John S. Philo & Tsutomu Arakawa, Alliance Protein Laboratories, 2009

1. 1
Challenges in measuring comparability of aggregation and conformation for
biosimilars (and often innovator products too!)
John S. Philo & Tsutomu Arakawa, Alliance Protein Laboratories
ABSTRACT: There is much current interest in analytical methods for demonstrating comparability of
biosimilars to the reference product. Heightened concerns about potential differences in
immunogenicity make it particularly important to show comparability of product molecular
conformation as well as aggregate content and distribution. However key biophysical tools such as
analytical ultracentrifugation, light scattering, and circular dichroism (CD) may not be applicable to
the final product due to interference from excipients. In such cases some biosimilar developers have
tried to re-create the innovator’s bulk drug substance by purifying the active ingredient out of the
reference final product. While this approach may give material suitable for some analytical methods,
we show that for erythropoietin (EPO) this creates a drug substance surrogate that is damaged and not
equivalent with respect to aggregation. Further, in this case subjecting two different final products to
the same re-purification protocol does not produce equivalent changes and thus would give a
potentially invalid comparison.
Some specifics about excipient interference with AUC, light scattering, CD, and/or SEC will be
discussed, with examples. Polysorbate 20 or 80 can be particularly troublesome when present at levels
above ~0.3% and may cause false peaks in SEC, sedimentation velocity, or DLS. Further, because the
detergent-associated impurities in product samples typically differ from those present in the
formulation buffers it is often not possible to correct for the detergent effects by running control
samples or buffer blanks. For CD the UV absorbance of simple salts such as citrate can cause
problems. The strong UV absorbance of bacteriostatic agents present in multi-dose formulations can
pose severe challenges for CD and AUC using absorbance detection.
© copyright 2009 Alliance Protein Laboratories

2. 2
Challenge 1: When bulk drug substance (DS) is not
available for comparability, trying to re-create it from drug
product may produce a compromised DS surrogate, as
happened for EPO
Amgen scientists purified EPO from Eprex® drug product (DP) for purposes of
comparison to the DS for Amgen’s EPO product, Epogen®, and reported
differences in conformation, aggregation, and spectroscopic properties between
these products.1 Subsequently in a collaboration with scientists at Centocor we
showed that this purification procedure had damaged the protein and created a DS
surrogate that was not equivalent to true Eprex DS. Subjecting either Eprex bulk
DS or Eprex DP to this purification protocol caused increased aggregate levels.
Further, the pH 8.4 elution used by Amgen caused degradation of some
component of the Eprex formulation, creating a new non-protein peak in the
elution profile. The fact that this peak was not seen when Amgen tried to re-create
an Eprex-like formulation shows that the components of that formulation were not
equivalent to those in the commercial EPREX product samples.
1
Deechongkit, S., Aoki, K. H., Park, S. S., and Kerwin, B. A. (2006). Biophysical comparability of the same protein from different
manufacturers: a case study using Epoetin alfa from Epogen and Eprex. J. Pharm. Sci. 95, 1931-1943.
2
Heavner, G. A., Arakawa, T., Philo, J. S., Calmann, M. A., and Labrenz, S. (2007). Protein isolated from biopharmaceutical
formulations cannot be used for comparative studies: Follow-up to "a case study using Epoetin Alfa from Epogen and EPREX". J.
Pharm. Sci. 96, 3214-3225.

3. 3
Sedimentation velocity shows that attempting to purify EPO
from either DS or DP causes increased aggregation
0 1 2 3 4 5 6 7 8 9 10
0
5
10
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0.000
0.025
0.050
bulk DS, 1.6% aggregates
DS after purification procedure, 3.6%
material purified from Eprex DP, 3.5%
normalizedc(s)
sedimentation coefficient, s20,w
(Svedbergs)
X 200

4. 4
The pH 8.4 elution buffer used to elute EPO in Amgen’s purification
protocol creates a new elution peak at 50 min that is due to chemical
alteration of components in the formulation buffer.
Hi-Trap elution profiles at 280 nm of EPREX bulk drug substance (blue), EPREX formulated product (red) and
EPREX formulation buffer (black). The elution buffer is the Tris pH 8.4 buffer used by Deechongkit et al. (ref. 1)
The non-protein, post-erythropoietin eluting peak observed by Deechongkit et al. at ca. 50 min is present in
EPREX formulated product and EPREX formulation buffer but not in EPREX bulk drug substance, indicating that
its source is the formulation buffer
New peak arises from the
formulation buffer
⎯ bulk DS
⎯ Eprex DP
⎯ Eprex formulation buffer

5. 5
Challenge 2: Interference from Tween
Tween-20 and/or Tween-80 are common excipients that can interfere with
comparability analysis or product characterization via AUC, light scattering, and
SEC.

