Protein Stability during Processing: Challenges and Strategies

1
Protein Stability during ProcessingChallenges
and Strategies

• Mark Cornell Manning
• Legacy BioDesign LLC
• May 17, 2007

Legacy BioDesign, LLC
2
Acknowledgments

• Legacy BioDesign Susan Manning James
  Matsuura Cody Van Pelt
• Charles S. Henry, Robert W. Payne, Joseph J.
  Valente (Colorado State University)
• W. William Wilson (Mississippi State Univ.)

Legacy BioDesign, LLC
3
Increased Demands on Biopharmaceuticals

• Stability for two years in an aqueous solution
• Move to higher concentration formulations (to
  increase the dose that can be given
  subcutaneously)
• Search for methods that predict aggregation
  behavior upon storage
• Rapid throughput needed during formulation
  development
• Need ability to develop multiple candidates at
  once

4
Process Development (PD) Weve Got Trouble

• Processing steps place an inordinate strain on
  proteins, causing both physical and chemical
  damage
• These stresses lower yields and reduce product
  quality
• Controlling aggregation is the major concern
• Each unit operation presents a different challenge

5
PD So, What Do We Do?

• We need a plan (importance of strategy)
• We need allies (draw upon knowledge in other
  groups)
• We need data (have better analytical methods in
  the right places PAT approaches)
• We need insight (seminars, short courses, and
  most of all, read a lot!)
• We need new tools (use emerging technologies)

6
The Enemy Within Aggregation

• How do we deal with aggregation? Better
  detection Better quantitation Better
  mechanistic understanding
• What drives aggregation? Loss of
  conformational stability Unfavorable colloidal
  stability Interfacial damage

7
Conformational vs. Colloidal Stability

• Both conformational and colloidal stability are
  important factors governing protein aggregation
• N D aggregate conformationa
  l shift equilibrium from D to N colloidal keep
  D from associating
• Minimizing aggregation depends on reducing
  protein-protein interactions and stabilizing the
  native state

8
Colloidal Behavior of Proteins

• Proteins are large enough to display colloidal
  properties
• Interaction between protein molecules can be
  repulsive or attractive
• These interactions affect important solution
  properties and processes, such as solubility,
  viscosity, crystallization and aggregation

9
Colloidal Behavior of Proteins

• Osmotic second virial coefficient (B22) provides
  quantitative measure of colloidal stability
• Positive value indicates repulsion, whereas
  negative value indicates attraction between
  protein molecules
• Interaction forces include hard-sphere,
  electrostatic, van der Waals and any other short
  range interactions

10
Methods of Measuring B22

• Typically, measured by static light scattering
  (SLS)
• SLS methods are labor-, time- and
  material-intensive
• Now B22 can be measured by self-interaction
  chromatographic (SIC) methods (Patro and
  Przybycien, Biotechnol. Bioeng. 1996, 52 193-203
  Tessier et al., Proteins 2003, 50 303-311)
• SIC provides a rapid throughput method of B22
  determinations to optimize solubility (Valente et
  al., Curr. Pharm. Biotechnol. 2005, 6 427-436)

11
Self-Interaction Chromatography (SIC)
Garcia et al., Biotechnol. Prog. 2003, 19575-579
12
B22 Measurements by SIC/SLS
Valente et al., Biophys. J. 2005, 89 4211-4218
13
pH-Solubility Curve for Ribonuclease A
pI value 3.5
2
6
10
Schmittschmitt and Scholtz, Protein Sci. 2003,
12 2374-2378
14
pH-Solubility Correlation
Payne et al., Biopolymers (Pept. Sci.) 2006, 84
527-533
15
Effect of pH on B22
pI 7.8
Six injections per condition SIC run time about 6
min. Entire pH profile can be done in one day
Henry, Payne, Ramsay, Wilson and Manning,
unpublished
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Effect of Colloidal Stability on Non-native
Protein Aggregation

• In general, conformational stability is the most
  influential factor in modulating aggregation
• Solution conditions (e.g., pH and ionic strength)
  can greatly affect protein aggregation rate,
  independent of effects on conformational
  stability by modulating intermolecular
  interactions
• References Krishnan et al., Biochemistry 2002,
  41 6422 Chi et al., Protein Sci. 2003, 12 903
  Ho et al., Protein Sci. 2003, 12 708

