipulation(s) required for cell processing and

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the physiological function of the cells shall be described. A flow diagram of the entire
process starting from biological fluid/tissue/organ or from cell banks should be prepared
indicating critical steps and intermediate products (e.g. intermediate cell batches), as well
as operating parameters, in-process controls and acceptance criteria. Manufacture of
combined medicinal products consisting of cells and matrices/devices/scaffolds, require
additional consideration regarding the cell-matrix/scaffold interactions and quality issues
raised there from. Attention should be paid to biodegradable materials, which may
possess the potential for environmental changes (e.g. raising pH) for the cells during the
manufacture or after administration.

Information on procedures used to transport material during the manufacturing process of

the product, including transportation and storage conditions and holding times, should be
provided.

The manufacturing area should be physically separated from the procurement area If
different tissues and cellular products are processed and stored in the same
manufacturing area there is an increased risk of cross contamination during each step of
the procedure, e.g. via processing equipment or in storage containers such a liquid
nitrogen tanks, and therefore, adequate control measures to prevent cross-contamination
should be put into place.

Equipment and premises used for manufacturing of CBMP should be suitable and
qualified for aseptic production. It is recommended that dedicated, product-specific or
single-use equipment are used in the production, whenever possible.

1. Cell preparation procedures

All cell preparation procedures should be justified in terms of their intended purpose.

Inappropriate handling and improper processing of cells/tissues must be avoided as they

can impair or destroy the integrity and/or function of the cells and thus result in
therapeutic failure. Microbiological control is a pivotal aspect of process control and
quality evaluation of all cell preparations. Monitoring of in vitro cell culturing at
selected stages of the production should be performed where feasible. The culture should
be examined for any microbial contamination in accordance with the culturing procedure

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and growth characteristics of the cells.

After appropriate controls have been performed / implemented, the biological fluid /
tissue /organ can undergo one or more of the following steps:

Organ/tissue dissociation

The procedure to obtain the cells from the organ/tissue has to be described (with
respect to the type of enzyme, media, etc.) and validated. Consideration should be given
to the degree of disruption applied to the tissue in order to preserve the intended
functional integrity of the cellular preparation and to minimize cell-derived impurities in
the product (cell debris, cross contamination with other cell types).

Isolation of the cell population of interest

Any procedure used to isolate and / or purify the cell population of interest should be
described. Its effectiveness should be addressed in relation to the intended use and the
method(s) should be validated.

Cell culture

During in vitro cell culture, consideration should be given to ensure acceptable growth
and manipulation of the isolated cells. The processing steps should be properly designed
to preserve the integrity and control the function of the cells. The procedures for any
manipulation should be documented in detail and closely monitored according to specific
process controls. The duration of cell culture and maximum number of cell passages
should be clearly specified and validated. The relevant genotypic and phenotypic
characteristics of the primary cell cultures, of the established cell lines 10 and the
derived cell clones should be defined and their stability with respect to culture longevity
determined. Consistency/repeatability of the cell culture process should be demonstrated
and the culture conditions including the media and the duration should be optimised
with respect to the intended clinical function of the cells.

Special consideration should be given to the growth potential of cells in response to

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growth factors since cell subpopulations may gain a growth advantage under defined in
vitro culturing conditions.

Cell modification

Various treatments (physical, chemical or genetic) can be applied to cells. The method
used to modify the cells should be fully described. In the case of genetic modification
of cells, requirements set up in the Note for guidance on Quality, preclinical and
clinical aspects of gene transfer medicinal products23 should be followed.

Cells cultured in or on a matrix/device/scaffold

If the cells are grown directly inside or on a matrix/device/scaffold, the quality of the
combined advanced therapy medicinal product relies predominantly on the properly
controlled manufacturing process. For such products, the cell culture process has to be
thoroughly validated and the effect of the device on the cell growth, function and
integrity has to be taken into account. The effect that the cells may exert on the device
(e.g. on rate of degradation) should also be considered (see also 4.2.6. Development
Pharmaceutics).

2. In-process controls

The manufacturing process needs to be controlled by several in-process controls at the

level of critical steps or intermediate products. Intermediate cell products are products
that can be isolated during the process; specifications of these products should be
established in order to assure the reproducibility of the process and the consistency of
the final product. Tests and acceptance criteria should be described. If storage occurs, it
is necessary to validate the storage conditions (e.g. time, temperature).

3. Batch definition

The purpose of the batch definition is to ensure consistency and traceability. A clear
definition of a production batch from cell sourcing to labelling of final container should
be provided (i.e. size, number of cell passages/cell duplications, pooling strategies, batch

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numbering system). In the autologous setting, the manufactured product should be
viewed as a batch.

4. Container and closure system

A description of the container closure system should be provided. Compatibility with the
product should be demonstrated. It should be indicated if the container closure per se
has a CE marking under the Medical Devices Directive 93/42/EEC24. Information on the
sterilisation procedures of the container and the closure should be provided.

The choice of packaging materials should be addressed as part of the development

pharmaceutics. Additional data may be required if packaging components are used in the
transport and/or application procedure.

4.2.3 Characterisation

The characterisation of a CBMP should encompass all the components present in the
finished product. Characterisation may prove particularly challenging for products
containing cells together with matrices, scaffolds and innovative devices. Characterisation
data are likely to be necessary for single components as well as for the combined final
product. Characterisation data could encompass data obtained throughout the development
and/or manufacturing process. It should be noted that in a combined product the
characteristics of both the cellular and the non-cellular components may be altered by
the process of integration.

An extensive characterisation of the cellular component should be established in terms

of identity, purity, potency, viability and suitability for the intended use, unless justified.
The expected biological function of a CBMP encompasses complex interactions that may
range from a biochemical, metabolic or immunological action to the structural
replacement of damaged tissue or organ. Therefore, the requirements for a complete
characterisation of the active substance in terms of biological function could be very
taxing. Moreover the specific mechanism of action is often difficult to pinpoint to
specific molecular entity but it is more dependent on the functionality of the cellular

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components acting in a “tissue-like” fashion as a whole. Therefore, when considering
the extent of characterisation, the following issues should be taken into account: i)
autologous cells vs. allogeneic cells, ii) extensively or minimally manipulated in vitro,
iii) immunologically active or neutral, iv) proliferative capacity of the cells, v) cell-like
or tissue-like organisation and dynamic interactions amongst cells and with the structural
component, vi) intended use.

Non-cellular components should be characterised in the context of their required function

in the finished product. This includes structural components designed to support the
cellular components such as scaffolds or membranes which should be identified and
characterised in chemical and physical terms such as porosity, density, microscopic
structure and particular size according to the type of substances and intended use
according to EN/ISO 10993-1826 and EN/ISO 10993-1927.

The characterisation should be designed to allow setting up the routine controls that will
be applied for release of the active substance and finished product as well as those to
be performed at several steps of the process to guarantee batch consistency.

If biologically active molecules (e.g. growth factors, cytokines etc.) are present as
components of the cell-based products, these have to be described adequately and their
interaction with the other components of the product and the surrounding tissues after
administration should be characterised. This should involve an appropriate range of in
vitro and where necessary in vivo methods.

1. Identity

Cellular Component

The identity of the cellular components, depending on the cell population and origin,
should be characterised in terms of phenotypic and/or genotypic profiles.

When addressing the phenotype of the cells, relevant markers could be used, where
justified. These markers may be based on gene expression, antigen presentation,
biochemical activity, response to exogenous stimuli, capability to produce biologically

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active or otherwise measurable molecules, etc. For adherent cells, morphological analysis
may be a useful tool in conjunction with other tests. Where applicable, a description of
the procedures which could lead to a modification of the characteristic of the product,
including adhesion, absorption, degradation, presentation of components of the culture
media, should be provided.

For cellular components of allogeneic origin, identity should include histocompatibility

markers, where applicable, and identification of genetic polymorphisms with specific
reference to the intended use.

Non-cellular Components of the active substance

All non-cellular components should be appropriately characterised as such and identity

parameters established.
Should the finished product contain a distinct active substance in addition to the cellular
component, then that active substance should be characterised with respect to identity in
accordance to relevant CHMP guidelines, depending on the nature of the active
substance, whether it be of chemical or biological origin.
Structural components designed to support the cellular components such as scaffolds or
membranes should be identified and characterised with respect to their composition and
structural characteristics.

