REVIEW

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Advances in Lentivirus Purification

Ana Sofia Moreira, David Guia Cavaco, Tiago Q. Faria, Paula M. Alves,
Manuel J. T. Carrondo, and Cristina Peixoto*

usually, the transduction of hematopoi-

Lentiviral vectors (LVs) have been increasingly used as a tool for gene and cell etic stem cells[6–11] or T-cells[12,13] —and in
therapies since they can stably integrate the genome in dividing and vivo—with gene delivery to the central ner-
nondividing cells. LV production and purification processes have evolved vous system[14] and the retina.[15] A compre-
substantially over the last decades. However, the increasing demands for hensive review of the clinical applications
of LV has been published recently.[16] The
higher quantities with more restrictive purity requirements are stimulating the
approval by the US Food and Drug Admin-
development of novel materials and strategies to supply the market with LV in istration (FDA), in 2017, of two chimeric
a cost-effective manner. A detailed review of each downstream process unit antigen receptor (CAR) T-cell therapies for
operation is performed, limitations, strengths, and potential outcomes being the treatment of B-cells lymphoma and
covered. Currently, the majority of large-scale LV manufacturing processes are leukemia evidences the advantages of these
viral vectors and is proof of its successful
still based on adherent cell culture, although it is known that the industry is
use at a clinical level.[17,18] For other types
migrating fast to suspension cultures. Regarding the purification strategy, it of cancer, the use of LV for immunization
consists of batch chromatography and membrane technology. Nevertheless, remains a promising application.[16,19]
new solutions are being created to improve the current production schemes The success of these clinical trials re-
and expand its clinical use. quires improved methods for purification
of LV both quantitively, increasing process
capacity, and qualitatively replacing tradi-
tional methods used at small scales that are
not economically and/or technically viable
1. Introduction for larger scales.[20,21]
Lentiviruses are enveloped viruses members of the Retroviradae The current production technologies commonly result in low
family that have become increasingly relevant for biopharmaceu- functional titers, ranging from 106 to 108 transducing units per
ticals since they lead to stable integration of the transgene(s) to mL (TU mL−1 ), depending on the production system, LV pseu-
be expressed in both dividing and nondividing cells.[1,2] Addition- dotype, harvest conditions, or even the titration technique.[22]
ally, their integration patterns seem less risky than 𝛾-retroviral Additionally, impurities vary with the production system. For
vectors[1,3] justifying their widespread usage from functional ge- example, the serum is present when LVs are produced by ad-
nomics to cell engineering studies, as well as, recombinant pro- herent cell lines, and the plasmid DNA is used for transient LV
tein production and clinical gene therapy.[4] production. Therefore, when developing a downstream process
Over the past years, the number of gene therapy clinical tri- (DSP), the concentration of LV is pursued along with increasing
als based on lentiviral vectors (LVs) has grown to attain 10.1% of LV purity, which must consider the impurities profile of the
the total gene therapy trials in 2019.[5] Their use in gene therapy production broth. Just like any other biological product, the de-
for several conditions has been reported both ex vivo—involving, gree of concentration and purification needed is fundamentally
connected to what the LVs are intended for. Research/small-scale
purposes do not generally require a very pure product.[23] How-
A. S. Moreira, D. G. Cavaco, Dr. T. Q. Faria, Prof. P. M. Alves,
Prof. M. J. T. Carrondo, Dr. C. Peixoto ever, when the product is intended for clinical use, the safety
iBET of the drug is a concern. The standard depends essentially on
Instituto de Biologia Experimental e Tecnológica the type of administration. For ex vivo applications the purity
Apartado 12, Oeiras, Portugal level of the drug product is more flexible.[24] Additionally, the
E-mail: peixoto@ibet.pt
multiplicity of infection (MOI) must be kept as low as possible to
A. S. Moreira, D. G. Cavaco, Dr. T. Q. Faria, Prof. P. M. Alves
Instituto de Tecnologia Química e Biológica António Xavier
reduce the tumorigenic potential and cytotoxicity.[20,100] For an in
Universidade Nova de Lisboa vivo approach doses of, at least, 1011 –1012 per patient are needed,
Av. da República, Oeiras, Portugal which will also vary according to target tissue.[4,27] In this case,
stricter control of the purity to reduce an immunogenic response
© 2020 The Authors. Biotechnology Journal published by Wiley-VCH
GmbH. This is an open access article under the terms of the Creative
by the patients is needed.[24,28–30]
Commons Attribution-NonCommercial License, which permits use, DSP must also consider the low stability of the LV func-
distribution and reproduction in any medium, provided the original work tional particles, which is affected by temperature,[31] ionic
is properly cited and is not used for commercial purposes. strength,[26,33] pH[31] and shear stress.[34,35] Beyond environmen-
DOI: 10.1002/biot.202000019 tal conditions, the stability is highly dependent on the particle

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itself, mainly on the envelope protein (pseudotype) but also on

the viral core.[26,35,36] Vesicular stomatitis virus G protein (VSV-
G) is the most used LV pseudotype due to its broad tropism
and robustness throughout the purification processes. Neverthe-
less, DSP schemes for other protein envelopes (e.g., baboon en-
dogenous virus (BaEV), RD114-TR, gibbon ape leukemia virus
(GaLV)) are emerging since they present higher tropism for spe-
cific target tissues per cells.[26,37,38]
The present article aims to comprehensively review the latest
technical developments in the purification of LV and trace pos-
sible future trends in the field. First, the traditional methods are
identified and then the more promising and easier scalable tech-
nologies are reviewed.

