Accepted Manuscript

Role of Buffers in Protein Formulations

Teddy J. Zbacnik, Ryan E. Holcomb, Derrick S. Katayama, Brian M. Murphy, Robert

W. Payne, Richard C. Coccaro, Gabriel J. Evans, James E. Matsuura, Charles S.
Henry, Mark Cornell Manning
PII: S0022-3549(16)41879-4
DOI: 10.1016/j.xphs.2016.11.014
Reference: XPHS 568

To appear in: Journal of Pharmaceutical Sciences

Received Date: 12 August 2016

Revised Date: 25 October 2016
Accepted Date: 17 November 2016

Please cite this article as: Zbacnik TJ, Holcomb RE, Katayama DS, Murphy BM, Payne RW, Coccaro
RC, Evans GJ, Matsuura JE, Henry CS, Manning MC, Role of Buffers in Protein Formulations, Journal
of Pharmaceutical Sciences (2016), doi: 10.1016/j.xphs.2016.11.014.

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 ACCEPTED MANUSCRIPT

ROLE OF BUFFERS IN PROTEIN FORMULATIONS

Teddy J. Zbacnik1, Ryan E. Holcomb1,2, Derrick S. Katayama1,2, Brian M. Murphy1,2,
Robert W. Payne1,2, Richard C. Coccaro1, Gabriel J. Evans1, James E. Matsuura1,3,
Charles S. Henry2, and Mark Cornell Manning1,2

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LegacyBioDesign LLC, Johnstown, CO, 80534 USA

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Department of Chemistry, Colorado State University, Fort Collins, CO 80523 USA
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Current address: Medivation, Inc., San Francisco, CA

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ABSTRACT
Buffers comprise an integral component of protein formulations. Not only do they
function to regulate shifts in pH, they also can stabilize proteins by a variety of
mechanisms. The ability of buffers to stabilize therapeutic proteins whether in liquid
formulations, frozen solutions or the solid state is highlighted in this review. Addition of
buffers can result in increased conformational stability of proteins, whether by ligand
binding or by an excluded solute mechanism. In addition they can alter the colloidal

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stability of proteins and modulate interfacial damage. Buffers can also lead to
destabilization of proteins, and the stability of buffers themselves is presented.
Furthermore, the potential safety and toxicity issues of buffers are discussed, with a

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special emphasis on the influence of buffers on the perceived pain upon injection.
Finally, the interaction of buffers with other excipients is examined.

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Introduction

One of the most common classes of excipients used in protein formulations are buffers.

While buffers are primarily intended to control the pH of the formulation, they have other

important properties that can contribute significantly by various mechanisms to the

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overall stability of a protein product. Despite their widespread use, there has not been a

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comprehensive overview of the role of buffers in protein formulations, with the possible

exception of the 2004 review article from Ugwu and Apte1, which covered a range of

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topics related to buffers. By comparison, this review is intended not only to provide an

up-to-date summary of the use of buffers, but also provide more detailed descriptions of

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how buffers can impact protein solubility and stability, both for liquid formulations and
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for freeze-dried (lyophilized) products.
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For decades, buffers have been an important component of any therapeutic protein
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formulation2-5. Indeed, buffers can impact the structure and stability of any protein, as
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illustrated in this review. Although there are many known buffers, a few tend to be more

widely used in approved protein therapeutics than others6-9. These include acetate,
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phosphate, histidine, and tris (also known as tromethamine). Still, the effects of a wide

range of buffers have been reported and will be presented herein. The diversity of buffer
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usage in a select number of marketed monoclonal antibody (MAb) products is provided

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in Table 1. In addition, a summary of buffers found in other protein biopharmaceuticals is

provided in Table 2. Most of the published articles regarding buffer effects on proteins

tend to employ this more select group of buffers, but the effects of all buffers is

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considered herein. The chemical structures of some of the more common buffer species is

shown in Scheme 1.