Combination Products

In a combination product the active substance may be formed by the integration of

cellular and non-cellular components to form a single entity. In such a case the identity
of both the cellular and the non-cellular components may be altered by the process of
combination. Consequently a distinctive way to define identity should be established for
the components in the combination, unless justified.

2. Cell purity

The cellular population of interest could contain other cells that are of different lineages
and/or differentiation stage or that may be unrelated to the intended population.

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Where a specific cell type is required for the indication, the unwanted cells should be
defined and their amount in the final product should be controlled by appropriate
specifications, i.e. acceptance criteria for the amounts of contaminating cells should be
set.

In cases, where the desired biological activity and efficacy of the product requires a
complex mixture of cells, the cell mixture needs to be characterised and its composition
controlled by appropriate in-process controls and release testing.

Irrespective of the cell type, the cell population can be contaminated with non-viable
cells. Since cell viability is an important parameter for product integrity and directly
correlated to the biologic activity, the ratio between non-viable and viable cells should
be determined and specifications should be set.

3. Impurities

Product or process-related

During the production of a CBMP, variable amounts of impurities, product- and

process-related, may be introduced into the final product. Any reagents known to be
harmful in humans should be analysed in the final product (or in individual components
if otherwise not possible) and acceptance criteria should be set. The specification limits
should be justified by levels detected in batches used for toxicological and/or clinical
studies.

Any material capable to introduce degradation products into the product during the
production, e.g. biodegradable materials, should be thoroughly characterised in this
respect and the impact of the degradation products to the cell component(s) should be
addressed.

If genetically modified cells are used in the product, any additional proteins expressed
from the vector, e.g. antibiotic resistance factors, selection markers, should be analysed
and their presence in the product should be justified.

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Adventitious agents
A critical aspect is to establish that CBMP are free from adventitious microbial agents
(viruses, mycoplasma, bacteria, fungi). The contamination could originate from the
starting or raw materials (see above), or adventitiously introduced during the
manufacturing process. A risk assessment should be performed to evaluate the possibility
of reactivation of cryptic (integrated, quiescent) forms of adventitious agents. A thorough
testing for the absence of bacteria, fungi and mycoplasma shall be performed at the
level of finished product. These tests should be performed with the current
methodologies described in the European pharmacopoeia for cell based products28. In
cases where the short shelf life of the CBMP is prohibitive for the testing of absence
of bacteria under the Eur Ph. requirements, alternative validated testing methods may be
acceptable, if justified.

4. Potency

It is strongly recommended that the development of a suitable potency assay be started

as soon as possible. Preferably, a suitable potency assay should already be in place
when material for the first clinical trial is produced and it should be validated prior to
pivotal clinical trials unless otherwise justified. Lot release and shelf life specifications
for potency should be determined and amended during product development, if
appropriate.

According to the ICH guideline 6QB29, potency is the quantitative measure of biological
activity based on the attribute of the product, which is linked to the relevant biological
properties. The assay demonstrating the biological activity should be based on the
intended biological effect which should ideally be related to the clinical response.

Basically, two types of potency assays can be envisioned: 1) in vitro assays using cell
systems and 2) in vivo assays using animal models. Major cellular functions as viability,
self renewal, death and differentiation are pivotal to the quality, function and
sustainability of the CBMP and may need to be monitored during production and at
release using surrogate markers and appropriate technology (e.g. gene expression profiles
by microarrays, flow cytometric immunofluorescent analysis, cell cloning, PCR and many
others). In vivo assays for potency may also be useful especially when experimental

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animal models are available.

Markers for purity and markers for potency should not be mixed in the same assay. .

Reference is made to the Guideline on “Potency testing of cell-based immunotherapy

medicinal products for the treatment of cancer”30. Although this guideline focuses on
cell-based immunotherapy medicinal products, the principles, including on reference
preparations, apply for all CBMP. As described in the referred guideline, a combination
of multiple methods may be needed to adequately define the potency of these products
during the development. Certain assays may be needed to control process changes,
whereas others are more suitable for release testing.

Tissue repair and regeneration

An in vivo test can either be performed in an animal model mimicking the intended
clinical tissue repair/ regeneration or can otherwise be based on the mode of action
(e.g. an ectopic model). An in vitro assay can be based on the expression of markers
that have been demonstrated to be directly or indirectly (surrogate markers) correlated to
the intended biological activity, such as cell surface markers, activation markers,
expression pattern of specific genes. Also a physiological response under defined
conditions such as differentiation in specific cell types and/or secretion of tissue specific
proteins (e.g. extracellular matrix components) can be used as a basic principle for a
potency test. The manufacturers should, however, ensure that the method of
characterisation is relevant for the intended biological effect in vivo.

The potency assay should be performed by using a specified number of cells and, when
possible, quantified against a qualified reference preparation. The potency should be
defined as the required time to obtain a predefined effect (e.g. restoration of function or
repair of anatomical structure) or the potency is calculated from the measured effect in
a defined time period.

Metabolic or pharmacological activity

Cells contained in a CBMP can be chemically treated or genetically modified in vitro to

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express certain desirable proteins like growth factors, cell surface antigens or other
molecules in order to sustain the biological response as long as needed in the new
microenvironment. Therefore, the potency assays to be developed should be able to
assess the activity-related aspects of the active substance that may be composed not
entirely of intact viable cells but also of other components.
If the intended biological function of the CBMP is mainly based on the capacity of
cells to secrete specific molecule(s) e.g. to repair a metabolic disorder, to promote
growth, to release a metabolite, then its potency assay will be based on the detection of
the active molecule(s) produced and the biological activity expected. This can be carried
out by conventional reliable qualitative and quantitative analytical methods (protein
analysis, nucleic acid identification, HPLC chromatography etc.). The same molecule can
be also assessed for function in animal model systems assuming that the active
substance is released from the cell-based medicinal product into biological fluids
(plasma, CSF, urine or interstitial fluid).

Immunotherapy

Potency assays of cell-based medicinal products intended for immunotherapeutic use will
be based on complex immune mechanisms which may be complicated by multi-antigen
formulations and inherent variability of the starting material. Special guidance for
cell-based immunotherapy medicinal products is provided in Guideline on “Potency
testing of cell-based immunotherapy medicinal products for the treatment of cancer”30.

5. Tumourigenicity

The tumourigenicity of CBMP differs from the classical pharmaceutics as the

transformation can also happen in the cellular component of the product (e.g.
chromosomal instability) and not only in the treated individual. If the risk of cellular
transformation and subsequent potential for tumourigenicity can be foreseen, the cellular
components should be evaluated for their tumourigenic potential by analysing e.g. their
proliferative capacity, dependence on the exogenous stimuli, response to apoptosis stimuli
and genomic modification. Testing of chromosomal integrity and tumourigenicity of cells
derived from a cell culture / cell banking system will be required. Reference is made to
the ICH Q5D10 and to the Ph. Eur. Monograph on cell substrates for the production of

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vaccines for human use31.

4.2.4 Quality control

For proper quality control, the active substance and/or the final product should be
subjected to release testing, whenever possible. If justified, it would be acceptable to
have reduced testing at one level provided an exhaustive control is performed at the
other. All release testing should be performed using methods validated at the latest at
the time of submission of an application.
1. Release criteria
The release specifications of the active substance and finished product should be
selected on the basis of parameters defined during the characterisation studies. Selection
of tests is product-specific and has to be defined by the manufacturer.
Specifications for release testing should include identity, purity, potency, impurities,
sterility, potency, cell viability and total cell number, unless otherwise justified. If the
structure is an essential characteristic of the product, the structural characteristics of the
active substance or finished product shall be defined and justified. In case the primary
function of the CBMP is the excretion of specific proteins, specifications regarding these
excreted proteins should be set.
If certain release tests cannot be performed on the active substance or finished product,
but only on key intermediates and/or as in-process tests, this needs to be justified. In
these cases an adequate quality control has to rise from the manufacturing process,
supported by the results of the clinical studies. These exceptions may include the
following:

§ Some release tests might not be feasible on the combined components of the
active substance/ finished product for technical reasons.
§ A complete release testing cannot be finalised before the product is
administered to the recipient due to time restrictions (e.g. in case of
autologous products, which are administered immediately after completion of
the production and initial testing). However, a critical set of essential tests
that can be performed in the limited time prior to clinical use must be
defined and justified. Whenever feasible, retention samples should be stored
for future analysis.