2. Traditional LV Purification and Concentration

Methods
The traditional purification and concentration techniques are
mostly used if the needs of LV can be satisfied by small-scale
production, for instance, scientific research. The centrifugation-
based methods can be used as concentration and/or purification
techniques. Viral pelleting and resuspension procedure is often
used as a concentration method.[39–41] The use of density gradi-
ent centrifugation or ultracentrifugation using sucrose[42,43] or
iodixanol[44] has been reported and can achieve some degree of
purification along with reasonable concentration.[41,45] Another
technique for small-scale purification schemes uses polyethy-
lene glycol (PEG). This polymer sterically excludes viral parti-
cles from the solution leading to their precipitation. Viral par-
ticles are recovered by centrifugation and high yields can be
achieved.[43,45]
Flaws of traditional methods include concerns with scalability
and the concentration of impurities in the product.[22] Although
a high degree of purification can be obtained, the use of ultracen-
trifugation reduces the infectiousness given the stress conditions
to which LVs are subject. Thus, the field is gradually moving away
to more versatile technologies.
3. Scalable Methods for LV Downstream
Processing
The development and use of DSP with chromatography steps and
membrane-based separation operations have been described over
the past years.[46] The processes can be divided into three main
phases: i) harvest and clarification; ii) intermediate purification,
with one or more concentration and purification steps; and iii)
polishing, formulation, and sterilization. The nucleic acid diges-
tion is usually required and can take place in different phases of
the process (see Figure 1). Currently, overall recovery yields for
large scale DSP range from 20% to 40%.[22]
3.1. Harvest and Clarification
LVs are extracellular bioproducts released by budding from the
producer cell. The majority of large-scale LV production pro-
cesses use transient transfection of human embryonic kidney
Figure 1. Downstream processing workflow for Lentiviral vectors purifi-
cation. This image illustrates three different sequences of unit operations
described for Lentiviral vector processing.[21,24,33] The nuclease used in
the represented processes was Benzonase that can be introduced in dis-
tinct stages of the process. For Valkama et al. the nuclease step is part of
the upstream harvest. NF, Normal flow.
(HEK) 293 or 293T adherent cells in batch mode and the harvest
occurs generally once or twice at 48–72 h post-transfection. Al-
though, more recently, the industry has been also transitioning
into suspension cultures.[47–53] The harvest quality is highly
dependent on cell viability which impacts the amount of host cell
(HC) derived impurities. Thus, the choice of the time of harvest
must consider not only the productivity but also the quality of
the supernatant.[46,54]
The clarification’s main goal is to remove cells and cell de-
bris from the LV containing media. This step generally com-
prises depth filtration,[21,33] normal flow microfiltration,[26,55] low-
speed centrifugation, or a combination of these operations. The
use of centrifugation followed by microfiltration[36,41,43] is a com-
mon strategy to reduce filter clogging and minimize fouling at
small volumes. With similar goals, but for larger scales, a mem-
brane cascade is commonly used,[26,49,55] the final pore size being
0.45 µm.[24]
The recoveries for these early steps are commonly not re-
ported, although, after optimization, small losses in functional
titer can be achieved.[21,26,33] Valkama et al. recently reported
functional recoveries close to 100% using a depth filter for clari-
fication of large supernatant volumes containing VSV-G pseudo-
typed LV.[21] However, the success of this operation can be highly
dependent on the pseudotype. Using a GaLV-TR pseudotyped LV,

