advancing biologics development programs with …standard approach to biologics development using...

10.5731/pdajpst.2018.009316Access the most recent version at doi: 264-27474, 2020 PDA J Pharm Sci and Tech

Paul Wu, Taymar Hartman, Louise Almond, et al. Stage Process and Product Consistency DataAddressing Clonality Concerns Prior to Availability of LateLines: Advantages and Limitations of Genetic Testing for Advancing Biologics Development Programs with Legacy Cell

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COMMENTARY

Advancing Biologics Development Programs with LegacyCell Lines: Advantages and Limitations of Genetic Testing forAddressing Clonality Concerns Prior to Availability of LateStage Process and Product Consistency Data

PAULWU1,*, TAYMAR HARTMAN2, LOUISE ALMOND3, JENNITTE STEVENS4, JOHN THRIFT1, JUHI OJHA1,CHRISTINA ALVES5, DAVID SHAW6, MICHAELW. LAIRD6, ROBYN EMMINS7, YUAN ZHU7, REN LIU8,ZHIMEI DU8, ROLF KOEHLER9, THOMAS JOSTOCK9, KARIN ANDERSON10, CHRIS CAMPBELL11,#, andHOWARD CLARKE12

1Bayer HealthCare LLC, Berkeley, CA; 2Abbvie Biotherapeutics Inc., Redwood City, CA; 3Allergan Biologics Ltd.,Liverpool, UK; 4Amgen Inc., Thousand Oaks, CA; 5Biogen Inc., Cambridge, MA; 6Genentech Inc., South San Francisco,CA; 7GSK, King of Prussia, PA; Stevenage, UK; 8Merck & Co., Inc., Kenilworth, NJ; 9Novartis Pharma AG, Basel,Switzerland; 10Pfizer Inc., Andover, MA; 11Takeda Pharmaceuticals, Cambridge, MA; and 12Seattle Genetics Inc.,Bothell, WA © PDA, Inc. 2020

ABSTRACT: The bioprocessing industry uses recombinant mammalian cell lines to generate therapeutic biologic drugs. To

ensure consistent product quality of the therapeutic proteins, it is imperative to have a controlled production process. Regula-

tory agencies and the biotechnology industry consider cell line “clonal origin” an important aspect of maintaining process

control. Demonstration of clonal origin of the cell substrate, or production cell line, has received considerable attention in

the past few years, and the industry has improved methods and devised standards to increase the probability and/or assurance

of clonal derivation. However, older production cell lines developed before the implementation of these methods, herein

referred to as “legacy cell lines,” may not meet current regulatory expectations for demonstration of clonal derivation. In this

article, the members of the IQ Consortium Working Group on Clonality present our position that the demonstration of pro-

cess consistency and product comparability of critical quality attributes throughout the development life cycle should be suf-

ficient to approve a license application without additional genetic analysis to support clonal origin, even for legacy cell lines

that may not meet current day clonal derivation standards. With this commentary, we discuss advantages and limitations of

genetic testing methods to support clonal derivation of legacy cell lines and wish to promote a mutual understanding with

the regulatory authorities regarding their optional use during early drug development, subsequent to Investigational New

Drug (IND) application and before demonstration of product and process consistency at Biologics License Applications

(BLA) submission.

KEYWORDS: Chinese hamster ovary (CHO), Production cell line, Cell line development, Genetic characterization,

Master Cell Bank (MCB), Working Cell Bank (WCB), Clonal derivation, Clonality, Clonal, IND, BLA.

Introduction

Over the past several years, demonstration of cell line

clonal derivation, colloquially referred to as clonality,

has been a topic of many industry and regulatory pre-

sentations and papers (1–4). Presentations from Food

and Drug Administration (FDA) colleagues have

established an expectation for sponsors to provide high

probability or assurance of clonality, based on the

potential of a nonclonal cell line to manufacture prod-

uct inconsistently, possibly resulting in product quality

changes that will affect the safety or supply of the

* Corresponding Author: Paul Wu, Bayer HealthCare

LLC, Berkeley, CA 94710, e-mail: [email protected]#Current affiliation: Sarepta Therapeutics, Cambridge,

MA, USA.