17
Summary B22 in Drug Development

• B22 correlates with protein and peptide
  solubility
• B22 appears to be important in controlling
  viscosity and aggregation behavior as well
• B22 values can be rapidly determined by SIC and
  these values are consistent with values from SLS
• Combined with experimental design, a B22 response
  surface could be mapped for various formulation
  parameters quite rapidly
• Much easier to investigate temperature effects
  than with SLS

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Summary B22 in Drug Development

• For peptides, SIC is the only method that will
  provide B22 values. The success will be dependent
  on adequate immobilization on the column
• Goal identify solution conditions that will
  provide adequate solubility/handling first
• So, it is a great early stage tool for
  formulation development. It is also an important
  tool throughout process development

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Membrane Fouling

• Membrane fouling refers to the build up of
  protein on a membrane (as during aseptic
  processing and UF/DF operations)
• Initial step is structural rearrangement (at the
  membrane or at the air-water interface)
• As the protein builds up on the membrane, the
  pressure required to maintain flux increases
• Significant literature on this topic

20
Membrane Fouling with hGH
Data suggest that more positive B22
values (further from pI) leads to less membrane
fouling the importance of colloidal stability
Maa and Hsu, Biotechnol. Bioeng. 1996, 50 319-328
21
Membrane Fouling with hGH
pH 7.3 plus 0.2 Tween
Surfactants retard fouling the importance of
interfacial stability
pH 6.8 plus 0.2 Tween
pH 7.3
pH 6.8
Maa and Hsu, Biotechnol. Bioeng. 1996, 50 319-328
22
Fouling of UF Membranes by BSA

• UF flux was measured at four different pH and
  three different salt levels
• Zeta potential of both the membrane and protein
  were measured
• Strong (negative) correlation of BSA zeta
  potential with flux suggests that colloidal
  stability plays some role in membrane fouling

Salgin, Chem. Eng. Technol. 2007, 30 255-260
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Membrane Fouling

• Conformational stability affects surface
  adsorption (cf. Karlsson et al., J. Biol. Chem.
  2005, 280 25558 Wendorf et al., Biotechnol.
  Bioeng. 2004, 87 565)
• Multiple pass through UF/DF pumps (Cromwell et
  al., AAPS J. 2006, 8(3) art. 66) and stirring
  speeds (Wan et al., Biotechnol. Bioeng. 2005, 90
  422) can increase protein aggregation
• Control of membrane fouling requires attention to
  all three types of protein stabilization
  (conformational, colloidal, and interfacial)

IS THE DAMAGE DUE TO SHEAR?
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What About Shear?

• There has been an ongoing debate about the
  effects of shear on protein structure and
  stability for the past 25 years
• The concerns are rising again with the use of
  high concentration formulations, faster filling
  machines, better analytical methodology, etc.
• What do we know for sure about shear effects?

25
Effect of Shear on Protein Denaturation

• Early literature suggested that shear could
  denature proteins (e.g., Charm and Wong, Enzyme
  Microb. Technol. 1981, 3 111-118 Reese and Ryu,
  Enzyme Microb. Technol. 1980, 2 239-240)
• More recent studies have demonstrated that
  proteins are too small to achieve sufficient
  force to unfold them (e.g., Maa et al.,
  Biotechnol. Bioeng. 1997, 54 503-512 Speigel,
  Int. J. Food Sci. Technol. 1999, 34 523-531 Yu
  et al., Eur. J. Pharm. Sci. 2006, 27 9-18)

26
Effect of Shear on Protein Denaturation

• Many shear studies are agitation studies (e.g.,
  Oliva et al., J. Pharm. Biomed. Analysis 2003,
  33 145-155 Byrne and Fitzpatrick, Biochem. Eng.
  J. 2002, 10 17-25 Colombie et al., Biotechnol.
  Lett. 2000, 22 277-283), where air-water
  interface is prevalent
• The interactions of the protein with solid
  surfaces (tubing, membranes, etc.) is often
  ignored
• Bottom line the literature is consistent with a
  view that shear denaturation does not occur
• HOWEVER.