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Boudeffa et al. obtained very low recoveries in the clarification
step later improved by supernatant dilution and stabilization.[26]
Nonetheless, to meet the LV’s increasing demand, recent
developments in upstream processing (USP) are emerging and
the integration of the early steps of DSP in USP can be highly
advantageous.[48,49,56] Recently, Oxford Biomedica reported the
use of Tangential Flow Depth Filtration (TFDF) to harvest and
clarify LV produced in suspension culture[57] resulting in high
recoveries and potentiating multiple harvests. Moreover, for
suspension cell cultures operating under perfusion, retention
devices, like the Virus Harvest Unit (VHU), allow the harvest
of clarified viral stocks.[48,58,59] Regarding adherent cell cultures,
several bioreactor configurations can be used for continuous
LV production.[60–63] Tangential flow microfiltration[64,65] and
continuous flow disk-stack centrifugation[65] are also continuous
techniques, although never reported for LV clarification.
3.2. Nucleic Acid Cleavage
To increase the overall biosafety of the final product[20] and com-
ply with the regulatory standards[66] a nucleic acid cleavage step
must occur. The use of Benzonase or Denarase reduces DNA size
and decreases bulk viscosity.[41,46] These enzymes require mag-
nesium ions as a co-factor and show optimum activity at 37 °C;
however, different experimental conditions (e.g., amount of en-
zyme, temperature, incubation time, salt concentration, pH, and
the presence of enzyme inhibitors) are reported regarding nu-
cleic acid cleavage step. Recently, Oxford Biomedica developed
a system (SecNuc) that avoids the addition of nucleases that are
co-produced with LV.[67]
Many times, nuclease step immediately precedes[21,68] or suc-
ceeds clarification.[24,69] In other processes, it is included in the
USP[26,55] or later DSP phases after some volume reduction.[70–72]
The early location of this step results in higher nuclease con-
sumption increasing the manufacturing costs which can be
counterweighted by easier subsequent DNA clearance and also
nuclease removal which must be accomplished if LVs are in-
tended for clinical use.[4]
3.3. Intermediate Purification
Following clarification, scalable purification steps for LV use
chromatography and/or Tangential Flow Filtration (TFF), both
techniques achieving some degree of concentration of the bulk
product.
Chromatography is a key technology in purification protocols
for the main biotherapeutic products offering scalability, purity,
high recovery rates, and reproducibility.[73] The classic resin
media were originally designed for protein purification; thus,
the large hydrodynamic radius of the LV raised some technical
constraints. The use of beads often restricts access to the bind-
ing sites, resulting in small dynamic binding capacities.[74,75]
Additionally, the high column backpressure also leads to limited
flow rates. The mass transfer in this kind of stationary phase is
predominantly limited by diffusion, whereas in membrane ad-
sorbers and monoliths it is mostly convective.[73,76] This allows a
quicker processing and larger throughput, making this supports
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ideal for virus capture. Anion-exchange and affinity chromatog-
raphy have been used in the early purification steps of LV
(Table 1).
3.3.1. Anion-Exchange Chromatography
Anion-exchange chromatography (AEX) is widely used for cap-
ture in large-scale bioprocesses. Briefly, the purification of these
viruses is performed in a bind-elute mode where the LV particles,
having a negative surface charge at working pH, are adsorbed by
the positively charged matrix.[41,54] The elution is performed with
buffer ionic strengths between 0.5–1 m NaCl.[4,56] As LVs are la-
bile, high salt concentration can lead to virus inactivation.[1,77] A
loss of 50% of LV biological activity after 1 h of exposure to 1 m
NaCl has been reported.[78] To reduce losses during the AEX step
it is often necessary to dilute the viral vector right after the elution
step.[79]
Membranes and monoliths stationary phases, as with conven-
tional chromatography resins, can be functionalized with strong
or weak ion ligands.[41,80] Strong ion exchangers with quaternary
amines as ligands have been used to produce high-quality
virus stocks,[81] in particular with Sartobind Q and Mustang
Q commercial membranes.[21,26] Conversely, weak exchangers,
for example, CIM Monolith with DEAE (diethylaminoethanol)
ligand, have also been reported in LV purification with high re-
covery yields.[33] In general, this technique results in functional
particle recovery yields ranging from 30% to 80% (Table 1).
Since binding is driven by electrostatic interactions, AEX ma-
trices are not selective for LV, and negatively charged nucleic acids
and proteins bind to the chromatographic media.[54,77] The high
salt concentration required to elute LV leads also to the release of
bound impurities. Merten et al. achieved around 99% and 86% of
protein and DNA removals.[24] However, additional purification
steps are usually required to obtain clinical-grade virus stocks.[81]
Novel stationary phases are continually being developed.
Ruscic et al. reported LV purification using regenerated cellulose
nanofibers derivatized with a quaternary amine and produced
by electrospinning. The authors claimed to achieve a volumetric
concentration factor of 100, with a recovery yield of 85%.[36]
3.3.2. Affinity Chromatography
The affinity chromatography (AC) is used to separate particles
from the broth-based on a highly specific interaction between
them and a selected immobilized ligand.[4,82] Unlike AEX, AC
presents a high selectivity, which potentially leads to higher
purity and concentration outcomes. Therefore, its incorporation
in a DSP can lead to a reduction in the number of unit operations
required and consequently greater overall recoveries.[4]
One of the first reported studies used the immobilized metal
affinity chromatography (IMAC). This technique explores the
binding between an immobilized metal ion and a molecule
capable of sharing electrons with it. For LV purification this
would involve the modification of the envelope protein with
the introduction of a histidine tag. Such insertion can result in
lower expression or compromise the protein’s function, with
loss in particle infectivity.[83–85] Furthermore, the desorption step
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Table 1. Anion exchange and affinity chromatography methods used for the purification of LV.

