26
Origins of Color Change in Biopharmaceuticals: Identification of Protein- and Excipient-Related Factors Alla Polozova Analytical Biochemistry Biopharmaceutical Development
Origins of Color Change in Biopharmaceuticals: Identification of Protein-and Excipient-Related Factors
Alla Polozova
Analytical Biochemistry
Biopharmaceutical Development
2
Outline
Color measurements and standards
Expected color of intact proteins
Case studies: change in color of protein formulations during storage and stress studies– Case study 1: interaction of protein and excipients– Case study 2: protein-related factors
3
Components of Color Assessment by Colorimetry
Light source:– white light is typically used for
measurementsTransmittance– will limit scope to clear solutions
Scale of spectral components– yellow-blue– red-green
D65 Day light spectrum
Identification of spectral components Transmittance spectra of colored solutions
Intensity scale
4
Expected Color of Protein Solutions: Comparison of Transmittance Spectra of Yellow and Brown-Yellow Color Standards and a MAb at Different Concentrations
400 500 600 700
Wavelength, nm
20
40
60
80
100
Rel
ativ
e tr
ansm
ittan
ce, %
• Some color is expected in protein solutions, especially at higher concentrations• Transmittance patterns in concentrated MAb solutions are consistent with yellow or yellow-
brown color standards
0
20
40
60
80
100
350 450 550 650 750Wavelength, nm
Rel
ativ
e tra
nsm
ittan
ce, %
10 mg/mL50 mg/mL100 mg/mL150 mg/mL
Yellow EP color standards Y7-Y1
Non-stressed MAb solutions
Brown-yellow EP color standards BY7-Y1
Rel
ativ
e tr
ansm
ittan
ce, %
400 500 600 700
20
40
60
80
100
5
Case study #1Change in color during
storage related to interaction of protein and excipients
6
Case Study #1 Background
MAb A solution kept in polypropylene vials at 25 ºC turned dark brown upon several weeks of storage
Color was associated with the protein fraction, as shown by ultrafiltration and organic phase extraction
There was no detectable change in intact MAb A mass measured by MS
4oC25oC
7
Analysis of Colored MAb A Solutions by Absorbance
MAB A stored at 4oC
MAB A stored at 25oC
Buffer control
• Presence of additional absorbance bands centered at 350 nm and 440 nmwas observed in colored MAb A solution
350 nm 440 nm
8
Change in MAb A color and Aggregate Formation
• HPSEC analysis showed that change in color to darker brown correlated with increase in aggregates
• Aggregate fractions had higher absorbance at longer wavelengths (440 nm)• Color and aggregation were partially reversible upon cooing to 4 oC
dark brown color changed to light brown and aggregate level dropped 50%
2 4 6 8 10 12
mAU
0
200
400
600
800
1000
DAD1 A, Sig=280,10 Ref=off (Q:\LBDEVICE\11\DATA\PC81208JB000006.D)
2 4 6 8 10 12
mAU
0
200
400
600
800
1000
DAD1 A, Sig=280,10 Ref=off (Q:\LBDEVICE\11\DATA\PC81208JB000008.D)
2 4 6 8 10 12
mAU
0
200
400
600
800
1000
DAD1 A, Sig=280,10 Ref=off (Q:\LBDEVICE\11\DATA\PC81208JB000010.D)
2 4 6 8 10 12
mAU
0
200
400
600
800
1000
DAD1 A, Sig=280,10 Ref=off (Q:\LBDEVICE\11\DATA\PC81208JB000012.D)
Dark brown
Light brown
Colorless
280 nm MonomerAggregates
2 4 6 8 10
mAU
02.5
57.510
12.515
17.520
DAD1 B, Sig=440,10 Ref=off (Q:\LBDEVICE\11\DATA\PC81208JB000006.D)
DAD1 B Si 440 10 R f ff (Q \LBDEVICE\11\DATA\PC81208JB000008 D)
440 nmMonomer
Aggregates
HPSEC chromatograms
Brown
9
Observations for Brown MAb A Samples and Maillard Reaction
MAb A formulation contained sugar and PS80
Brown samples had new absorbance bands at 350 nm and 440 nm
Brown sample contained high levels of partially reversibleaggregates
Brown samples had oily and sticky appearance
Maillard reaction:
Cross-linking reaction between proteins/peptides and sugars
Results in formation of brown melanoidin compounds
Initial cross-linking reaction steps (Schiff base, Amadori products) are reversible
Is it possible that brown substance in stressed MAb A samples was a product of Maillard reaction?