All listed companies are member companies of the IQ

Consortium Clonality Working Group.doi: 10.5731/pdajpst.2018.009316

264 PDA Journal of Pharmaceutical Science and Technology

on August 5, 2020Downloaded from

product. Increased attention and scrutiny on this aspect

of cell line development from regulatory agencies has

resulted in companies evaluating the relative importance

of clonality in the larger context of product quality and

process consistency (1). Many have implemented proce-

dures and technology intended to achieve acceptable

assurance of clonality for products entering clinical de-

velopment today (5). This move has strengthened the

industry as a whole, but sponsors can still face a chal-

lenge with older legacy cell lines, created in a manner

that did not meet the current expectations for demonstra-

tion of clonal derivation, with requests from regulatory

authorities to provide additional evidence to support clo-

nal derivation of these cell lines.

Current regulatory guidance (6) instructs cloning the

cell substrated “from a single cell progenitor” during

cell line development. The FDA has recommended that

two rounds of limiting dilution cloning (LDC) at suffi-

ciently low seeding densities (≤0.5 cells/well) provideacceptable probability that a cell line is clonallyderived. More recently, one round of cloning through avalidated flow cytometry method or LDC with suffi-cient supporting justification, such as use of sensitiveimaging technology on Day 0, has provided acceptableassurance of clonal derivation when using validatedmethods (7±9). However, some ongoing clinical pro-grams use legacy cell lines that were created before theindustry had such practices and methods in place.Sponsors of these programs, including some of the cur-rent authors, face the challenge of addressing theheightened focus on clonal origin without introducingchanges to the production cell line that could signifi-cantly impede the availability of life-impacting medi-cines. Introduction of a new production cell line orgenerating a subclone is a last-resort option with

considerable risk, cost, and time needed to demonstrateprocess and product comparability. Given this, it hasbeen the authors’ collective experience that regulatorshave proposed two alternate paths to commercializationusing such legacy cell lines: 1) providing additionalassurance of clonality through genetic testing, or 2)augmenting control strategies to manage a potentiallynonclonal cell line (3, 4). Neither path is straightfor-ward, as additional genetic testing may yield inconclu-sive results given the plasticity of Chinese hamsterovary (CHO) genomes (10±12), making it difficult todetermine clonal origin, and implementation of aug-mented process controls (additional control strategyelements to improve process consistency) can put on-erous constraints on operational manufacturing flexi-bility, especially after stable process and productconsistency of the cell line through limit of in vitrocell age (LIVCA) has been demonstrated.

The choice of if, when, or how to provide supplemental

assurance of clonality is something each sponsor must

evaluate individually based on available process and

product data, product stage, and individual experience.

The typical stages in which data are obtained for justi-

fying that a cell line is appropriate for commercial

production are provided in Figure 1. Ultimately, the

Biologics License Applications (BLA) will include

data from extensive process characterization studies

performed to demonstrate consistent product quality

and cell culture performance from qualified scale-

down, pilot, and commercial scales, as well as cell line

genetic characterization studies to show stable trans-

gene integration profiles.

Demonstration of robust process control to maintain

consistent product quality is essential to ensure patient

Figure 1

Standard approach to biologics development using cell lines with high probability of clonal derivation.

Vol. 74, No. 2, March--April 2020 265


safety and product efficacy. Production robustness

requires that process parameters are monitored and

controlled throughout the entire manufacturing cam-

paign, for example, measuring cell growth, productiv-

ity, metabolite consumption and production rates, as

well as controlling pH and dissolved oxygen profiles.