Column/matrix PT Load BV Des. reagent Buffer pH Rec. (%) Ref.

Anion Exchange
Resin
HiTrap Q HP VSV-G 1 mL 1 mL LG: up to 1 m NaCl 100 × 10−3 m Tris 7.5 89.6 [41]

DEAE Sepharose FF – 30 mL 16 mL SW: 0.65 to 1 m NaCl – – 95 [116]

DEAE resin VSV-G 48 L – 750 × 10−3 m NaCl PBS – – [24]

Poros DM/50 GaLV-TR 45/29 mL 8 mL SW: elution at 0.7 m 20 × 10−3 m Bis–Tris 6.0 61.5 [26]

NaCl + 5% sucrose +
2 × 10−3 m MgCl2
Poros D 50 GaLV-TR 20 mL 4 mL 0.65 m NaCl 20 × 10−3 m Bis–Tris 5.5 100 [91]

Membrane
a)
Sartobind Q VSV-G 11 kg 400 mL SW: 0.3–1.50 m NaCl 50 × 10−3 m HEPES 7.5 22.4 [21]

b)
Mustang Q XT5 BaEV-R-less 1L 5 mL 2 m NaCl 50 × 10−3 m Tris 8.0 >33 [49]
b)
Mustang Q XT5 VSV-G 1L 5 mL 2 m NaCl 50 × 10−3 m Tris 8.0 >55 [49]

Nanofiber (RC w/Q) RDPro 400 mL 0.1 mL LG: elution at PBS + Tween 20 7.45 85.4 [36]

0.6–0.9 m NaCl
Mustang Q VSV-G 6L 60 mL 1.2 m NaCl 25 × 10−3 m Tris–HCl 8 – [55]

Mustang Q XT5 VSV-G 1.5 L 5 mL 1.5 m NaCl PBS 7.2 – [39]

Mustang Q Acrodiscs MVG 540 mL 0.8 mL LG: elution at 25 × 10−3 m Tris–HCl 7.4 64.36 [117]

0.2–0.4 m NaCl
Mustang Q XT – 3L – SW: 0.5 and 1.2 m 20 × 10−3 m Tris + 7.6 50–60 [68]

NaCl 2 × 10−3 m MgCl2

Sartobind Q75 VSV-G – 75 mL SW: elution at 1.2 DMEM (or TSSM) – – [118]

NaCl
Mustang Q capsule VSV-G – – 1.5 m NaCl 50 × 10−3 m Tris–HCl 8 – [96]

c)
Microcapillary film (EVOH VSV-G 15 mL 1.5 mL 1.3 m NaCl 25 × 10−3 m Tris–HCl 8.0 11/10 [119]

w/Q)
LentiSELECT 40/500/1000 VSV-G 40 mL – – – – 52 [79]

LentiSELECT 40/500/1000 VSV-G 500 mL – – – – 38 [79]

LentiSELECT 40/500/1000 VSV-G 1000 mL – – – – 37 [79]

Mustang Q VSV-G 36–52 L 60 mL 1.5 m NaCl – 7.5 >60 [72]

Monolith
CIM DEAE VSV-G – – SW: elution at 10 × 10−3 m Tris 8.0 80 [33]

0.65 m NaCl
Affinity
CIMac RDPro 50 mL 0.1 mL 15 × 10−3 m biotin + X-VIVO 15 – 20 [85]

BSA
Pierce monomeric avidin VSV-G 12 mL 2 mL 2 × 10−3 m biotin PBS – 68 [86]

coated column
d)
Fractogel EMD Heparin VSV-G 35 mL 1 mL 0.35 m NaCl 20 × 10−3 m Tris–HCl 7.5 53 [47]

PT, Pseudotyping; BV, Bed volume; Des. reagent, Desorbing reagent; Rec., Recovery of functional particles; SW, Stepwise elution; LG, Linear gradient elution. Calculated by
a) b) c)
the authors based on the article’s data. Recovery for a peak at 444 × 10−3 m NaCl. Recovery of more than one unit operation, including chromatography. Capture of
d)
clarified LV/capture of unclarified LV. Fractogel EMD Heparin had been discontinued.

requires the use of imidazole resulting in LV inactivation.[4,84]
Finally, another major drawback of IMAC for LV purification is
the possible adverse effects promoted by His-tag in clinical trials,
thus ensuring its current rejection.[77]
The use of heparin AC in LV purification enables an elution
under mild conditions.[47] However, its low selectivity can result
in a binding competition of host cell proteins and DNA with LV
and consequently co-elution.[1,4,47] Given the animal origin of
heparin, its use will be avoided in industrial applications.[22]
Another strategy involves LV labeling with desthiobiotin[86] or
biotin mimics[85] that binds streptavidin columns (or magnetic
beads) with low affinity. LV desorption is made with biotin. Chen
et al.[86] modified a cell line to generate desthiobiotin-labeled
LV further purified in monomeric avidin-coated chromatography
columns. More recently, Mekkaoui et al.[85] reported the purifica-
tion of an RDPro pseudotyped LV labeled with cTag8. This biotin
mimic was genetically encoded, and the technique is envelope-
protein independent.