10
Expected Hallmarks for Maillard Reaction
Presence of a reducing sugar– Presence of glucose was detected in brown-colored MAb A samples, but not in
controlsWhere did glucose come from? - Initial formulation contained non-reducing sugar
– PS-80 purity levels in brown MAb A samples were <50%– High level of peroxides were detected in brown MAb A solutions (~6-16 nM
compared to none in controls)– Possible reaction pathway:PS-80 degradation → peroxides → sugar oxidation → Maillard reaction*
Involvement of primary amines in the reaction– Level of Lys-containing peptides dropped as much as 75% in peptide map of brown
MAb A sampleIncrease in brown color with increase in high molecular weight products
Progressive increase in aggregate fraction correlated with increase in brown color
– Aggregates were partially reversible; dark brown color disappeared after aggregates dissociated
*Related published references:Hoffman, T., J. Agric. Food Chem. 46, 3891-5, 1998Nagaraj, R., et al., J. Biol. Chem. 271, 19338-45, 1996
11
Case Study #1 Summary
Reversible increase in brown color was associated with increase in high molecular weight aggregates
PS-80 degradation and presence of peroxides correlated with oxidation of sugar in MAb A formulation
Significant loss of peptides with Lys residues was observed in peptide maps of brown samples
We speculate that oxidized sugar reacted with primary amines in MAb A as in Maillard reaction– Brown color was specifically associated with high molecular weight
aggregate fraction– Bridging melanoidin protein-sugar adducts are known to confer dark
brown color, larger aggregates have higher color intensity*
*Hoffman, T., J. Agric. Food Chem. 46, 3891-5, 1998
12
Case Study #2
Change in color during photo stress accelerated stability studies
13
Case Study # 2 Background
MAb B is an IgG formulated at 150 mg/mL
MAb B slightly brown-yellow color changed to brown under intense visual light during photostability study
During accelerated stability studies at higher temperature (25 oC for > 12 months) MAb B slightly yellow/yellow-brown color increased in intensity
Analysis was carried out to determine factors responsible for change in color of MAb B solutions
14
Color of MAb B Samples Stressed by Intense Visual Light
Intense brown B1**Day 14
Intense brown yellow BY1*Day 4
Slightly brown yellow BY5*Initial
ColorTime point
Results of visual appearance test
Day 14 Day 4Initial
Color of MAb B samples stressed by intense visual light (9500 lux***), progressively changed from slightly brown-yellow to intense brown
* EP brown-yellow standard** EP brown standard*** Typical lab/office illumination is 400-1000 lux
15
Change in MAb B Absorbance Pattern with Increased Exposure to Intense Visual Light
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
300 400 500 600 700 800
Wavelength, nm
Abs
orba
nce
InitialDay 1Day 2Day 4Day 7Day 10Day 14Day 14 (dark control)
Shoulder
430 nm
Exposure of MAb B to intense visual light resulted in increased absorbance at longer wavelengths:
Increase in shoulder between 310 and 390 nmNew band at ~430 nm
16
RP-HPLC Analysis of Photo Stressed MAb B Samples