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 § The amount of available product is limited to the clinically necessary dose
(e.g. due to very limited cell numbers at collection or low proliferation rates).
The release of the product should be justified by the validation of the cell
manipulation process and the in-process controls.

2. Stability testing

A shelf life for the cells under specified storage conditions shall be determined for the
following materials: i) all intermediates subject to storage if applicable, ii) components
of the combined CBMP, iii) the active substance, iv) the finished product. Furthermore,
a valid in-use shelf life (after opening from the transport container) should be assigned
to the CBMP. Also, all storage conditions including temperature range should be
defined. Transportation and storage conditions should be supported by experimental data
with regard to the maintenance of cell integrity and product stability during the defined
period of validity. If relevant, appropriate methods for freezing and thawing should be
documented.

Due to the complex nature of the active substance of a CBMP, requirements for
stability should be defined on a case-by-case basis. Whenever possible, stability should
be assessed for both the cellular as well as the non-cellular component prior to
combination and together as a finished product in the final packaging.

3. Special quality requirements for cell-based medicinal products containing genetically

modified cells

If cells have been genetically modified, quality control must be performed in compliance
with guidance available on gene transfer medicinal products23. This information is in
addition to control of the cells according to the guidance presented elsewhere.

4. Special quality requirements for combination products

Specifications for structural components of the product shall be defined. Impurities and
degradation product that originate from the structural component (matrix, scaffold,

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device) shall be described and specifications for the relevant impurities should be set.
Testing of the structural/mechanical properties and biological activity with reference to
the anticipated conditions for use and potential for degradation may be difficult to
conduct as part of release testing. Thus, it is anticipated that these parameters could be
explored through proper testing of raw materials and characterisation studies of the final
product. In extremely limiting conditions (e.g. for autologous products with small cell
numbers), the analysis of structural/functional characteristics of a combination product
may necessitate the development of a model product composed of same non-cellular
components combined with cell component(s) of equal characteristics but with proven
availability.

4.2.5 Validation of the manufacturing process

The entire manufacturing process, including cell harvesting, cell manipulation processes,
maximum number of cell passages, combination with other components of the product,
filling, packaging, transport, storage etc., should be validated. Validation of the
production process of a combined product should encompass all steps from separate
components up to the final combination to ensure consistent production.

It should be demonstrated that each step of the manufacturing process of the active
substance, supportive components and final product is well controlled. The selection and
acceptance criteria of the operational parameters and the in-process controls should be
justified. Putative variability, related to starting materials and biological processes, should
be taken into account in the validation. Furthermore, the critical points of the
manufacturing process should be defined and validated, especially the aseptic processing.
Any preservation steps, holding periods and/or transportations of the active substance,
final product, supportive structures or intermediate products during the manufacturing
process should be validated.

In case of limited sample sizes (e.g. autologous preparations for one single
administration), it is recommended that a more extensive validation is performed with
cell preparations of comparable characteristics but available in sufficient amounts for
validation purposes. It is recommended that validation of such a manufacturing process
is performed depending on the product characteristics, for adventitious agents, identity,

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potency, viability, purity/impurities and other product specific parameters.

4.2.6 Development Pharmaceutics

The general principles set out in Note for Guidance on Development Pharmaceutics for
Biotechnological/Biological Products32 can be applied to human CBMP. The potential
complexity of composition and the dynamic nature of a product containing living cells
will result in very specialised pharmaceutical and biopharmaceutical requirements for
each development programme from the individual cell components into the final product.

1. Cellular Components

The development programme should address the choices of materials and processes to
be used in production. This should be addressed from the point of view of the
biological/therapeutic function, the maintenance and the protection of the cell population.

Integrity of the cellular component is most critical for the CBMP and must be assessed
by the ability of cells to survive, and maintain the genotype or phenotype needed for
the intended functions. However, detection of possible changes in cellular nature that
may influence the intended function, can be feasible by analysis of cellular surface
antigens, proteomics and functional genomics analysis (e.g. microassay for gene
expression profile, flow cytometry etc.). Cell viability can be easily assessed in culture
by employing widely applied assays. For combined products, where structural
components are an integral part of the active substance, such assays may be more
difficult to apply. Alternative approaches could be sought such as combination of other
suitable assays (e.g. detection of pH and O2 / CO2), where needed.

The ability of cells to continue to produce or express products should be evaluated as

part of the stability programme. Such stability studies should be carried out as long as
the defined period of validity requires.

2. Non-Cellular Components

A CBMP may contain non-cellular components, such as biomaterials, bioactive

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molecules, proteins or chemical entities. These may supply structural support, suitable
environment for growth, biological signalling or other functions. They may also be used
during the ex vivo manipulation process.

Matrices, scaffolds, devices, biomaterials or biomolecules which are not an integral part
of the active substance, are considered as excipients of the finished product. For
excipient(s) used for the first time in combination with cells and/or tissues, the
requirements for novel excipients, as laid down in part I, Annex I of Directive
2001/83/EC1, do apply. Conventional excipients should also be characterised with respect
to their combination with cells.
Information on the choice of excipients, their properties, their characteristics and the
design and testing of a final scaffold/matrix should be provided in the dossier as part
of the development pharmaceutics.
Should the finished product contain components that act to modify the delivery or
ensure the local retention of cells after administration, the scientific rationale should be
provided and supported by adequate development data. The evaluation of individual
non-cellular components is required although aspects of this evaluation may be
incorporated into studies designed to assess the product as a whole. Where the safety of
a non-cellular component has previously been established for other applications, for
example in support of the approval of a particular material for a medical device or
medicinal product application, elements of that evaluation may be applicable to an
evaluation of its safety and suitability when used in a cell-based medicinal product, if
justified.
The relevance of the structural and functional characteristics of the non-cellular
components in a combination product should be discussed. Interaction of the cellular
component and any additional non-cellular components with the device should be
evaluated and the development and characteristics of the combined product as a whole
should be presented.
Tissue differentiation and functionality are highly dependent on the local environment
and thus on the choice of biomaterials and cell signalling biomolecules (e.g. growth
factors). Therefore, studies should be carried out to verify critical aspects of the
character and performance of biomaterials and other non-cellular components used in the
CBMP, for example biocompatibility and mechanical strength.
In particular, to confirm that the properties of a biomaterial permit the growth and

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proper function of the tissue/cells with which it is in contact and support the overall
performance of the product, assurance should be provided in relation to the following:

- absence of components or leachables that might be toxic to cell growth

and/or to the intended performance;
- characterisation of features (e.g. topography, surface chemistry, strength)
critical to structural support, optimisation of viability and cellular growth or
other functional characteristics;
- biocompatibility of the structural material with the cells or tissues to confirm
that the system maintains the desired cell differentiation, functionality and
genotype during production and until use;
- release kinetics and/or rate of degradation of any bioactive molecules, to
verify that they are appropriate for the achievement of the intended effect.

To establish biocompatibility, it is necessary to specify the nature of biological

responses that a biomaterial is required to elicit from the host tissue or cell-based
components, and to provide evidence that the desired tissue response is achieved in a
relevant model.

The stability of the non-cellular components should be assessed in the presence and
absence of cellular components in order to determine whether the non-cellular
component undergoes degradation, or physico-chemical alterations (e.g. aggregation,
oxidation) that may impact on the quality of the product by affecting cellular behaviour
and survival. The effect of the cellular component or of the surrounding tissues on the
degradation (rate and, if appropriate, products) or stability of the structural component
should be assessed, considering also the effect of the non-cellular components throughout
the expected lifetime of the product. The general principles that are applied to the
biological evaluation of medical devices can also be applied to the evaluation of
biomaterials intended for use in CBMP. Such an evaluation involves a programme of
characterisation, testing and review of existing data to assess the potential for an
adverse biological reaction to occur as a result of exposure to the biomaterial. These
principles are set out in international standard ISO 10993 Part 133. Other parts of the
ISO 10993 series of standards specify methods that may be relevant to the assessment
of material characteristics, biological safety and degradation of biomaterials used in

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cell-based medicinal products. Additional studies (e.g. cell adhesion studies, growth
studies) may be necessary to demonstrate aspects of biocompatibility specific to
cell-based applications.

3. Final Product

Once the “formulation”, i.e. the delivery system of a combined product has been
established, the parameters for determining the role of constituents and appropriateness
of composition should be presented within a justification of the composition of the
product.