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Shear Does Damage Some Proteins

• There are clear cases where shear does appear to
  increase aggregation
• If it is not due to shear denaturation, there
  must be other mechanisms involved
• WHAT ARE THEY?
• Shear (possibly via cavitation) can lead to
  radical formation and aggregation via S-S
  linkages (e.g., Morel et al., Biomacromolecules
  2002, 3 488-497 Maa and Hsu, Biotechnol.
  Bioeng. 1996, 51 458-465)

28
Shear Does Damage Some Proteins

• Turbulent flow dislodging unfolded protein from
  surfaces (e.g., Santos et al., J. Food Eng. 2006,
  74 468-483), possibly producing prenuclei
• Increased mixing by shear leads to increased
  growth rate for aggregates (e.g., Belmar-Beiny et
  al., J. Food Eng. 1993, 19 119-139 Simmons et
  al., J. Food Eng. 2007, 79 517-528)
• Shear could dislodge foreign matter from
  surfaces. Many of these could lead to
  nucleation-dependent aggregation

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Nucleation-dependent Aggregation

• Observation For some protein systems, during
  isothermal incubation, there is no detectable
  protein precipitation for certain period of time
  (lag phase), followed by a rapid appearance of
  visible particles
• Prenuclei can be damaged protein (oxidized,
  dimers, etc.) or foreign matter
• Precipitation of numerous particles coated with
  adsorbed protein is a recipe for increased
  immunogenicity

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Container-Induced Aggregation Pafase

• Aggregation appears after prolonged storage (gt 6
  months) in liquid formulations
• Not due to agitation
• Not seen in lyophilized formulations
• Effect of container and container handling
  (depyrogenation)
• Aggregation is pH dependent
• Glass shedding or delamination (demonstrated by
  seeding with glass nanoparticles)

Chi et al., J. Pharm. Sci. 2005, 94 256-274
31
Pafase Adsorption to Silica Nanoparticles
Chi et al., J. Pharm. Sci. 2005, 94 256-274
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Issues with Frozen Solutions

• Sounds simple to do
• Many issues to consider with frozen solutions
• Question Is the solution really frozen at 20
  C? Tg is usually below 20 C for most
  formulations
• Product distribution issues (i.e., cold chain
  integrity)
• Characterization (analytical) issues
• Freeze concentration effects (up to 15- or
  20-fold). Franks (Cryo-Letters 1990, 1193)
  reports a 24-fold increase for freezing isotonic
  saline.

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Issues with Frozen Solutions

• Oxygen content can be more than 1000-fold higher
  in a partially frozen solution than at 0º C (and
  it is higher at 0 º C than at room temp.) cf.
  Wisniewski, BioPharm 1998, 11 50-60
• Interfacial damage at water-ice interface
  (proportional to ice-water surface area cf.
  Chang et al., J. Pharm. Sci. 1996, 85 1325-1330)
• Temperature dependence of cmc for surfactants
  (may not have enough Hillgren et al., Int. J.
  Pharm. 2002, 237 57-69)

34
Freezing Issues

• Cold denaturation? A few reports catalase
  (Shikama and Yamazaki, Nature 1961, 190 83)
  ovalbumin (Koseki et al., J. Biochem. 1990, 107
  389)
• Freezing rate effects on stability have been
  reported (Hsu et al., Pharm. Res. 1995, 12 69
  Sarciaux et al., J. Pharm. Sci. 1999, 88 1354
  Jiang and Nail, Eur. J. Pharm. Biopharm. 1998,
  45 249)

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Degradation during Chromatography

• Stabilization during chromatography is rarely
  considered
• Focus on stabilizing buffers and pH
• Interfacial damage?

Bondos and Bicknell, Analytical Biochem. 2003,
316 223-231
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Degradation during HIC

• Both HIC and RP are known to be denaturing to
  proteins
• In the case of a-lactalbumin, the protein
  denatures on the surface, so both conformational
  stability and adsorption kinetics are important
• Absence of salt leads to great adsorption and
  greater destabilization

Xiao et al., Biotechnol. Bioeng. 2006, 93
1177-1189
37
Degradation during IEC

• Increased aggregation of g-globulin at pH 3.5-4.0
  is due to later eluting IgG2
• IgG2 appears to be more aggregation prone at low
  pH than other IgGs
• IgG2 also appears to be more hydrophobic

Lewis and Nail, Process Biochem. 1997, 32 279-283
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Degradation during Elution from Protein A Columns

• Protein A chromatography is an important method
  for purifying antibodies
• Elution requires acidic conditions
• Campath (alemtuzumab) aggregates extensively (
  25) when eluted at pH 3.2 (citrate buffer)

Phillips et al, Cytotherapy 2001, 3 233-242
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Summary

• Development of protein products is challenging,
  but new approaches can help address the most
  pressing PD challenges
• Nearly all unit operations have the potential to
  damage your protein
• Interfacial damage can range from imperceptible
  to catastrophic via nucleation-dependent
  aggregation
• Conformational, colloidal and interfacial
  stability all impact the extent and rate of
  aggregation at membranes and surfaces