S a m p l e N a m e : C A T - 3 5 4 _ D E V 1 1 9 6 2 6 _ J F 2 1 S e p 1 2 D a t e A c q u ir e d : 9 / 2 2 / 2 0 1 2 1 0 : 0 5 : 1 9 A M E D T C h a n n e l D e s c r i p t i o n : A C Q U IT Y T U V C h A 2 8 0 n m S a m p l e N a m e : C A T - 3 5 4 _ D E V 1 1 9 6 4 2 _ J F 2 1 S e p 1 2 D a t e A c q u ir e d : 9 / 2 2 / 2 0 1 2 1 2 : 2 2 : 2 9 P M E D T C h a n n e l D e s c r i p t i o n : A C Q U IT Y T U V C h A 2 8 0 n m S a m p l e N a m e : C A T - 3 5 4 _ D E V 1 1 9 6 3 8 _ J F 2 1 S e p 1 2 D a t e A c q u ir e d : 9 / 2 2 / 2 0 1 2 1 : 0 8 : 1 8 P M E D T C h a n n e l D e s c r i p t i o n : A C Q U IT Y T U V C h A 2 8 0 n m S a m p l e N a m e : C A T - 3 5 4 _ D E V 1 1 9 6 5 2 _ J F 2 1 S e p 1 2 D a t e A c q u ir e d : 9 / 2 2 / 2 0 1 2 2 : 3 9 : 3 6 P M E D T C h a n n e l D e s c r i p t i o n : A C Q U IT Y T U V C h A 2 8 0 n m
AU
0 . 0 0
0 . 0 2
0 . 0 4
0 . 0 6
0 . 0 8
0 . 1 0
0 . 1 2
0 . 1 4
0 . 1 6
0 . 1 8
0 . 2 0
0 . 2 2
0 . 2 4
0 . 2 6
0 . 2 8
0 . 3 0
M in u t e s0 . 0 0 0 . 5 0 1 . 0 0 1 . 5 0 2 . 0 0 2 . 5 0 3 . 0 0 3 . 5 0 4 . 0 0 4 . 5 0 5 . 0 0 5 . 5 0 6 . 0 0 6 . 5 0 7 .0 0
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
Day 14
Initial
Day 4
280 nm
330 nm
Day 7
• Analysis of RP-HPLC profiles showed that species absorbing at longer wavelengths were associated with protein
• Longer light exposure correlated with increased absorbance at longer wavelengths
Initial
Day 4
Day 7
Day 14
Absorbance spectra of individual peaks
Day 14Day 4
Day 7
Initial
17
Peptide Mapping Profiles of Mab A Samples Exposed to Intense Visual Light for 14 Days
min10 20 30 40 50 60 70
mAU
0
5
10
15
20
25
DAD1 B, Sig=290,4 Ref=500,40 (JF2012092722C\JF2012092722C13.D)
min*10 20 30 40 50 60 70
mAU
0
5
10
15
20
25
*DAD1 B, Sig=290,4 Ref=500,40 (JF2012092722C\JF2012092722C10.D)
min10 20 30 40 50 60 70
mAU
0
1
2
3
4
DAD1 F, Sig=355,4 Ref=500,40 (JF2012092722C\JF2012092722C13.D)
min*10 20 30 40 50 60 70
mAU
0
1
2
3
4
*DAD1 F, Sig=355,4 Ref=500,40 (JF2012092722C\JF2012092722C10.D)
M Oxi
Initial
Day14
Initial (dark control was similar)
Day14
H Oxi
W Oxi
W Oxi W Oxi
• Multiple oxidation products were identified (M, H & W)
• Only peptides containing oxidized W were detectable at 355 nm absorbance
H Oxi
355 nm
220 nm
220 nm
355 nm
18
min10 11 12 13 14
mAU
0
50
100
DAD1 A, Sig=220,4 Ref=500,40 (JF2012092722C\JF2012092722C10.D)
min10 11 12 13 14
mAU
0
2
4
DAD1 B, Sig=290,4 Ref=500,40 (JF2012092722C\JF2012092722C10.D)
min10 11 12 13 14
mAU
0.250.5
0.75
DAD1 D, Sig=310,4 Ref=500,40 (JF2012092722C\JF2012092722C10.D)
min10 11 12 13 14
mAU
0.51
1.5
DAD1 E, Sig=330,4 Ref=500,40 (JF2012092722C\JF2012092722C10.D)
min10 11 12 13 14
mAU
0123
DAD1 F, Sig=355,4 Ref=500,40 (JF2012092722C\JF2012092722C10.D)
355 nm
H9+32Da
Hx+4Da
Intact peptide Hx
330 nm
310 nm
290 nm
220 nm
Hx +32DaHx +16Da Hx +4Da
Hx+4Da
Multiple Oxidation Forms of One of the Peptides in Peptide Map of MAb B Sample Exposed to Intense Visual Light for 14 Days
Three different W oxidation forms of peptide Hx have different absorbance properties
19
Absorbance Spectra of Three Detected W Oxidation Forms of Peptide Hx in Peptide Map Of Photo Stressed MAb B
DAD1, 11.787 (23.7 mAU, - ) of JF2012092722C10.DDAD1, 11.967 (6.6 mAU, - ) of JF2012092722C10.DDAD1, 14.154 (60.1 mAU, - ) of JF2012092722C10.D
nm250 300 350 400 450
DAD1, 10.800 (23.5 mAU, - ) of JF2012092722C10.D
Intact HxHx +16DaHx +32DaHx +4Da
• Individual W oxidized forms have different absorbance properties