Product consistency is often defined in terms of ac-

ceptable variation in a product’s critical quality attrib-

utes (CQAs). By definition, a CQA is “a physical,

chemical, biological, or microbiological property or

characteristic that should be within an appropriate

limit, range, or distribution to ensure the desired

product quality” (13). The product quality analysis

can monitor post-translational modifications, amino

acid substitutions, product isoforms, product-related

impurities, high molecular weight forms, and other

product quality attributes. All of these attributes can

become part of the target product profile, which is the

“prospective and dynamic summary of the quality

characteristics of a drug product that ideally will be

achieved to ensure that the desired quality, and thus

safety and efficacy, of a drug product is realized”

(13). Range testing of manufacturing set-points and

other aspects of the product quality control strategy

will ensure that drug substance and product manufac-

tured in the future will be consistent. Qualified scale-

down bioreactors are commonly used for this pheno-

typic analysis at the process characterization stage.

The ultimate goal is to identify and predict the pro-

cess and product variability that may arise owing to

control or material inputs. The decision to implement

augmented control strategies such as reducing LIVCA

may arise due to actual process inconsistencies or pre-

dicted future genetic or process drift. In situations

wherein product or process inconsistencies are

observed, augmented control strategies should be

implemented to ensure consistency.

Consistent process and product quality data are re-

quired for approval but are typically not available until

late in product development. During earlier stages of

product development, before these data are available,

sponsors face a challenge in assessing and mitigating

any potential risks that might be associated with a leg-

acy cell line not being clonally derived. Therefore, a

product manufactured from a legacy cell line that does

not meet the current regulatory expectations for new

cell lines may benefit from genetic testing to provide

additional assurance of clonal derivation and help

inform later stage development and filing strategies.

However, the authors believe supplemental genetic

work performed to investigate clonal origin should be

considered optional, based on the limitations in using

this type of data to assure a high probability of clonal

derivation.

The authors believe comparable product quality, con-

sistent process performance, and acceptable genetic

profiles (based on ICH Q5B guidance) from clinical

[Master Cell Bank (MCB)] to commercial process

[Working Cell Bank (WCB)] are necessary and suffi-

cient to enable the approval of a legacy cell line for

manufacturing a biotherapeutic. General agreement

between sponsors and regulatory agencies on this posi-

tion should provide a pathway to approval, independent

of clear demonstration of clonal derivation, provided

the other existing aspects of process control are in

place and satisfactory for ensuring product consistency.

In the absence of a current consensus understanding of

how best to proceed with legacy cell lines, we state our

position on, and review the utility and limitations of,

possible genetic testing methods for analyzing cell line

clonality to support early-stage programs before com-

plete genetic characterization.

1. Opinion on Additional Genetic Testing

New molecular technologies provide an ability to ana-

lyze a cell line’s genetic profile in great detail. The FDA

has indicated a willingness to accept different types of

genetic data as additional assurance of clonality, includ-

ing characterizing individual analytical subclones from

the MCB (3, 4) to show they are genetically similar.

In this context, we present methods for analyzing cell

lines to provide additional assurance of clonality, and

discuss technical challenges, limitations, and anecdotal

experiences when applying these methods to produc-

tion cell lines. Many of the techniques and examples

we present may pose a challenge to companies, as new

questions arise concerning how the findings could

affect a product or program. Given the well-docu-

mented plasticity of CHO cells, detailed genetic exami-

nation of legacy cell lines runs the risk of generating

data that are not easily explained, or even dispute clon-

ality of a cell line that is, in fact, clonal in origin

(14–17). This additional information may contradict

the breadth of knowledge that supports a cell line’s

commercial use, such as extensive characterization, or

phenotypic and genotypic stability evaluations. As

such, some sponsors may choose not to undertake such

genetic testing depending on their experience with



regulatory authorities and specific knowledge of cell

line and process consistency. Instead, they may wait

for process and product consistency data to be avail-

able at a late stage or elect to implement augmented

control strategies if they believe they would be

required, to provide additional assurance based on the

totality of data. Figure 2 attempts to simplify these

complex decisions that are grounded in the authors’

belief that consistent late-stage product quality and

process performance should obviate the need for addi-

tional assurance of clonal derivation.