The key parameters for performance testing of the completed product should be justified
in relation to the development data and the final quality requirements. It may be
appropriate that in vitro and in vivo testing of the formulation/delivery system/combined
product during development are included.

4.2.7 Traceability

A system allowing complete traceability of the patient as well as the product and its
starting materials is essential to monitor the safety and efficacy of cell-based medicinal
products. The establishment and maintenance of that system should be done in a way to
ensure coherence and compatibility with traceability and vigilance requirements laid
down in Directive 2004/23/EC4 and in Directives 2006/17/EC5 and 2006/86/EC6 and Art.
15 of the Regulation (EC) No 1394/20072.

Article 15 of the Regulation (EC) No 1394/20072 defines a two tiered system connecting
the required traceability from cell donation and procurement (Dir 2004/23/EC) to the
manufacturer and user (hospital or practice). This means that anonymity can be
guaranteed. At the tissue establishment there has to be a link between the donor and
the donation. At the manufacturing side, there has to be a link between donation and
product and at the hospital/practice side there has to be a link between the product and
the recipient. The systems should allow full traceability from the donor to the recipient
through anonymous coding systems. Manufacturers should establish their coding systems
in a rational way, building from the coding system of the tissue establishment, and

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designing it to facilitate the tracing of the donation to the product and to the patient.
Bar coding and peeling labelling systems could be suitable tools for the purpose of
patient management.

4.2.8 Comparability

Development of a cell-based medicinal product may encompass changes in the

manufacturing process that might have an impact on the final product. Given the
complex and dynamic nature of CBMP it is particularly important that all stages of
development are fully evaluated and tracked within the dossier. This is especially
significant once clinical studies have commenced. Data on the behaviour and
characteristics of developmental prototypes should be retained as it could provide
background information relevant to the evaluation of the final product. During the
pivotal clinical studies changes should not be introduced to the manufacturing process
and the final product.

Materials used in the clinical studies should be sufficiently characterised in order to

allow the demonstration of consistency in the production. The manufacturers should
consider the critical parameters drawn from the characterisation of their product to
establish the analytical tools necessary for the required comparability studies throughout
development. Comparability studies with the product resulting from those changes should
be performed in relation to clinical trial batches that were used. Appropriate guidance
can be found in ICH Q5E Comparability of Biotechnological/Biological Products34 and
related guidance documents.

Whenever comparability at the analytical and/or non-clinical level cannot be established,

it must be demonstrated by clinical data.

4.3 Non-clinical development

The scrutiny applied during non-clinical testing should take into account the nature of
the Cell-based medicinal product (CBMP) and be proportional to the risk expected to be
associated with clinical use.

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The variability of cell-based medicinal products should be reflected in the non-clinical
studies. Conventional requirements as detailed in Module 4 for pharmacological and
toxicological testing of medicinal products may not always be appropriate. Any deviation
from these requirements shall be justified. If cells in a CBMP have been genetically
modified, non-clinical development must be performed in compliance with guidance
available on gene transfer medicinal products23.

The objectives of the non-clinical studies are to demonstrate proof-of-principle, define

the pharmacological and toxicological effects predictive of the human response, not only
prior to initiation of clinical trials, but also throughout clinical development. The goals
of these studies include the following: to provide information to select safe doses for
clinical trials, to provide information to support the route of administration and the
application schedule, to provide information to support the duration of exposure and the
duration of the follow-up time to detect adverse reactions, to identify target organs for
toxicity and parameters to monitor in patients receiving these therapies.

The non-clinical studies should be performed in relevant animal models. If relevant

animal models cannot be developed, in vitro studies may replace animal studies. The
rationale underpinning the non-clinical development and the criteria used to choose a
specific animal model must be justified. Expression level of biologically active
molecules, the route of administration and the dosages tested should reflect the intended
clinical use in humans.

The recommendations of the ICH S6 Guideline35 should be considered. The number of

animals, their genders, the frequency and duration of monitoring should be appropriate
to detect possible adverse effects.

The safety and suitability of all structural components for their intended function must
be demonstrated, taking into account their physical, mechanical, chemical and biological
properties. (See section 4.2.6 Development pharmaceutics).

4.3.1. Pharmacology

Primary pharmacodynamics

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Non-clinical studies should be adequate to demonstrate the proof of principle of the
CBMP. The principal effects should be identified in non-clinical studies in a suitable
model in vitro or in vivo.

Reasonably justified markers of biological activity should be used to adequately identify

the pharmacodynamic action of the CBMP in the host.

If the intended use of the CBMP is, for example, to restore the function of deficient
cells/tissue (tissue regeneration), functional tests should be implemented to demonstrate
that function is restored. If the intended use is, for example, adoptive immunotherapy in
cancer patients, the biological effect should be supported by data describing the
immunologic action of the CBMP.

The chosen animal model may include immunocompromised, knockout or transgenic

animals. Homologous models may be advantageous, since the in vivo behaviour of the
applied cells or tissue in heterologous models could be altered due to species-specific
mismatches. Homologous models should be considered for the study of stem cell
differentiation. In vitro studies, addressing cell and tissue morphology, proliferation,
phenotype, heterogeneity and the level of differentiation may be part of the primary
pharmacodynamic analyses.

If possible, studies should be conducted in order to determine the minimal or optimal

effective amount of CBMP that is needed to achieve the desired effect.

Secondary pharmacology

Potential undesirable physiological effects of human CBMP including their bioactive

products should be investigated in an appropriate animal model. Cells may migrate from
their intended location and, after a systemic administration, may home to other organs
beside the intended location. Also, somatic cells may secrete additional biologically
active molecules besides the protein of interest. Also, the protein(s) of interest can have
additional targets beside the desired one.

Safety pharmacology

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Safety pharmacology should be considered on a case-by-case basis depending on the
characteristics of the CBMP. Cells may secrete pharmacologically active substances
resulting in CNS, cardiac, respiratory, renal or gastrointestinal dysfunctions. Alternatively,
cells by themselves could be envisaged to induce such consequences for example stem
cells or muscle cells transplanted to infarcted regions of the heart.
For further guidance see ICH S7A Guideline36 when applicable.

Kinetics, migration and persistence

Conventional ADME studies are usually not relevant for human CBMP. However,
studies should be carried out to demonstrate tissue distribution, viability, trafficking,
growth, phenotype and any alteration of phenotype due to factors in the new
environment.

Cells may migrate within the host, thus presenting clinical concerns regarding adverse
reactions deriving from displaced, possibly differentiating cells. This should be evaluated
in animals using appropriate methods for specific identification of the cells.

Regarding biodistribution, the use of small animals allows meticulous cell detection,
which will be practically more difficult in larger animals.

For human CBMP producing systemically active biomolecules, the distribution, duration
and amount of expression of these molecules and the survival and the functional
stability of the cells at target sites should be studied.

Interactions

The interaction of the applied cells or surrounding tissue with the non-cellular structural
components and other bioactive molecules as well as the integration of the CBMP with
the surrounding tissue should be monitored.

4.3.2. Toxicology

The need for toxicological studies depends on the product. However, as conventional
study designs may not be appropriate, the scientific justification for the models used, or

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the omission of studies, shall be provided.

Toxicity may evolve, for example, due to unknown cellular alterations developing during
the manufacturing process such as altered excretion patterns and in vivo behaviour due
to differentiation of the cells. Other potential factors that may induce toxicity include
the allogeneic use of the product, the presence of components that are used in the
manufacturing process or are part of a structural component, or proliferation of the
applied cells in an unwanted quantity or in an unwanted location.

Conventional toxicology studies might nevertheless be required, for example for complex
regimens where CBMP are combined with other medicinal products or treatments such
as adjuvants/cytokines or irradiation, respectively. The need for drug interaction studies
is dependent on the intended use and the type of the cell-based product and should be
discussed.

The induction of an immune response against the cells themselves and/or towards
cell-derived pharmacologically active substances might modulate the efficacy of the
CBMP. Therefore, the possible immunogenicity of a CBMP should be considered. For
guidance on immunogenicity of excreted substances see ICH S6 Guideline35.

Auto-immunity should be considered when cells are used for immunotherapy purposes,
e.g. cancer immunotherapeutic products.