6. 6
In sedimentation velocity with absorbance detection, only those
Tween micelles that contain UV-absorbing impurities are detected
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9
Absorbance(280nm,1.2cmpathlength)
Radius (cm)
0 1 2 3 4 5 6 7 8 9 1 0
0 .0
2 .5
5 .0
9 2 .8 % ,
1 .9 8 S
0 .1 % T w e e n -8 0 in w a te r
normalizedc(s)
s e d im e n ta tio n c o e ffic ie n t, s 2 0 ,w
(S v e d b e rg s )
Some velocity scans at 280 nm
and 60,000 rpm for a Tween-80
lot containing relatively high
levels of impurities at 0.1%
(wt/vol) in water.
Tween-80 micelles typically
sediment at about 2 S, a rate
comparable to that for a protein
of about 20 kDa. As we see here
minor amounts of faster-
sedimenting forms are also
sometimes detected.

7. 7
Because it is really the UV-absorbing impurities in Tween that are detected
(possibly including species mobilized from container surfaces), and because
those impurities affect the micelle sedimentation coefficient, it is quite
difficult to provide a matching reference sample to cancel out the Tween
signals
6.0 6.2 6.4 6.6 6.8 7.0
-0.2
0.0
0.2
0.4
0.6
OD
radius (cm)
Some sedimentation velocity
scans early in the run for a
protein containing relatively
high levels of Tween-20 (more
than 0.2%). The buffer sample
loaded into the reference
channel, whose OD is subtracted
point-by-point during data
acquisition, was a poor match
for the actual protein sample,
leading to the negative OD
readings seen here.

8. 8
Therefore for SV analysis of samples containing 0.1% or more of Tween it is
not unusual to see a peak at a sedimentation coefficient near that expected for
the micelles (~2 S). Thus for a peptide product the micelles could be mistaken
for an aggregate, while for antibodies they could be mistaken for a product
fragment, as shown here.
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0.0
2.5
5.0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0.00
0.05
0.10
normalizedc(s)
sedimentation coefficient (Svedbergs)
Tweenmicellesorproductfragment?
a monoclonal antibody containing > 0.3% Tween-80
X 50

9. 9
In SV the Tween micelles sediment like a small protein, but in dynamic light
scattering (DLS) they have a hydrodynamic size near that for an antibody
monomer. The extent of Tween interference for DLS depends strongly on the
product molecular weight and concentration.
The micelles in this formulation buffer
containing 0.1% Tween-80 show up clearly
at a mean radius of 5.06 nm, corresponding
to a globular protein of about 150 kDa.
Clearly the micelle peak overlaps
somewhat with this product peak (mean
radius 7.59 nm). However in this case the
distortion of the product peak should be
quite small because the scattering intensity
from the product is ~25X higher.

10. 10
Tween can also cause significant interference for SEC and SEC-
MALS, often eluting in many regions of the chromatogram.
RI signal, formulation buffer with Tween 80
RI signal, antibody product
volume (mL)
5.0 6.0 7.0 8.0 9.0 10.0
relativescale
0.00
0.01
0.02
0.03
0.04
0.05
0.06
MALS signal, formulation buffer with Tween 80
MALS signal, antibody product
volume (mL)
5.0 6.0 7.0 8.0 9.0 10.0
relativescale
0.0
0.2
0.4
0.6
0.8
1.0
The RI detector shows that
Tween 80 is eluting across
the whole chromatogram,
producing signals
comparable to those from
the minor components in
the product.
The Tween contributions to
the light scattering signals
are even larger. The high
level of Tween in this
product makes it impossible
to identify the stoichiometry
of the aggregates via
MALS, and even distorts
the data for the main peak.