• All oxidized forms show shift to longer wavelengths, some of them to greater extent, with +4 Da form exhibiting the biggest red shift
20
Time-Dependent Accumulation of Major Oxidized Tryptophan Species of MAb B Peptide Hx
+16Da +32Da +4Da
Known W oxidation pathways
• Larger amounts of W oxidation forms absorbing at longer wavelengths accumulate with prolonged MAb B exposure to intense visual light
• Accumulation of these W oxidation products correlates with change in color to more intense yellow and brown
Peptide H9 Oxidation Distribution
0.0
5.0
10.0
15.0
20.0
25.0
0 2 4 6 8 10 12 14
Light Exposure Time (Day)
% O
xida
tion
H9(W104+4Da,11.6 min)
H9(W104+16Da,10.6 min)
H9(W104+32Da,11.8 min)
Different W oxidation forms in peptide Hx
Hx+4 Da
Hx+16 Da
Hx+32 Da
21
Comparison of Visual Light Absorbing Species in Photo Stressed and Heat Stressed MAb B Samples
min10 20 30 40 50 60 70
mAU
0
1
2
3
4
DAD1 F, Sig=355,4 Ref=500,40 (JF2012092722C\JF2012092722C12.D)
min10 20 30 40 50 60 70
mAU
0
1
2
3
4
DAD1 F, Sig=355,4 Ref=500,40 (JF2012092722C\JF2012092722C10.D)
min*10 20 30 40 50 60 70
mAU
0
1
2
3
4
*DAD1 F, Sig=355,4 Ref=500,40 (JF2012092722C\JF2012092722C11.D)
Day14 photo stress
Accelerated stability
(+25 oC for > 12 months)
W oxi
W Oxi2W Oxi
W Oxi
Initial (dark control was similar)
W Oxi
• In both photo- and heat-stressed samples only oxidized W species were responsible for absorbance in visual spectrum
• Some differences in location of oxidized W were observed for heat-stressed MAb B
355 nm
355 nm
355 nm
22
Case 2 Study Summary
Photo degradation of MAb B under intense visual light results inoxidation of multiple Met, Trp and His residues
Only Trp oxidation products have absorbance in visual spectrum contributing to MAb B color
Multiple Trp oxidation forms with different absorbance properties are present – Accumulation of forms absorbing at longer wavelengths correlates with
change in MAb B color to brown after longer exposure to intense visual light
Similar factors were responsible for change in color intensity in heat stressed MAb B– Differences in location of Trp residues susceptible to oxidation,
compared to photo stressed MAb B, were observed
23
Conclusions
Proteins are expected to exhibit some color, especially in highly concentrated solutions
Multiple factors can lead to change in color during storage and accelerated stability studies:– Interaction of protein and excipients influenced by environmental factors
(exposure to oxidants and light)– Accumulation Trp oxidation products can lead to increase in color
intensity and change to brown color
24
Acknowledgements
Jose Casas-Finet
Jenny Feng
Flaviu Gruia
Arun Parupudi
Hung-Yu Lin
Sophia Levitskaya
Methal Albarghouthi
Yiming Li
Chris Barton
Bob Strouse
Mike Washabaugh
Pat Cash
Mark Schenerman
25
Back up slide
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Correlation between Color Change and Potency Loss in MAb B Case Study
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14
Time (days)
%
% Trp oxidation
% Potency
% Transmittance at 430 nmSlightly
brown-yellow
Intense brown-yellow
Intense brown
• Change in MAb B color to intense brown correlated with potency loss• Trp oxidation responsible for color change was a contributing factors to
potency loss, but not the only one