Single and repeated dose toxicity studies

Toxicity studies should be performed in relevant animal models. If the human cells are
not immediately rejected, the studies may be combined with safety pharmacology, local
tolerance, or proof of concept and efficacy studies. Sufficiently characterized analogous
animal-derived cells may be used for some allogeneic CBMP when not immediately
rejected.

The duration of observations in such studies might be much longer than in standard
single dose studies, since the cells are supposed to function for long times, or induce
long term effects, which should be reflected in the design of these studies. The route

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and dosing regimen should reflect the intended clinical use. Repeated dose toxicity
studies are only relevant if the clinical use includes multiple dosings.

Local tolerance studies

Local tolerance studies may be required in an appropriate species. Most often, local
tolerance, tissue compatibility and tolerance to excreted substances can be evaluated in
single or repeated dose toxicity studies.

Other toxicity studies

The risk of inducing tumourigenesis due to neoplastic transformation of host cells and
cells from the CBMP should be considered, as appropriate, on a case-by-case basis.
Conventional carcinogenicity studies may not be feasible. Tumourigenesis studies should
preferably be performed with cells that are at the limit of routine cell culturing or even
beyond that limit. Tissues found to contain applied cells or expressed products during
the biodistribution studies should also be analysed with special emphasis during
tumourigenicity studies.

Genotoxicity studies are not considered necessary for human CBMP, unless the nature
of any expressed product indicates an interaction directly with DNA or other
chromosomal material.

The need for reproductive studies is dependent on the CBMP and should be considered
on a case-by-case basis.

4.4 Clinical development

4.4.1 General aspects

In general, when a Cell-based medicinal product (CBMP) enters the clinical development
phase, the same requirements as for other medicinal products apply. The clinical
development plan should include pharmacodynamic studies, pharmacokinetic studies,

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mechanism of action studies, dose finding studies and randomised clinical trials in
accordance to the Directive 2001/20/EC37 and to the existing general guidances and
specific guidances for the condition evaluated.
Due to specific biologic characteristics of CBMP, alternative approaches to Phase I to
Phase III clinical trials might be required and acceptable for clinical development, if
justified. The relevant non-clinical studies, previous clinical experience of the treated
pathology, and initial clinical studies could be applied for demonstration of the “proof
of principle” and the choice of clinically meaningful endpoints for safety and efficacy
evaluation. European scientific advice is strongly recommended in case of uncertainties
in rationale of CBMP development at the earliest phase.
CBMP might require administration through specific surgical procedures, method of
administration or the presence of concomitant treatments to obtain the intended
therapeutic effect. The biological effects of CBMP are highly dependent on the in vivo
environment, and may be influenced by the replacement process or the immune reaction
either from the patient or from the cell-based product. These requirements coming from
the clinical development should be taken into account for the final use of these
products. Their standardisation and optimisation should be an integral part of the clinical
development studies. The therapeutic procedure as a whole, including the method of
administration and required concomitant medication, such as immunosuppressive regimens
need to be investigated and described in the product information, notably in the
Summary of Product Characteristics (SPC).

4.4.2 Pharmacodynamics

Even if the mechanism of action is not understood in detail, the main effects of the
CBMP should be known. When the purpose of the CBMP is to correct the function of
deficient or destroyed cell/tissue, then functional tests should be implemented. If the
intended use of the CBMP is to restore/replace cell/tissues, with an expected lifelong
functionality, structural/histological assays may be potential pharmacodynamic markers.
Suitable pharmacodynamic markers, such as defined by microscopic, histological, imaging
techniques or enzymatic activities, could be used.
When CBMP includes a non cellular component, the combination should be assessed
clinically for compatibility, degradation rate and functionality.

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4.4.3 Pharmacokinetics

Conventional ADME studies are usually not relevant for human CBMP. Study
requirements, possible methodologies and their feasibility shall be discussed, attention
being paid to monitoring of viability, proliferation/differentiation, body distribution /
migration and functionality during the intended viability of the products.
If multiple (repeated) administrations of the CBMP are considered, the schedule should
be discussed in view of the expected in vivo life span of the CBMP.

4.4.4 Dose finding studies

The selection of the dose should be based on the findings obtained in the quality and
the non-clinical development of the product and it should be linked with the potency of
product. Even though the dosage for cell-based product might be determined by
individual characteristics of the intended patients (i.e. cell mass density per body weight/
volume of missing tissue/ missing surface), the dose to be tested in the confirmatory
trial should be supported by the evidence provided by the phase I/II studies.
Phase I/II studies should be designed to identify a Minimal Effective Dose, defined as
the lowest dose to obtain the intended effect or an Optimal Effective Dose Range,
defined as the largest dose range required to obtain the intended effect based on the
clinical results for efficacy and tolerability. If possible, also the Safe Maximal Dose,
defined as the maximal dose which could be administered on the basis of clinical safety
studies without acceptable adverse effects, should be investigated.

4.4.5 Clinical Efficacy

Clinical efficacy studies should be adequate to demonstrate efficacy in the target patient
population using clinically meaningful endpoints, to demonstrate an appropriate
dose-schedule that results in the optimal therapeutic effect, to evaluate the duration of
therapeutic effect of the administered product and to allow a benefit – risk assessment
taking into account the existing therapeutic alternatives for the target population.
Confirmatory studies should be, as stated before, in accordance to the existing general
guidelines and specific guidelines for the condition evaluated.
Deviations from these will need a justification. For example, the fact that the nature and

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the mechanism of action of the CBMP may be entirely novel does not mean necessarily
that the therapeutic benefit should be measured by different endpoints from those
recommended in the current disease-specific guidelines (e.g. medicines vs. cell implants
for Parkinson’s disease).

For new therapeutic applications of CBMP where limited guidance exists, consultation of
regulatory authorities on the clinical development plan, including the confirmatory
studies, is highly recommended.

The use of previously validated or generally accepted surrogate endpoints is possible

provided that a correlation-between clinical meaningfully endpoints and efficacy can be
established. Sometimes, the desired clinical endpoint, such as prevention of arthrosis, can
be observed only after a long follow up. In such cases, the marketing authorisation can
be based on surrogate markers. If the efficacy is dependent on the long-term persistence
of the product, a long-term follow up plan of the patients should be provided. Thus, the
use of novel meaningful endpoints, clinical or other, is acceptable if justified.

4.4.6 Clinical Safety

The safety database should be able to detect common adverse events. The size of the
database might be decided also in the light of previous clinical experience with similar
products.

The risk of the therapeutic procedure as a whole, e.g. the required surgical procedures
to administer the CBMP or the use of immunosuppressive therapy, shall be evaluated
and used to justify the clinical studies and the choice of the target patient population
All safety issues arising from the preclinical development should be addressed, especially
in the absence of an animal model of the treated disease or in the presence of
physiologic differences limiting the predictive value of homologous animal model.

Particular attention should be paid to those biological processes including immune

response, infections, malignant transformation and concomitant treatment during
development and post-marketing phase of cell-based medicinal products.

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For products with expected long term viability, patient follow-up is required in order to
confirm long term efficacy and safety issue related to the product.

Clinical safety studies on repeated administrations should be performed as required by

the risk analysis. The definition of Maximal Safe Dose should take into account also
the possibility of repeated administration.

4.4.7 Pharmacovigilance and Risk Management Plan

The routine pharmacovigilance and traceability of the product should be described in the
EU Risk Management Plan (RMP) as described in Guideline on risk management
systems for medicinal products for human use7. CBMP may need special long-term
studies to monitor specific safety issues, including loss of efficacy.

The long-term safety issues, such as infections, immunogenicity/immunosuppression and

malignant transformation as well as the in vivo durability of the associated medical
device/biomaterial component should be addressed in the RMP. Special
pharmacoepidemiological studies may be needed. The specific requirements are linked to
the biologic characteristics of the cell-based product. Traceability in the
donor-product-recipient axis, or of the product-recipient for autologous products, is
required in all circumstances as described in Directive 2004/23/EC4 and in Regulation
(EC) No. 2007/1394/EC on Advanced Therapy Medicinal Products2.