11. 11
Tween can even cause problems for standard SEC with
absorbance detection at 280 nm
UV signal (280 nm), formulation buffer with Tween 80
UV signal (280 nm), antibody product
volume (mL)
5.0 6.0 7.0 8.0 9.0 10.0
relativescale
0.000
0.005
0.010
0.015
0.020
0.025
0.030
potential false peak
near void volume
See also:
Hermeling, S., Schellekens, H., Crommelin, D. J. A., and Jiskoot, W. (2003). Micelle-
associated protein in epoetin formulations: A risk factor for immunogenicity? Pharm.
Res. 20, 1903-1907.
Heavner, G. A. (2006). Reaction to the paper: Interaction of polysorbate 80 with
erythropoietin: A case study in protein-surfactant interactions - Rebuttal letter. Pharm.
Res. 23, 643-644.

12. 12
Challenge 3: Excipient interference with
circular dichroism (CD) due to high
background absorbance
Circular dichroism spectra, as well as thermal unfolding measured by CD, are often
used to establish comparability of protein conformation. However even NaCl
produces significant absorbance that may preclude collecting far-UV data below
200 nm, and common buffers such as acetate and citrate absorb strongly at much
higher wavelengths.
The absorbance of impurities present in Tween are also potentially problematic,
particularly if Tween is present at levels above ~0.2%. Common anti-microbials
such as benzyl alcohol or phenol not only introduce significant absorbance, but if
they actually bind to the protein it is possible that an asymmetric binding
environment will induce CD signals arising from the bound preservative.
To some extent this interference can be reduced by using CD cells with very short
pathlengths (0.01-0.02 cm), but that then requires correspondingly higher protein
concentrations (which may not be available for high-potency drugs like cytokines).

13. 13
For 50 mM citrate buffer the far-UV CD spectra are noisy even
when using a cell pathlength of only 0.02 cm; for PBS 0.05 cm
gives good data.
-18000
16000
-10000
0
10000
Mol. Ellip.
270
530
400
198 260220 240
HT[V]
Wavelength[nm]
50 mM citrate, 0.05 cm cell (scan stopped
at 210 nm)
50 mM citrate, 0.02 cm cell
8 mM phosphate, 137 mM NaCl, 0.05 cm cell
Note: this graph
monitors the
photomultiplier
voltage, which
rises as the OD
rises.

14. 14
-50
30
-20
0
CD[mdeg]
200
900
400
600
800
240 340260 280 300 320
HT[V]
Wavelength [nm]
Effects of Tween-20 and preservatives on near-UV CD for an antibody:
high excipient absorbance gives high noise and non-linear CD signals.
⎯ 1 mg/mL in PBS, 1 cm
⎯ + 0.2% Tween-20
⎯ + 0.2% benzyl alcohol
⎯ + 0.05% phenol
While the impurities in the Tween-20 do slightly increase the background absorbance, the CD
spectrum is unaffected. Benzyl alcohol blocks nearly all the light from ~240-265 nm, while phenol
does so from 250-282 nm, but the protein CD spectrum is identical outside those blocked regions.

15. 15
By using a 0.01 cm cell for far-UV spectra 0.2% Tween-20 has no effect, and it
is possible to acquire essentially full spectra with 0.05% phenol. However with
0.2% benzyl alcohol it is only possible to go down to 210 nm. The excipients do
not alter the spectra over the regions that can be acquired.
-4
4
-2
0
2
CD[mdeg]
200
600
300
400
500
195 260220 240
HT[V]
Wavelength [nm]
⎯ 1 mg/mL in PBS, 0.01 cm
⎯ + 0.2% Tween-20
⎯ + 0.2% benzyl alcohol
⎯ + 0.05% phenol
Due to the high absorbance for the samples containing phenol or benzyl alcohol
the scans were terminated at the wavelengths indicated by the arrows.

16. 16
-13
0
-10
-5
CD[mdeg]
380
500
400
450
20 6030 40 50
HT[V]
Temperature [C]
sample at ~0.1-0.2
mg/ml
25 mM Bis-Tris; 225 nm
50 mM Tris; 222 nm
Higher OD
causes higher
noise.
Unfortunately the shortest pathlength cells cannot be temperature
controlled* so high excipient absorbance is more of a problem for
thermal unfolding studies. Here is an example where 25 mM Bis-
Tris causes problems for a thermal unfolding study in a 0.1 cm cell.
*For room temperature studies we have cells with 0.01, 0.02, 0.05, 0.2, and
1 cm pathlength; temperature-controlled cells are 0.1 or 1 cm pathlength.

17. 17
Acknowledgements
• The EPO studies were done in collaboration with George
Heavner (Centocor R&D), Melissa Calmann (Global
Biologics Supply) and Steven Labrenz (Global Biologics
Supply)