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Table 2. Tangential-flow filtration for concentration and/or diafiltration of LV.

PT MWCO Pressure data Area Feed VCF Rec. (%) Ref.

Cassette
5 × Sartocon Hydrosart (Sartorius) Cellulose VSV-G 100 kDa TMP ≤ 0.1 bar 3.0 m2 126.8 kg 13 70–80 [21]

2 × Sartocon Hydrosart + Cellulose VSV-G 100 kDa TMP ≤ 0.2 bar 4.2 m2 178.4 kg 18 – [21]

1 × Sartocube Hydrosart (Sartorius) Cellulose VSV-G 100 kDa TMP ≤ 0.2 bar 4.2 m2 178.4 kg 18 – [21]

a)
Sartocon Slice Hydrosart (Sartorius) TMP ≤ 0.2 bar 0.1 m2 855 mL 1.5 ≈60
Vivaflow 50 (Sartorius) – VSV-G 100 kDa – – – – >55 [49]

BaEV R-less >33

Centramate cassette (PALL) Omega PES VSV-G 50 kDa ΔP = 0.3 bar – 300 mL 11–12 100 [43]
b)
Centramate cassette (PALL) Omega PES VSV-G 100 kDa PFeed = 1 bar – 300 mL 11–12 91.4 [43]

Centramate cassette (PALL) Omega PES VSV-G 300 kDa – 300 mL 11–12 81.3 [43]

ΔGP Jenv 50 kDa 184 mL 7.4 97.9

Omega T series (PALL) Omega PES VSV-GI 100 kDa – 0.1 m2 – 40 – [68]

Omega T series (PALL) Omega PES VSV-GNJ 100 kDa – 0.1 m2 – 40 – [68]

Omega T series (PALL) Omega PES Cocal 100 kDa – 0.1 m2 – 40 – [68]

Pellicon-2 mini filter (Merck PVDF VSV-G 500 kDa – 0.1 m2 – 10 – [96]

Millipore)
Vivaflow (Sartorius) – VSV-G 100 kDa PFeed = 1 bar – – 72 [33]

Omega screen channel-cassette Omega PES VSV-G 100 kDa PFeed = 1 bar – 67 100 [88]

(PALL) 300 kDa

Hollow fiber
(Spectrum Labs) mPES RDPro 500 kDa TMP = 0.075 bar – – – ≈20 [36]

(Spectrum Labs) – VSV-G 500 kDa TMP = 0.35 bar 190 cm² 1.2 L 8 – [55]

c),d)
(Spectrum Labs) mPES GalV-TR 500 kDa PFeed = 0.5 bar 115 cm² – – 40 [ 26,91]

c)
(GE Healthcare) PES 750 kDa 110 cm² 190 11.2 64–74
c)
410 cm² 550 36.6 86
c)
(GE Healthcare) PES VSV-G 750 kDa 110 cm² 190 11.2 100
FlexStand (GE Healthcare) PS VSV-G 100 kDa TMP = 0.5 bar – – – – [71]

(GE Healthcare) – VSV-G 500 kDa – – – 100 – [70]

(GE Healthcare) – VSV-G 500 kDa – – – 20 – [69]

(GE Healthcare) – VSV-G 750 kDa – – – 20 L – [24]

e)
Tandem TFF (Spectrum Labs) – VSV-G 500 kDa PFeed 615 + 40 cm2 5.5 L 2000 94–100 [120]

FP1: < 0.45 bar

FP2: < 0.6 bar
a)
PT, Pseudotyping; MWCO, Molecular weight cut-off; VCF, Volumetric Concentration Factor, Recovery of functional particles. Calculated by the authors based on the article’s
b) c)
data. The authors consistently reported a recovery increase when operating at higher feed and retentate pressures, maintaining the differential pressure constant. pH of
d) e)
the DF buffer has a considerable impact on vector recovery. Before optimization. Uses a system with two sequential and integrated HF filters, FP1, and FP2.

Despite the encouraging results at a lab-scale for LV purifica-
tion using AC, much has to be improved to translate this tech-
nique into an industrial setting. Particularly, modifications on the
LV envelope may compromise the viral biologic activity, by vector
inactivation or tropism alteration.
3.3.3. Tangential Flow Filtration
Ultrafiltration/diafiltration is the gold standard for virus large-
scale concentration and buffer exchange since it is a fast and ro-
bust technique, easily scaled-up and, transferred to good manu-
facturing practices (GMP).[4] Briefly, TFF is used to concentrate
viral particles in the retentate, whereas small impurities such as
proteins and DNA fragments are removed throughout the pores
into the permeate.[46] Membrane fouling can be a major limita-
tion leading to permeate flow rate decrease and transmembrane
pressure (TMP) increase during the concentration process. TFF
can be applied at different stages of the downstream process.
First, it can be used for concentration of the feed stream, after
virus clarification, reducing the feed volume for the later steps,
and if diafiltration is performed, exchanging the buffer to an
appropriate composition for subsequent operation units.[77] Sec-
ond, it is also commonly employed in the downstream polishing
steps for concentration and buffer exchange or formulation[87]
(see below).
As previously mentioned, one of the major challenges in
LV purification is the maintenance of their biological activity.
Pressure variations, high shear forces, and foaming can lead
to loss of infectivity or even envelope disruption.[21] Membrane
chemistry and pore size are selected by experimental screening
followed by optimization of the critical process parameters
such as flow rate, TMP, membrane throughput, and process
time to assure a smooth operation. Flat sheet cassettes[21,88] or