REFERENCES (scientific and / or legal)

1. Directive 2001/83/EC of the European Parliament and of the Council of 6 November
2001 on the Community code relating to medicinal products for human use.
2. Regulation (EC) No 1394/2007 of the European Parliament and of the Council on
advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation
(EC) No 726/2004.
3. Ph. Eur. General Chapter 2.7.29: Nucleated cell count and viability. (01/2008:20729)
4. Directive 2004/23/EC of the European Parliament and of the Council of 31 March
2004 on setting standards of quality and safety for the donation, procurement, testing,
processing, preservation, storage and distribution of human tissues and cells.
5. Commission Directive 2006/17/EC of 8 February 2006 implementing Directive

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2004/23/EC of the European Parliament and of the Council as regards certain technical
requirements for the donation, procurement and testing of human tissues and cells.
6. Commission Directive 2006/86/EC of 24 October 2006 implementing Directive
2004/23/EC of the European Parliament and of the Council as regards traceability
requirements, notification of serious adverse reactions and events and certain technical
requirements for the coding, processing preservation, storage and distribution of human
tissues and cells.
7. EMEA/CHMP Guideline on risk management systems for medicinal products for
human use (EMEA/CHMP/96268/2005)
8. Directive 2003/94/EC laying down the principles and guidelines of good
manufacturing practice in respect of medicinal products for human use and
investigational medicinal products for human use.
9. Annex 2 of Directive 2003/94/EC: Manufacture of Biological Medicinal Products for
Human Use.
10. ICH Q5D, Derivation and Characterisation of Cell Substrates Used for Production of
Biotechnological/Biological Products (CPMP/ICH/294/95)
11. ICH Q5A Guideline on Quality of Biotechnological Products: Viral Safety
Evaluation of Biotechnology Product Derived From Cell Lines in of human or animal
origin (CPMP/ICH/295/95)
12. EMEA/CPMP Note for Guidance on Virus Validation Studies: The Design,
Contribution and Interpretation of Studies validating the Inactivation and Removal of
Viruses (CPMP/BWP/268/95)
13. Eudralex Vol. 2 B, Notice To Applicant, part II-V : virological documentation
14. Ph. Eur. General Text 5.1.7: Viral safety (01/2008:50107)
15. EMEA/CPMP/CVMP Note for guidance on minimizing the risk of transmitting
animal spongiform encephalopathy agents via human and veterinary medicinal products
(EMEA/410/01 rev.2)
16. Guideline on Production and Quality Control of Medicinal Products Derived by
Recombinant DNA Technology. (3AB1A)
17. EMEA/CHMP Note for Guidance on Production and quality control of Monoclonal
Antibodies (CHMP/BWP/157653/07)
18. EMEA/CPMP Note for guidance on plasma-derived medicinal products
(CPWP/BWP/269/95, rev.3)
19. EMEA/CHMP Points to consider on Xenogeneic Cell Therapy Medicinal Products

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(CPMP/1199/02)
20. EMEA/CHMP Note for Guidance on Use of Bovine Serum in the Manufacture of
Human Biological Medicinal Product (CPMP/BWP/1793/02)
21. Eudralex Vol. 7Blm10a Table of extraneous agents to be tested for in relation to
the general and species specific guidelines on production and control of mammalian
veterinary vaccines
22. EMEA/CPMP Note for Guidance on Production and Quality Control of Animal
Immunoglobulins and Immunosera for Human use (CPMP/BWP/3354/99)
23. EMEA/CHMP Note for Guidance on the quality, preclinical and clinical aspects of
gene transfer medicinal products (CPMP/BWP/3088/99)
24. Council Directive 93/42/EEC of 14 June 1993 concerning Medical Devices
25. Council Directive 90/385/EEC of 20 June 1990 on the approximation of the laws of
Member States relating to Active Implantable Medical Devices
26. EN/ISO 10993-18:2005 Biological evaluation of medical devices- Part 18: Chemical
characterization of materials
27. EN/ISO 10993-19:2006 Biological evaluation of medical devices- Part 19:
Physico-chemical, morphological and topographical characterization of materials
28. Ph.Eur. Text 5.1.6: Alternative methods for control of microbiological quality
(01/2008:50106) and General Method 2.6.27: Microbiological control of cellular products.
29. ICH Q6B Note For Guidance on Specifications: Test Procedures and Acceptance
Criteria for Biotechnological/Biological Products. (CPMP/ICH/365/96)
30. EMEA/CHMP guideline on potency testing of cell-based immunotherapy medicinal
products for the treatment of cancer (CHMP/BWP/271475/06).
31. Ph. Eur. General Text 5.2.3: Cell substrates for the production of vaccines for
human use (01/2008:50203)
32. EMEA/CPWP Note for Guidance on Development Pharmaceutics for
Biotechnological and Biological Products (CPMP/BWP/328/99)
33. EN/ISO 10993-1, Biological evaluation of medical devices - Part 1: Evaluation and
testing
34. ICH Q5E, Comparability of Biotechnological/Biological Products
(CPMP/ICH/5721/03)
35. ICH S6; Preclinical safety evaluation of biotechnology derived products
(CPMP/ICH/302/95)
36. ICH S7A, Safety pharmacology studies for human pharmaceuticals

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(CPMP/ICH/529/00)
37. Directive 2001/20/EC of the European Parliament and Council of 4 April 2001 on
the approximation of the laws, regulations and administrative provisions of the Member
States relating to the implementation of good clinical practice in the conduct of clinical
trials on medicinal products for human use.

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암치료를 위한 수지상 세포치료제 평가시 고려사항

목 차

암치료를 위한 수지상 세포치료제 평가시 고려사항 ............................... 1

1. 일반적 사항 ................................................................................ 1
2. 수지상 세포의 품질에 관한 사항 .................................................... 1

1) 제조과정에 관한 사항 ................................................................ 1
2) 확인: 표면항원 ......................................................................... 2

3) 순도 ........................................................................................ 2
4) 세포 생존율 ............................................................................. 3
5) 역가 ........................................................................................ 3

6) 수지상 세포의 분화 유도에 사용하는 항원 ................................... 3

3. 비임상 평가 ................................................................................ 4
1) 효력 평가 ................................................................................ 4

2) 독성 평가 ................................................................................ 4
3) 용법·용량 설정시 고려사항 ......................................................... 4
4. 임상 평가 ................................................................................... 5
1) 용법?용량 설정시 고려사항 ........................................................ 5

2) 안전성 평가 ............................................................................. 6
3) 유효성 평가 ............................................................................. 6
5. 적응증을 변경할 경우 고려사항 ..................................................... 7
6. 참고문헌 ..................................................................................... 7

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 암치료를 위한 수지상 세포치료제 평가시 고려사항

1. 일반적 사항

본 가이드는 최근 암치료를 위해 개발되고 있는 자가 유래 수지상 세포치료제의

평가시 부분적으로 고려할 항목에 대해, 수지상 세포치료제 개발자 혹은
평가자에게 유용한 정보를 제공함을 목적으로 작성되었다.

수지상 세포치료제 역시 세포치료제의 한 종류로서, '의약품등기준및시험방법심사의뢰서심사규정

(식품의약품안전청 고시 2005-57호, 2005.10.17)’, ‘생물학적제제등허가및심사에관한규정
(식품의약품안전청 고시 2005-29호, 2005.6.1)’ 및 ‘의약품임상시험계획승인가이드라인
(식품의약품안전청 고시 제2004-51호, 2004.7 .19)’에 따라 평가되어야 한다.

본 가이드에서 논의되는 다음의 사항들은 수지상 세포치료제 평가시 특이적으로

고려해야 할 사항으로, 현시점에서 가능하면서 과학적으로 최선으로 생각되는
공통된 사항을 도출한 것으로 추후 과학기술의 발전에 따라 추가적으로 수정되어야
할 것으로 생각된다. 본 가이드는 직접적인 평가가이드라인은 아니며, 많은 부분
협의체의 의견을 반영한 것으로 법적인 구속력은 없음을 밝혀둔다.

2. 수지상 세포의 품질에 관한 사항

1) 제조과정에 관한 사항

○ 일반적으로 말초혈액에서 혈액단구 (CD 14+ 세포)나 CD 34+ 세포를 분리하여

Granulocyte-macrophage colony stimulating factor (GM-CSF) 와
interleukin-4 (IL-4) 등 적절한 싸이토카인을 처리하고 배양함으로써 미성숙
수지상 세포가 유도된다.
○ 다양한 자극인자를 처리하고 더 배양함으로써 성숙된 수지상세포를 유도할 수도
있다.
○ 수지상 세포는 항원제시 세포로서 T 세포를 자극하여 면역반응을 유도하는
것으로 알려져 있으므로, 적절한 단계에서 수지상 세포의 항원제시능력 및
림프구 증식이 증가된 것을 확인하여야 한다.