2. Current Genetic Testing for BLA

The regulatory authorities have provided guidance on

when and how to apply genetic testing to cell lines to

demonstrate genotypic stability. For integrated expres-

sion constructs, ICH Q5B (18) prescribes “restriction

endonuclease mapping or other suitable techniques

should be used to analyze the expression construct for

copy number, for insertions or deletions, and for the

number of integration sites.” This testing is applied to

the MCB and an end of production (EOP) cell bank

derived from cells at the limit of in vitro cell age,

which is the maximum cell age allowed for product

manufacturing, to demonstrate genetic consistency.

Southern blotting is used to demonstrate transgene

integration integrity and consistency over time in cul-

ture. All this testing will need to be performed before,

and included in, a BLA submission.

3. Genetic Technologies Supporting Assurance of

Clonality

With the advent of new molecular technologies and

methods, detailed genetic information may be gener-

ated to potentially provide evidence of clonal deriva-

tion. Table I outlines the methods we reviewed for

clonality assessment of legacy cell lines. In reviewing

these methods, we assume legacy cell lines were gener-

ated by random transgene integration, as many of the

methods would not be useful to support clonality inves-

tigation with cell lines generated using newer site-spe-

cific, targeted integration techniques. The Southern

blot method, discussed above, is traditionally included

in BLA submissions to provide production cell line

genetic stability data. Other methods, not typically

included in BLA submissions, could provide supple-

mental characterization data to confirm genetic consis-

tency of the transgene in support of clonal derivation.

These include fluorescence in situ hybridization (FISH),

additional Southern blotting, next-generation sequenc-

ing (NGS) technologies, and polymerase chain reaction

(PCR)-based assays. These genetic tests differ based

on their sensitivity, throughput, or limit of detection

for potential low-level presence of a different clone.

Several of these methods can be used to provide infor-

mation that is useful in subsequent higher-throughput

assays. For example, based on NGS sequence informa-

tion, high-throughput PCR can be used to map integra-

tion sites to provide supportive assurance of clonality.

These technologies are detailed, along with guidance

on analytical subclone generation, in the following

sections.

3.1 Derivation of Analytical Subclones

To identify genetic heterogeneity in a production cell

line, the hallmark of a nonclonal cell line, one must be

able to compare individual cells within the population.

Of the genetic methods discussed below, only FISH is

capable of analyzing single cells to assess the homoge-

neity within the parental population. The remaining

methods (Southern blotting, NGS, PCR-based assays)

require subcloning to derive cell lines that can be com-

pared against each other and the parental production

cell line population (application of these methods for

single cell-based analysis is not discussed herein). This

analytical subcloning can provide an assessment of

genetic consistency to support the assurance of clonal-

ity of the production cell line. However, inconsistent

results must be scrutinized by investigators, as recent

publications have demonstrated that analytical sub-

clones from clonally derived production cell lines can

display phenotypic, genotypic, and/or epigenetic heter-

ogeneity (14–17).

3.2 FISH

FISH is a molecular cytogenetic technique used to

study genetic rearrangement and target sequence inte-

gration in cells based on selective binding of the

hybridization probes to cellular DNA followed by mi-

croscopic analysis. This technology can detect the spe-

cific location of the integrated recombinant plasmid at

the chromosomal level in single cells of the MCB.

Given that the CHO genome comprises 2.45 Gb of

genomic sequence with 24 383 predicted genes on 21

chromosomes (19), it is highly unlikely that the same

plasmid integration event would happen randomly in

two independent clones. Therefore, this approach for

cell line clonality is recommended only for cell lines



Figure 2

Decision tree for implementing additional genetic testing as assurance of clonal derivation.