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Figure 2. General purification scheme of lentiviral vectors for ex vivo or in vivo therapies. Impurities eliminated in each step and added elements during
the process are also represented.
hollow fibers[24,26,72] are used for LV concentration and different
materials are available—polysulfone (PS), polyethersulfone
(PES), polyvinylidene fluoride (PVDF), or regenerated cellulose
(RC).[46,56] The selected molecular weight cut-offs (MWCO)
range between 100 and 750 kDa. A higher impurity clearance
and decrease of process time can be achieved for devices with
larger pore size; however, this can lead to viral loss, as observed
by Valkama et al.[21] These non-recovered LV can leave the system
in the permeate or be entrapped in the membrane. Table 2 lists
the high recovery yields that have been reported by different
groups using LV pseudotyped with VSV-G. This technique
started to be employed for more envelope types as described by
different research teams.[26,43,49] The maximum concentration
factor achieved and the number of diafiltration volumes should
be established considering the final clinical use of the viruses.
3.4. Polishing, Formulation, and Sterilization
The polishing and formulation of LV preparations can be
achieved either by using chromatographic methods (based on
size exclusion) or by TFF.
Some large-scale processes explore the difference in the size
of the LV compared to remaining impurities for the polishing
step.[22,24,70,72] In the size exclusion chromatography (SEC), viral
vectors are excluded from the internal pores of the packing ma-
terial while other biomolecules migrate slowly as they are more
likely retained in the pores.[4,73] A maximum MWCO of 750 kDa
(equivalent to 50 nm) is commonly used to prevent titer loss.[22]
This method enables the efficient removal of small-sized particles
with well-reported recoveries, from 50% to 80%.[4,26,33,72] How-
ever, low flow rate and sample size limitations make this tech-
nique not ideal for the processing of large volumes.[89,90] Besides,
sample dilution by two- to fourfold hampers the production of
highly concentrated viral stocks.[30] Nevertheless, a scalable pro-
cess for purification of GaLV-TR pseudotyped LV was reported
adopting SEC for polishing.[26]
Alternatively, multimodal chromatography, like Capto Core
700, can be used for polishing.[26,41,46,91] This kind of flow-
through chromatography combines the properties of SEC and
AEX. The chromatographic media is composed of particles with
a functionalized core, to which larger particles cannot access,
and a non-functionalized outer layer.[92] Boudeffa et al. reported
recovery of 86% in TU for an LV pseudotyped with GaLV-TR.[91]
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As mentioned above, TFF is frequently used to concentrate
and formulate LV.[21,55,93] A proper virus formulation is cru-
cial to provide maximum stability and guarantee therapeutic
success.[26] The LV must remain biologically active after freeze-
thaw or lyophilization[4,30] and during its clinical use.[56,70] For
ex vivo applications, there is some flexibility in the formulation
that leads to the use of protein-containing media such as Lonza’s
X-VIVO and CellGenix’s CellGro media.[8,24,26,49] Traditionally,
the stabilization of LV has been accomplished with the use of
different buffers systems—PIPES,[70,94] HEPES,[21] HIS-HCl,[95]
Tris-HCl,[68] or PBS[26,96] —coupled with stabilizer adjuvants.
The more common excipients include sugars (sucrose[21,26,70] or
trehalose[95,97] ), or proteins such as recombinant human albumin
and gelatin.[32,95,98]
Finally, sterilization of the drug substance is achieved by fil-
tration through a 0.22 µm filter, before vialing. This is a standard
regulatory requirement[22] and a critical step regarding vector
losses. Valkama et al. reported losses between 30% and 50% in
the last step of their large-scale DSP.[21] The recoveries on this
step are dependent on the membrane material[4] and the LV titer.
Oxford BioMedica verified that sterilization after the final concen-
tration step resulted in a low LV functional recovery. Therefore,
in their DSP protocol, the sterile filtration is placed before the
final TFF which is done under aseptic conditions.[93] Sterilization
can be skipped if the process is performed aseptically in a semi-
closed system.[22,69] The final product is then stored at –80 °C to
protect the virus from thermal inactivation[4,13] or lyophilized.[68]
4. Analytical Approaches and Regulatory
Requirements
From the impure LV containing supernatant to a final prod-
uct, an increase of the vector titer and a decrease of the total
amount of impurities is expected (Figure 2).[30,56] Those impu-
rities are commonly distinguished between process-related and
product-related that were extensively reviewed by Segura et al.[4]
Analytical methods play a key role in process development
and operation, as well as in the characterization of the produced
lots.[61] This characterization is required in GMP manufacturing
and to enter clinical trials.[4] However, like other advanced ther-
apeutic medicinal products, there are still no strict guidelines
for LV manufacturing.[56,99] Efforts in standardization have been
made resulting in a standard for LV analytics[100] and an LV
reference material that will be released soon.[21,25] Each lot
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Table 3. Assays for quality assessment of LV preparations.