2) 확인 : 표면항원

○ 수지상세포의 확인을 위해서는 유세포 분석 등을 이용한 세포의 표면항원

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 발현의 측정이 유용할 것으로 생각되며, 공통적으로 확인 가능할 것으로
생각되는 표면항원은 다음과 같다.
- 발현이 최소로 감소되는 표면항원 : CD14 (유래에 따라 고려), CD3, CD19
- 발현이 증가되는 표면항원 : HLA ClassⅠ/Ⅱ, CD80/CD86
- 성숙지표로 활용가능 할 것으로 생각되는 표면항원 : CD83

○ 다만 이들 항원의 발현 정도, 수지상세포의 성숙도 및 기타 adhesion

molecule인 CD54/CD50, chemokine receptor CCR7, CD25, CD11c 등은
제품별 특성에 맞게 평가하여야 할 것으로 생각된다.

3) 순도

○ 세포치료제의 미생물 오염에 대한 검사로 일반세균, 진균, 마이코플라스마 오염

여부는 필수적으로 확인해야 하며, 엔도톡신 시험 및 바이러스 등 기타
외래인자에 대한 적절한 시험도 수행되어야 한다.

○ 제조과정 중 사용한 싸이토카인등 제품 중 잔존이 우려되는 물질에 대한 분석이

수행되어야 한다.

○ 수지상 세포는 가능하면 혈청이 없는 배지를 사용하는 것이 좋으며, 사용할

경우 사람 혈청이나 사람 혈청 알부민 등을 사용하는 것이 권고된다. 특히
동물유래 혈청배지를 사용한 경우에는 소해면상뇌증 (Bovine spongiform
encephalopathy, BSE) 또는 소유래바이러스와 같은 동물유래 병원체에
감염되지 않았음을 증명하는 자료가 요구된다.

4) 세포 생존율

○ 세포 생존율의 허용기준은 최소 70 % 이상이며, 일반적으로 trypan blue

exclusion 방법으로 시험한다. 기타 가능한 방법으로 평가한 경우에는
시험방법에 대한 타당성 자료가 첨부되어야 한다.

5) 역가

○ 미국 FDA, 유럽 EMEA, 일본 PMDA의 최신 가이드라인에 따라 제품의 특성에

맞는 역가시험법을 설정하여야 한다. (2010년 Version)

6) 수지상 세포의 분화 유도에 사용하는 항원

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○ 수지상 세포의 분화 유도를 위하여 항원을 사용한 경우에는 사용 방법에 따라
사용항원의 자가 품질 기준을 명확히 설정한다. 예를 들어 Peptide 항원을
사용한 경우, 순도, 물질적 특성에 따른 용해도, dimer 형성 여부, 분해 정도
등을 평가하여야 한다. Tumor lysate를 사용한 경우에는 생산방법, 오염도,
단백질 함량 등을 평가하여야 한다.

3. 비임상 평가

1) 효력 평가

○ 면역기능조절제로 사용되는 수지상세포 치료제의 효력시험 동물모델 선택 시,

적절한 동물모델을 선정하여 효능을 입증하는 것이 바람직하다. 면역체계가
불완전한 nude mouse와 같은 종을 선택하거나, 사람세포를 마우스에 투여하는
것과 같은 xenogenic model은 효능을 입증하는데 적절하지 않을 수 있다.

○ 적절한 효력 평가를 위해서는 동물에서의 효력시험 결과와 사람세포를 이용한

in vitro에서의 효력시험 결과 간의 비교가 필요하다.

○ 동물의 세포를 이용하여 동물에서의 효력시험을 수행하는 경우, 사용하는

동물세포는 사람에서의 것과 유사 또는 동등한 제조방법을 통해 확보된 것을
사용하여야 한다.

2) 독성 평가

○ 독성시험에 사용하는 동물종을 선택할 경우에는 ‘효력평가모델’과 동일한 사항을

고려하여야 하며, 독성 평가에 보다 적절한 동물모델을 선정하는 것이
바람직하다. 또한 효력시험과의 인과관계 고찰이 용이한 동물종의 선택이
고려될 수 있다.

3) 용법•용량 설정시 고려사항

○ 비임상 시험시 투여경로는 임상과 동일한 투여경로를 원칙으로 한다. 다만,

시험동물에서 투여경로의 특이성이 과학적 사실에 의거하여 타당하다고
판단되는 경우, 임상 사용시 투여 예정경로와 다르게 적용할 수 있다.
예) 마우스의 경우는 전신작용을 나타내기 위해 복강주사를 통해 면역반응을
유도할 수 있음.

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○ 투여 일정의 경우에는 살아있는 세포가 생체 내에서 세포면역을 유도할 수
있도록 적절한 시간간격을 두고 투여일정 확립을 위한 시험을 실시하여야 하며,
면역반응과 관련한 세포의 형성 유도를 위해 반복투여에 대한 사항이 충분히
고려되어야 한다.

○ 비임상시험 결과를 토대로 임상시험시 예측 투여용량을 결정하기 위해서는,

용량-반응곡선을 나타낼 수 있는 적절한 투여용량을 설정하여 시험을
실시하여야 하고, 임상시험 프로토콜을 뒷받침할 수 있어야 한다.

4. 임상 평가

1) 용법․용량 설정시 고려사항

○ 일반적으로 수지상 세포의 임상 시험 투여경로는 다음과 같으며, 대상 질환 및

제품별 특성을 고려하여 적절한 투여경로를 선택하여야 한다.
- 피내 (intra-dermal) 또는 피하 (sub-cutaneous) 투여
- 림프절내 (intra-nodal) 투여
- 정맥내 (intra-venous) 투여
- 종양내 (intra-tumoral) 투여

○ 수지상 세포의 투여용량은 비임상시험을 통해 결정하여야 한다. 현재 알려진

배양 기술이나 배양원료 확보 등을 고려할 때 107 ~ 108 cells/회가 가장 많이
시도되고 있으나, 각 임상 프로토콜별로 적합한 용량 결정을 위한 기초자료
제시가 필요하며, 각 연구의 특이성을 적절히 고려하여야 한다.

○ 투여 일정의 경우 비임상시험과 임상시험이 반드시 동일하게 적용될 수는

없다고 판단되나, 면역반응과 관련된 세포의 형성을 유도하기 위해 반복투여에
대한 사항이 충분히 고려되어야 한다. 또한 살아있는 세포가 생체 내에서
세포면역을 유도할 수 있도록, 적절한 시간 간격을 두고 투여하는 것이
바람직하다.

○ 투여횟수는 환자의 상태, 반응성 등을 고려하여 결정하여야 하며, 3회 이상이

적절할 것으로 생각된다. 투여간격은 1-4주 간격으로, 암종의 성장 속도등에
따라 각 임상 프로토콜별로 적절한 자료를 첨부함으로서 차별화될 수 있다.

- 86 -
2) 안전성 평가

○ 임상시험 진행과정에서는 다음의 사항들이 안전성 평가를 위하여 측정해야 할

항목으로 고려될 수 있다.
- 일반적인 임상시험의 안전성 평가지표
- 치료효과로 인한 이상반응 : 종양괴사에 수반되는 과다 출혈이나 파괴
- 자가면역 관련 반응 : 자가항체 생성 검사 등. 자가 면역반응이 나타나는
경우에는 실제 질병이 나타나는지 여부를 확인할 필요가 있다.
- 지나친 면역증진에 따른 생리적 이상
- 세포치료를 중단하거나 지속하기 위한 객관적인 기준

3) 유효성 평가

○ 일반적인 사항은 항암제의 임상평가 기준을 따른다.

○ 암치료를 위한 임상시험의 단기적인 유효성 평가지표로는 일정 시간 내의 종양

반응성을 측정하는 것이 적절할 것으로 생각되며, 가능하다면 종양표지자의
변화 유무를 측정하는 것이 고려될 수 있다.

○ 임상시험 단계 (Phase Ⅰ/Ⅱ/Ⅲ)에 따라 암환자의 생존기간 및 생존률,

질병진행까지의 시간과 질병진행이 없는 시간(Time to Progression and
Progression-Free Survival), 반응률(Objective Response Rate) 등 적절한
유효성 평가지표를 관찰하여야 한다.