The ultimate requirement for all commercial cell lines, regardless of cell line development history, is demon-

stration of consistent process performance and product quality during process characterization (Process Con-

trol) and conformance (Assurance). These data are not typically available at an early clinical stage to support

the consistency of a process utilizing legacy cell lines. As such, some legacy cell lines may receive IND nonhold

questions regarding clonality and a request to provide additional assurance of clonal origin (Additional Assur-

ance). The authors propose that if additional genetic testing is performed and provides assurance of clonal ori-

gin, then no further evidence of clonality or augmented controls should be required (dark gray YES arrow). If

genetic testing does not support clonal derivation or is inconclusive, then augmented control strategies may be

required (dark gray NO arrow), depending on the totality of data, including product quality, process perform-

ance, and stability. Based on the availability of these data, sponsors may decide to bypass additional genetic

analysis (dashed arrows) and rely on a robust commercial application package to satisfy regulatory concerns

regarding clonal origin. Sponsors choosing not to perform additional genetic tests (dashed arrows) may opt to

1) proceed with standard process control strategies, or 2) implement augmented control strategies, as advised

in Welch (4), based on project status. The expectations at this decision junction would benefit from further elu-

cidation from health authorities, as some authors have experienced requests for augmented control strategies

in addition to genetic testing that could demonstrate cell line clonal derivation.



developed as a result of random plasmid integration

rather than site-specific integration because the out-

come of site-specific integration Cell Line Develop-

ment (CLD) should preferentially produce a pool of

clones with one integration site. FISH can be per-

formed using fluorescently labeled probes targeting

the integrated genes, for example, the antibody light

chain and heavy chain or any other transgene

sequence. If a consistent FISH pattern is observed in

analyzed metaphase cells, the cell line can be deemed

of clonal origin. The same slide can also be counter-

stained using multicolor-FISH (M-FISH) (20). M-

FISH consists of “painting” the arrested metaphase

chromosomes with a specific mixture of fluorochrome

probes for the different Chinese hamster chromo-

somes. The resultant color pattern can be used to

identify the specific hamster chromosome and chro-

mosome region where the target sequences reside.

In the case that two or more FISH patterns are found in

the MCB, either the cell line is nonclonal in origin or

chromosomal rearrangement has occurred during prop-

agation from the point of clone isolation to MCB prep-

aration. CHO cell lines are known to have an unstable

karyotype (11). Therefore, the potential for genetic

drift and the occurrence of subpopulations are high

when developing a CHO-derived cell line. In such sit-

uations, the application of M-FISH can enable a differ-

entiation between a cell line of nonclonal origin and a

cell line in which secondary genetic populations have

derived from a single progenitor cell by processes such

as chromosome deletion, amplification, or transloca-

tion. An example from an author: An MCB was found

to contain two populations of cells using dual-color M-

FISH—one with two chromosome integrations (chro-

mosomes A and B) and another having only one inte-

gration on chromosome A. From M-FISH analysis, the

cells having one integration were confirmed to carry

the same integrated chromosome A as the cells carry-

ing both chromosome A and B integrations. This sug-

gests that the cell line was of clonal origin but

generated a second population by gene or chromosome

loss owing to plasticity of the CHO genome.

Genomic plasticity of the CHO cells might impose li-

mitation of this technique. Multiple probes binding

per cell, or differential data in M-FISH owing to com-

plex genomic rearrangements, might be challenging to

obtain enough resolution to parse out the genetic events

and conclude on clonality. As such, these methods may

TABLE I

Genetic Methods for Characterizing Clonal Origina

Method Production Cell Line Analytical Subclones

Southern

Blots

Traditionally used to compare MCB with EOP

cells. Accepted methodology for BLA filing.

A single shared hybridization band in a suitable

number of analytical subclones can provide

assurance of clonal origin.

FISH Karyotype analysis of individual cells from the

MCB can identify unique consistent integration

sites to support clonal origin.

Not necessary, as integration site consistency can

be detected in MCB.

