Human Gene Therapy - April 2023 - 268

268
OVERTON ET AL.
Figure 6. A chromatogram from a typical run using POROSTM
absorbance units. CIP, clean-in-place.
CaptureSelectTM
AAVX Resin. Cond: conductivity; mS/cm: millisiemens per centimeter; mAU: milli
immunosorbent assay (ELISA, specifically the Progen
AAV2 titration ELISA). The students use the data to calculate
step yield, as required by the U.S. Code of Federal
Regulations (21 CFR 211.103)12 and a mass balance, with
the following equations:
samples (not shown here) and the elevated ultraviolet
absorbance at 280 nm during the load step clearly indicate
that significant purification (e.g., separation of
AAV2 capsids from host cell proteins) occurs during
this chromatography step.
% step yield¼
+i ¼fraction N
i ¼fraction 1 fraction volume mL
load volume mL
load FT volume mL
ðÞi ·AAV2 concentration in fraction capsids=mL
ðÞ·AAV2 concentration in load capsids=mL
ðÞ
ðÞþ
wash FT volumn mL
eluate volume mL
% mass balance¼
ðÞ· AAV2 concentration in load FT (capsids=mL
ðÞ·AAV2 concentration in wash FT capsids=mL
ðÞ·AAV2 concentration in eluate capsids=mL
ðÞþ
cleaning FT volume mL
load volume ·AAV2 concentration in load capsids=mL
ðÞx AAV2 concentration in cleaning FT capsids=mL
ðÞ
ðÞ)
(4)
ðÞþ
ðÞi
(3)
The data provided to the course participants results
in a >95% step yield and a mass balance of just >100%.
However, based on our experience, we see that step
yields vary between 50% and 95%. This variability
suggests that capsids could be partially degraded (denatured)
by low pH elution conditions, although this
has not been proven for the BTEC process. Typically,
no capsid is detected in the load or wash FT samples.
Furthermore, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) of load and elution
The final laboratory in the downstream section of the
course covers UF for the concentration and buffer exchange
of viral vectors. It is performed in TFF mode using
a bench-top PendoTECH filtration unit that houses a
MilliporeSigma Pellicon 3 UF cassette with an Ultracel
membrane of a molecular weight of 30, 100, or 300 kDa.
The laboratory explores the impact of certain process parameters
on UF performance. For example, the effect of
transmembrane pressure on permeate flux is measured,
and the relationship between diafiltration volumes-the

Human Gene Therapy - April 2023

Table of Contents for the Digital Edition of Human Gene Therapy - April 2023

Contents
Human Gene Therapy - April 2023 - CT1
Human Gene Therapy - April 2023 - CT2
Human Gene Therapy - April 2023 - Cover1
Human Gene Therapy - April 2023 - Cover2
Human Gene Therapy - April 2023 - 239
Human Gene Therapy - April 2023 - 240
Human Gene Therapy - April 2023 - 241
Human Gene Therapy - April 2023 - 242
Human Gene Therapy - April 2023 - 243
Human Gene Therapy - April 2023 - 244
Human Gene Therapy - April 2023 - Contents
Human Gene Therapy - April 2023 - 246
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Human Gene Therapy - April 2023 - Cover3
Human Gene Therapy - April 2023 - Cover4
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