○ 다음과 같은 면역반응시험은 수지상 세포치료제의 유효성 평가시 세포치료제의

효과를 판단할 수 있는 2차적 평가지표로 고려될 수 있다.
- 지연형 과민반응 검사 (Delayed-type hypersensitivity, DTH): 종양 항원 또는
면역보조제 (Keyhole limpet hemocyanin, KLH)에 대한 반응
- Antigen-specific proliferation assay : 말초혈 단핵세포(Peripheral Blood
Mononuclear Cells, PBMC) 또는 T 세포
+
- IFN-γ 등 effector molecule 분비 측정 또는 cytotoxic CD8 cell 확인

5. 적응증을 변경할 경우 고려사항

○ 새로운 적응증에 대한 임상시험을 통해 각각의 암종에 따른 유효성을 평가하는

것이 필요하며, 투여경로가 변경되는 경우 등은 추가적인 비임상시험이

- 87 -
 고려되어야 한다. (예 ; 전신노출 정도가 커진 경우 피하주사 → 정맥주사 등)

○ 수지상세포치료제의 적응증을 변경하기 위하여 암특이적 항원만을 변경한 수지상

세포치료제의 경우에는 비임상시험을 통한 고찰이 필요하며, 과학적으로
타당성이 인정되는 경우에 한하여 비임상시험이 필요하지 않을 수 있다.

6. 참고문헌

○ Carl G Figdor et al. Dendritic cell immunotherapy: mapping the way, Nat.
Med. 10, 475-480(2004)

○ Jacques Banchereau & Ralph M. Steinman, Dendritic cells and the control
of immunity, Nature 392, 245-252(1998)

○ Vincezo Cerundolo et al. Dendritic cells: a journey from laboratory to clinic,

Nat. Immunol. 5, 7-10(2004)

○ Theresa L. Whiteside & Christine Odoux, Dendritic cell biology and cancer
therapy, Cancer Immunol Immunother. 53, 240-248(2004)

○ Lee D. Cranmer et al. Clinical applications of dendritic cell vaccination in

the treatment of cancer, Cancer Immunol Immunother. 53, 275-306(2004)

- 88 -
 용역연구개발과제 최종보고서
과제번호 응용연 09172 652

단위과제명 첨단기술 응용기반 연구

3)

국문 줄기세포치료제의 GVHD (graft-versus-host

과 제 명 역가시험법 검증연구
disease)

영문 The Efficacy and Potency of Mesenchymal Stem

Cells for the Treatment of GVHD

주관 기관명 소재지 기관장

연구기관 이화여자대학교 서울서대문구대현동 조지형
산학협력단 11

주관 성 명 소속 및 부서 전 공
연구책임자 유경하 소아과학교실 혈액종양
총연구기간 년 월 일 ～ 년 월 일 개월
2009 3 1 2009 11 30 ( 9 )

총 연구개발비 천원 70,000

연구년차 연구기간 연구개발비

차년도 년 월 일 ～
2009 3 년 월1
천원 2009 11
1
일 30
70,000

2차년도 년 월 일 ～ 년 월 일 천원
관계규정과 모든 지시사항을 준수하면서 이 용역연구개발연구사업을
성실히 수행하고자 다음과 같이 용역연구개발과제최종보고서를
제출합니다 .

2009 년 월 11 일
주관연구책임자 : 유경하 (서명 또는 인)
주관연구기관장 : 조지형 (직 인)

식품의약품안전평가원장 귀하

- 89 -
Ⅰ. 총괄연구개발과제 요약문 (국문 요약문)
과 제 명 줄기세포치료제의 역가시험법 검증연구
GVHD(graft-versus-host disease)
중심단어 이식편대숙주병 줄기세포치료기술 지방조직유래줄기세포 역가 평가
, , , ,
주관연구기관 이화여자대학교 산학협력단 주관연구책임자 유 경 하
연구기간 2009-03-01 ~ 2009-11-30
연구목표
1. 본 연구는 중간엽줄기세포 면역조절능력을 이용한 이식편대숙주병(GVHD)의 치료 시 발생
하는 세포 수의 부족을 줄기세포 공급원과 이식기술을 다양화하여 해결하고자 한다.
2. 이를 위해 선행 연구에서 제시된 GVHD in vitro 역가 시험법을 이용하여 지방조직 유래 중간
엽줄기세포의 치료 역가를 측정하고, 마우스 동물모델에 이식하여 반복투여 및 면역억제제
와의 병용투여 시 in vivo 효능과의 상관성 입증함으로써 줄기세포치료제의 GVHD역가 시험
법을 검증하고 이를 확립하고자 한다.
연구내용 및 성과
1. 지방조직 유래 중간엽줄기세포(AD-MSCs) 이용 GVHD 치료 기술 개발
(1) AD-MSCs의 면역표현형 및 다분화능 확인
- AD-MSCs를 분리하여 배양하였고, 면역학적으로 CD34, CD45에는 음성이고, 줄기세포
표지자인 CD73, CD90, CD105에 양성으로 표현되는 MSC임을 확인하였으며 BM-MSC
과 유사하였다.
- 이 세포들은 지방세포 (oil red o), 뼈세포 (Alizarin red S), 신경세포 (NSE, TUJ1,
MAP-2, Nestin, GFAP), 근육세포 (Myosin) 및 연골세포 (toluidine blue O)등으로 다
양한 분화능력이 있음을 확인하였다.
(2) AD-MSC의 면역조절 능력
- AD-MSC의 T-세포 억제 효과는 AD-MSC 주입용량에 비례하였다.
- BM-MSC과 비교하였을 때 AD-MSC의 면역조절능력은 MSC:T 비율이 1:1 이하에서
는 T-세포 증식억제효과가 낮았으며, 1:1 비율 이상에서는 같은 효과를 보였다.
- AD-MSC에 의해 T-reg cell의 발현이 증가하였고 이는 BM-MSC와 일치하는 소견이
었다.
(3) AD-MSC 이용 GVHD 치료기술 개발
- 8Gy 용량의 방사선 조사후 T세포 이식 방법으로 GVHD 마우스 모델을 제작하였고 이
식받은 마우스의 골수, 흉선, 비장에서 donor 세포가 착생됨을 바이오이미징으로 확인
하였다.
- GVHD 마우스 군은 지속적으로 육안적 평가점수가 심각한데 비해 대조군과 MSC 주
입군에서는 시간이 지날 수록 호전됨을 확인하였다.
- AD-MSC 이식 후 조직학적점수가 소장, 피부, 대장에서 호전됨을 확인하였다.
2. MSC 이용 GVHD 치료효과 극대화 기술 개발
(1) 단회 및 반복 이식 효과 분석
- AD-MSC 이식은 전체적으로는 생존율 향상에 도움이 되지만 BM-MSC에 비해 효과
는 미미하였R고 특히 생존율 분석에서는 단회이식에 비해 반복이식의 효과가 미미하였
다.
(2) 기존 면역억제제와 병합사용 효과 분석
- 스테로이드의 용량은 5 mg/kg 군보다는 1 mg/kg가 더 적절한 용량임을 확인하였고
단회이식이 반복 이식 군에 비해 효과가 우수하였다.
- 그러나 MP만 사용한 경우보다는 중간엽줄기세포를 같이 이식한 군은 마우스 모두 생
존하는 높은 생존율을 보였다.
기대효과
-지방흡인술 후 버려지는 지방조직으로부터 MSC을 얻어 GVHD를 치료 할 수 있다면 어렵
게 골수채취를 해야 하는 번거로움과 경제적인 문제를 한꺼번에 해결할 수 있는 무한한 가
치를 가질 수 있다.

- 90 -
 세포치료제 역가시험 참고 자료집
══════════════════════════════════════════
발 행 일 : 2010년 12월
편 집 위 원 장 : 바이오생약국장 이정석
편 집 위 원 : 식품의약품안전청 바이오생약심사부
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권오석
식품의약품안전평가원 의료제품연구부
박순희, 정승태, 오일웅, 류시형, 박기대, 최민정
발 행 부 서 : 바이오생약국 첨단제제과
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연 락 처 : 식품의약품안전청 바이오생약국 첨단제제과

전 화 번 호 : 043) 719-3512
팩 스 번 호 : 043) 719-3500
- 92 -