NGS Whole genome sequencing and TLA can provide

detailed transgene and integration site DNA

sequence.

Unique transgene integration sites or sequence

variant markers can be used as clone-specific

markers for subsequent PCR-based assays.

TLA provides greater sequence coverage of the

targeted transgene and flanking genome and can

detect low frequency gene of interest sequence

variants.

Markers identified in a suitable number of

analytical subclones can provide assurance of

clonal origin. These NGS methods could be

performed directly on analytical subclones but

are data and cost intensive compared with PCR-

based assays.

PCR Inverse and splinkerette PCR can be used to

identify transgene genomic integration fusion

junctions and flanking sequences.

Genomic integration site junctions are unique

identifiers that can be used for comparing a

suitable number of analytical subclones to

provide assurance of clonal origin.aFor Random Integration Expression System.



be useful only to support clonal derivation depending

on the extent of genetic drift over time.

3.3 Southern Blotting

Southern blotting is a standard practice for testing

genetic stability of cell lines during the cell line devel-

opment process. The principle is to assess recombinant

biologic transgene hybridization banding patterns in

electrophoresed host cell genomic DNA. For antibody

expressing cell lines, heavy (HC) and light chain (LC)

labeled probes can be used to screen a number of sub-

clones, comparing the hybridization patterns with pa-

rental MCB DNA. Presence of one or more consistent

hybridization bands in the genomic samples would sup-

port a position of clonal derivation of the cell line that

generated the MCB.

A genetically stable cell line shows the maintenance of

HC and LC banding pattern over extended culture genera-

tions. Whereas maintenance of the banding pattern is a

strong indication of stability, inconsistent banding pattern

does not necessarily indicate a nonclonal cell line, as in-

herent heterogeneity could increase with generational age

and may be indicative of an unstable cell line (17). Incon-

sistent banding patterns can be observed owing to the

genomic plasticity of the CHO cells, while deletion, dupli-

cation, amplification, translocation, transversion, or other

complex genetic events cause disappearance of expected

bands and/or appearance of new bands. A recent publica-

tion (14) used Southern blotting to identify loss of HC-

hybridizing bands in two of six analytical subclones, in

two distinct antibody-producing cell lines. The subclones

originated from documented single-cell progenitor host

cell lines using their characterized methods. These results

demonstrate that transgene-related heterogeneity revealed

by analytical subcloning can occur from clonally derived

parental cell lines, highlighting our claim that genetic con-

sistency can help provide clonality assurance, but hetero-

geneity does not mean the parental cell line was not

clonal. It is even possible that the process of subcloning

subjects cells to extreme growth conditions for extended

periods, leading to the observed genetic drift and change

in the banding patterns seen by Southern blotting (14).

One of the frequently cited criticisms of Southern blot-

ting is the low sensitivity of the technique. Low-level

sensitivity presents limitations in detecting transgene

integration site changes for low copy number cell lines.

Therefore, in the scenario that Southern blotting cannot

identify common genetic events in subclones and

MCB, techniques with higher sensitivity and resolu-

tion, described below, can perhaps be utilized.

3.4 NGS-Based Technologies

NGS technologies have revolutionized the field of

genomics studies, including completion of a draft CHO

genome sequence that serves as a foundation for CHO

cell genetic characterization studies (21). The follow-

ing are examples of assays based on NGS that can be

used to interpret clonality of the cell line.