Target Assay(s) Ref.

Identity
Confirm identity Western Blot, vector sequencing, restriction enzyme mapping, [ 100]

reverse phase HPLC, SDS-PAGE

Purity
Total protein Bradford, BCA protein assay [ 24,68]

Host cell protein ELISA [24]

Residual endonuclease ELISA [24]

Residual BSA ELISA [24]

Total DNA Fluorimetry (PicoGreen) [24]

Host cell DNA qPCR, fluorimetry [36,121]

DNA size distribution Agarose gel eletrophoresis, capillary electrophoresis [69,122]

E1A and SV40 LTA sequences in the vector qPCR, Southern blot [24]

Transfer of plasmid (vsv-g and gag-pol), and host-cell (EIA qPCR, Southern blot [4,24]

and SV40 LTA) sequences to target cells

Potency
Total viral particles p24 ELISA, PERT, RT qPCR [ 24,69,100]

Functional viral particles (analysis of transduced cells) Flow cytometry, qPCR, ddPCR [ 24,69,100]

Safety
Recombinant competent lentiviruses (RCL) RCL amplification in suitable cell line by serial passages [4,24]

followed by quantitation assay (e.g., PCR, p24 ELISA)

Adventitious agents In vitro testing, PCR-based methods [4,69]

Mycoplasma Culture-based methods, PCR-based methods [4]

Endotoxins/pyrogens LAL test, rabbit pyrogen test [4]

Sterility Bacterial and fungal sterility [4]

Ref., reference; BCA, bicinchoninic acid; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; PERT,
product-enhanced reverse transcriptase; RT qPCR, quantitative reverse transcription-polymerase chain reaction; ddPCR, droplet digital polymerase chain reaction; LAL, Limu-
lus amebocyte lysate.

must be tested for identity, purity, potency, and safety.[1,4] Some
examples of assays used for LV release are depicted in Table 3.
The identity can be confirmed using Western Blot for gag pro-
teins, vector sequencing, restriction enzyme mapping, reverse
phase high performance liquid chromatography (HPLC), or
sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE).[100]
Regarding purity, both process-related and product-related im-
purities should be well characterized in the final product. The
quantification of total proteins can be acquired[101] using colori-
metric assays, like Bradford or BCA.[4,13,24] Other impurities such
as HC proteins, nuclease, and bovine serum albumin can be
quantified by ELISA.[4] The DNA must be present at a concen-
tration below 10 ng per dose and the DNA size under 200 base
pairs.[101] Their quantification can involve spectrofluorimetry and
agarose gel electrophoresis.[1,4,24,69] Since HEK293T has a tumori-
genic phenotype, the LV produced with these cell lines must be
analyzed to quantify E1A and SV40 LTA DNA sequences. If the
production process involves transient transfection, the number
of copies of plasmid sequences (vsv-g and gag-pol) must also be
assessed, using qPCR or Southern Blot.[4,24,69,101]
A good indication of vector quality is the ratio between total
and active particles, correlated with its potency. If this parameter
is too high, the lot must be rejected.[1,4] No reference value is
defined by current guidelines but ratio values in the 103 order
of magnitude are reported.[24,69] The number of total particles
is commonly inferred by p24 titration using ELISA kits.[4,56,100]
Other analytical methods for quantification of total particles
may include the assessment of reverse transcriptase activity
and determination of genomic RNA.[4,102] Physical methods are
commercially available and enable a quicker quantification of
total particles and characterization of size distribution. Those
methods include field-flow fractionation multiple-angle laser
light scattering (FFF-MALLS), nanoparticle tracking analysis
(NTA), flow cytometry, and tunable resistive pulse sensing
(TRPS).[56,103,104] The number of active particles is estimated
by a gene transfer or gene expression assay in a suitable cell
line.[4] For research purposes, when using GFP as the transgene,
the functional titer is often estimated by flow cytometry of the
fluorescent cells.[56,102] However, for clinical purposes, it should
be quantified by the number of reverse-transcribed viral genomes
integrated into target cells using droplet digital PCR (ddPCR)
or quantitative PCR (qPCR) via classical TaqMan of genomic
DNA[100] or by another method suitable for the used transgene.[61]
Also, each vector batch must be evaluated regarding sev-
eral safety parameters, mainly analyzing for recombinant
competent lentiviruses (RCL), endotoxins, mycoplasma, adven-
titious agents, and sterility.[4,105] Physicochemical parameters
like pH, osmolarity, and appearance complement product
characterization.[4]