3.4.1 Whole Genome or Exome Sequencing: Whole

genome sequencing (WGS) of a legacy CHO cell line

can determine transgene copy number, arrangement,

and genomic integration site. However, the complexity

and cost of complete genome sequencing make it

somewhat impractical for assessing clonality across

multiple CHO samples. Whole exome sequencing

(WES) is a targeted NGS approach that focuses specifi-

cally on protein-coding genes. WGS and WES are

powerful tools for generating a wealth of transgene

sequence information that could be used for identifica-

tion of clonal cell lines. However, genome- or exome-

level sequencing is not a suitable tool to assess clonal-

ity of cell lines without comparison with other clones

or analytical subclones as a reference. NGS-based

assays provide high sensitivity data, but the prohibitive

drawback of the technique is the complexity of the

dataset and time required to comprehensively interpret

the data especially for complex cell lines with large

copy numbers and multiple integration sites. Further, it

is unclear if WGS could provide assurance of clonality

across subclones, given the degree of genetic drift and

potential chromosome rearrangements that can occur

during clonal outgrowth and subsequent subcloning

(22). Therefore, NGS may be best suited to design mul-

tiplexed PCR reactions to detect transgene fusion junc-

tions or integration sites that can further be used to

understand clonality of the cell line.

3.4.2 Targeted Locus Amplification: Targeted locus

amplification (TLA) is a technique that crosslinks

DNA, to constrain loci that are in proximity, and then

uses a combination of inverse PCR and deep sequenc-

ing to obtain data targeting the transgene integration

sites in the cell line (23). TLA provides consensus

sequence information across the entire integrated ex-

pression vector, as well as fusion junction sequences to

other transgene concatemerization sites, if they exist.

This yields a more complete analysis of the transgene



integration architecture for designing PCR screens.

Comparison of MCB and subclone TLA data, for

example, integration site sequences and approximate

number of integration sites, could support clonality.

The throughput is lower than PCR-based assays and

could limit the number of subclones to be analyzed and

compared. Also, the potential for genetic rearrange-

ments applies and should be considered when design-

ing experiments and interpreting data. However, TLA

can be very useful to generate markers for clonality,

such as the unique transgene to CHO genomic DNA

(TG-CHO) fusions that arise at integration (16). These

TG-CHO fusion junctions can be detected by simpler

PCR-based methods to follow clonal lineages, as

detailed in Section 3.5.

3.5 PCR-Based Methods

PCR-based methods use a targeted approach and provide

a practical tool for assessing clonality in CHO clones

and cell lineages. Methods such as inverse PCR (iPCR)

(24) and splinkerette PCR (spPCR) (25–27) can be used

to identify the TG-CHO genomic sequence junctions in

subclones and individual production cell lines. Both

methods work to extend the sequence coverage outside

the integrated transgene into the adjoining host genome

without previous knowledge of the integration site. The

iPCR and spPCR methods provide only junction sequen-

ces biased to those near the plasmid PCR primer-binding

site. Once the TG-CHO junction sequences are identi-

fied, it is straightforward to design PCR genetic finger-

printing methods to characterize and compare cell banks

and subclones to provide assurance of genetic consis-

tency in the production cell line. These technologies are

useful to assess clonality in legacy cell lines derived

through random integration of transgenic expression cas-

settes, wherein the genomic insertion sites are unknown

and expected to be unique in lineages derived from inde-

pendent integration events. PCR genetic fingerprinting

can support clonal origin by showing that cell lines with

the same TG-CHO sequence junctions derive from the

same single-cell progenitor. However, it will not yield in-

formation about gene rearrangements that do not disrupt

the PCR amplicon. Conversely, rearrangements can occur

in subclones derived from single-cell progenitors (14),

and this would inhibit amplification if the rearrangement

disrupted the PCR target sequence.

McVey and others (27) recently showed the elegant

application of spPCR in the instance where Southern

blotting showed disappearance of bands in some of the

subclones. Specifically, a splinkerette was used to iden-

tify various integration sites in the cell line. Further

detailed characterization of the integration sites was

performed in various subclones as well as the MCB.

This confirmed the presence of a common genetic

event (integration site in this specific case) across vari-

ous subclones and the MCB. Such integration site anal-

ysis and confirmation of common genetic events could

further support clonality of the cell line.

As another example, diagnostic PCR was tested on pre-

production cell lines that were identified as clonally

derived through cell imaging. Unique TG-CHO junc-

tions identified by TLA in each of three independent

cell lines were found to be identical by PCR in all re-

spective subclones, demonstrating high probability that

the cell lines each derived from a single cell (16). In

cases such as this, when all tested subclones were iden-

tical, the results provide assurance of clonal derivation

for each of the pre-MCBs. However, negative results

would not exclude the possibility of single-cell origin,

as discussed in the Southern blot example above.

4. Further Discussion and Conclusions

Production cell line development methods have

evolved considerably over the past 30 years since com-

mercializing the first CHO cell-produced biologic (28).

A concerted focus by the industry to improve cloning

methods has led to more rapid single-cell cloning tech-

nologies with higher probability of clonality. Sponsors

are on a path to utilization of these improved methods

for new programs. However, legacy cell lines face

increased scrutiny, as they were generated using out-

dated cell cloning methods, resulting in lower probabil-

ity of clonal derivation. In these cases, health authority

questions regarding clonal origin are being received at

initial Investigational New Drug (IND) filing, preced-

ing process and full genetic characterization efforts by

sponsors. It is difficult for sponsors to select an appro-

priate path to address these questions at this stage, even

though some may wish to address them expediently to

derisk the program as soon as possible.

In the absence of current regulatory guidance, the

authors have presented our position that additional

genetic testing does not fully mitigate risk, as genetic

demonstration of clonal origin does not ensure process

consistency and, conversely, nonclonal cell lines can

produce consistent process/product. No single or com-

bined genetic technique(s) can conclusively demonstrate



clonality owing to the plasticity of the CHO cells and

high rate of genetic drift. They, at best, provide support-

ing data that the cell line was clonally derived. The

methods described herein can be useful if they support

additional assurance of clonality and can derisk develop-

ment of legacy programs and may strengthen regulatory

filings. However, if they do not provide clear assurance

of clonality, the cell lines still can be suitable for com-

mercial manufacturing depending on demonstration of

process and product consistency.

The current authors advocate that demonstration of pro-

cess and product consistency should be sufficient to

approve a license application without further evidence

of clonality. If sponsors can provide a data package at

the time of the marketing application to regulatory

authorities that a legacy cell line has robust process per-

formance and the product consistently meets acceptance

criteria for its CQAs, we believe additional proof of

clonality through genetic testing or augmented control

strategy, beyond ICH Q5B specified, should not be

required, as the risk associated with the probability of a

nonclonal bank would have been addressed through the

totality of data. Thus, the true utility of additional

genetic testing would be the option to perform tests early

in the development cycle that could mitigate the spon-

sors’ business risk of developing a therapeutic biologic

in a legacy cell line through to commercial application.

We strongly believe that mutual agreement and under-

standing must be reached on the utility and possible out-

comes of genetic testing for supporting clonal derivation

of legacy cell lines. Without mutual agreement with

health authorities, companies may not undertake such

resource-intensive studies, as the decision to pursue addi-

tional testing to satisfy clonality concerns will ultimately

belong to the companies striving to develop therapeutic

biologics. The authors all recognize that commercializ-

able manufacturing processes must demonstrate process

and product consistency, and we should strive to put con-

trols in place to ensure this reproducibility. Clonal deriva-

tion of manufacturing cell lines can contribute to this

process and product consistency, but it does not guaran-

tee consistent production of a safe and efficacious prod-

uct, which is the ultimate goal for process development,

and is in line with regulatory expectations.

Acknowledgments

We would like to acknowledge the IQ consortium

leadership group for their support.

This manuscript was developed with the support of the

International Consortium for Innovation and Quality in

Pharmaceutical Development (IQ; www.iqconsortium.

org). IQ is a not-for-profit organization of pharma-

ceutical and biotechnology companies with a mission

of advancing science and technology to augment the

capability of member companies to develop trans-

formational solutions that benefit patients, regulators,

and the broader research and development community.

Conflict of Interest Declaration

There are no conflicts of interest to declare for any of

the authors.

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