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5. Concluding Remarks
Over the past decade, lentiviral vectors have become a funda-
mental tool for gene and cell therapy, explaining the increasing
number of clinical trials that use these viruses. The approval
of Kymriah[17] and Yescarta[18] cell therapeutic products have
reinforced the urgent need for simple, fast, and robust manu-
facturing processes of viral vectors. In recent years, this field has
witnessed a tremendous effort to develop and optimize large-
scale production and purification processes capable to meet the
demanding number of viral doses necessary for clinical use.
In this article, a detailed review of each downstream operation
unit was performed, and significant strengths and limitations
were highlighted. For chromatography, the use of convective
driven devices is effective in LV purification schemes and new
stationary phases are emerging with very promising results.[36]
Additionally, affinity chromatography using tailor-made ligands
might prove valuable due to its high selectivity allowing to de-
crease the number of purification steps and increasing recovery
yields.[82,106]
LVs purification processes have evolved significantly, however,
recovery yields remain far from their maximal potential, and the
amount of virus required cannot be efficiently obtained using
these with current technologies. The transition to new produc-
tion systems may overcome these hurdles but can introduce dif-
ferent challenges into the downstream processing of LV.[107] The
increase of the production titer will of course benefit the purifica-
tion train, but the shift for a suspension system will increase the
cell mass during the clarification step. The impurity profile will
also change and an increase in process-related impurities will be
observed (e.g., genomic DNA, HC proteins). Therefore, the DSP
has to engage with those challenges to achieve the same high-
quality viral stocks.[108]
The biopharma industry is now moving to new opera-
tion modes trying to reach fully continuous and integrated
bioprocesses.[109] These improvements are very appealing be-
cause they allow high purity and productivity, reducing the
overall costs.[46] Much has been done to improve these continu-
ous processes, more specifically in continuous chromatography
for other viral particles such as adenovirus,[110] influenza,[111]
and extracellular vesicles.[112] However, the production of LV
using continuous processes may be hampered due to LV cy-
totoxicity, particularly for VSV-G pseudotyped vectors. Until a
robust LV manufacturing process is established, the transition to
continuous production and purification will be difficult to apply
to a larger scale but research at a lab-scale is already committed
to these new processing operation modes.[113–115]
In this review, some future directions were identified, and the
progress made in the manufacturing of LV was described. Some
success stories have already been reported and novel applications
and technological developments are thriving. The trend is likely
to continue, as several groups and companies are committed to
investing more funding and time to push Lentiviral vectors into
broader clinical use.
Acknowledgements
The authors would like to acknowledge funding from the iNOVA4Health
Research Unit (LISBOA-01-0145-FEDER-007344), which is co-funded by
Biotechnol. J. 2021, 16, 2000019 2000019 (8 of 11)
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www.biotechnology-journal.com
Fundação para a Ciência e Tecnologia/Ministério da Ciência e do En-
sino Superior (FCT/MCES). Funds by FEDER under the PT2020 Part-
nership Agreement is also acknowledged. The authors Ana Sofia Mor-
eira and Tiago Q. Faria acknowledge FCT/MCES for the Ph.D. fellowship
PD/BD/135501/2018 and the project PTDC/EQU-EPQ/29306/2017, re-
spectively.
Conflict of Interest
The authors declare no conflict of interest.
Author Contribution
A.S.M. and D.G.C. contributed equally to this work. A.S.M.: Conceptual-
ization, data curation, investigation, methodology, visualization, writing-
original draft, writing-review, and editing. D.G.C.: Methodology, visual-
ization, writing-original draft, writing-review, and editing. T.Q.F.: Concep-
tualization, data curation, methodology, supervision, validation, writing-
original draft, writing-review, and editing. P.M.A.: Funding acquisition,
project administration, supervision. M.J.T.C.: Funding acquisition, project
administration, resources. C.P.: Conceptualization, project administra-
tion, resources, supervision, writing-review, and editing.
Keywords
bioseparation, chromatography, downstream processing, lentiviral vec-
tors
Received: May 29, 2020
Revised: October 15, 2020
Published online: December 8, 2020
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Cristina Peixoto graduated in Applied Chemistry (Branch Biotechnology) New University of Lisbon
and she holds a Ph.D. in Engineering Sciences (ITQB-NOVA). Since 2009, she is the Head of the
Downstream Process Development lab at Animal Cell Technology Unit of iBET. Meanwhile Cristina
coordinated more than 12 research contract projects with Industrial partners, some of the processes
developed are nowadays in GMP for phase I. More recently, the activities were expanded to stem cells
purification and to development of new purification trains for viral based biopharmaceuticals using
advanced materials in integrated and continuous process.

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