Buffer Properties

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Buffer systems are typically comprised of two chemical species that are related by a

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change in protonation state. Thus, one may have an acid and its conjugate base or a base

and its corresponding conjugate acid as the buffer system. One such example would be

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acetic acid and sodium acetate. The effective pH of a buffer is dependent on the relative

proportions of the two species and their overall concentrations, as indicated by the

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Henderson-Hasselbalch equation (equation 1).
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(1)
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This seminal equation describes the interrelationship between the concentrations of

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various buffer species and the effect on pH, given the pKa of the corresponding acidic
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species. The pKa value, or the ionization constant, corresponds to the pH where the

molecule is 50% ionized. These ionization constants can be measured by a variety of

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techniques, including by titration, electrophoresis, electrochemistry, and NMR10, 11.

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The primary reason for using a buffer system in a pharmaceutical product is to limit

changes in pH during storage and handling. There are number of excellent reference

works on the properties of various buffer systems and their ability to modulate variations

in pH12-18. These references include tabulation of pKa values of various buffer systems

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(Table 3). Many buffer species have more than one ionizable group, resulting in two or

more distinct pKa values (Table 3). This means that some buffers (e.g., phosphate, citrate,

etc.) will have some appreciable buffering capacity over more than one distinct pH range

that may be of interest to the formulation scientist. For buffers with multiple ionization

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constants, there are multiple peaks corresponding to the specific pKa values. For

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phosphate buffer, the buffering capacity is maximal near pH 7, but quite low at pH 5 and

9. It increases again to be significant at pH 3 and 1119. Spreadsheets, algorithms, or

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programs for calculating buffering capacity are currently available20, 21.

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Thermodynamic parameters for ionization of various buffer species, including
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temperature dependence, have been tabulated18. The effect of temperature on a buffer is

worth noting, as degree of ionization can change with temperature, meaning the pH of the
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buffer will vary as the sample is cooled or warmed. For the formulation scientist, this
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means the pH of the system measured at room temperature may not be the exact pH when
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stored at 2-8º C. In particular, N-based buffers (tris, HEPES, etc.) are well known to

display this behavior. Tris shows a change in pKa (dpKa/dT) of -0.028/° C 13, 18 or -0.031/
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°C , which is among the highest seen for any commonly used buffer system. For

example, a Tris-buffered solution measured to be 8.05 at 25° C rose to pH 8.85 at 0° C12.

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By comparison, a 50 mM sodium phosphate buffer changes only from pH 7.00 to 7.02

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over the same temperature range22. Table 3 also lists dpKa/dT values for a number of

common buffers (phosphate, His, Tris) that were measured all the way down to -30° C22.

More detailed information on the temperature dependence of MOPS23 and HEPES24 can

found. The temperature dependence of a series of noncomplexing tertiary amine buffers

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has been investigated and they appear to provide suitable buffering capacity across a pH

range from 3 to 1125. A temperature independent buffer system was developed

comprising 60% HEPES and 40% potassium phosphate, which holds pH across a very

wide temperature range26. In addition to changes in the ionization constants, the effects

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of temperature on the density, refractive index, and conductivity of tris buffer have been

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reported27. Finally, shifts in pH as a function of temperature has also been reported not

only for buffers but also for formulated food products28.

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When selecting a buffer, one typically considers the buffering capacity of such an acid-

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base system when selecting a buffer. Buffering capacity is a measure of how well the
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buffer will maintain the pH. Buffering capacity was described nearly a century ago by

Van Slyke29. The calculated buffering capacity is symmetrical around the pKa value when
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plotted against pH. Determining the buffering capacity of a monoprotic acid/conjugate

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base is relatively straightforward, displaying a single peak centered at the pKa. The
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analysis of titration curves to obtain pKa values and assessment of buffering capacity has

been reviewed21. The usual rule of thumb is that a buffer will exhibit some measurable
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buffering capacity if the pH is within one pH unit of the pKa of the corresponding acid

form. For example, the pKa of acetic acid is ~4.8. This means that an acetate buffer
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system should be effective at controlling pH at values approximately between pH 3.8 and

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5.8. Specific calculations of buffering capacity can be found in the literature for various
14, 15, 19, 20, 29
buffers , and data on buffering capacity are even available for the proteins

themselves30-32.

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For polyprotic compounds like proteins, the pKa values can vary, depending on the local

environment around the ionizable group. The average pKa values for folded proteins has

been tabulated33-35 (Table 4). This summary illustrates how the degree of ionization for

various amino acids can vary widely, depending on the surrounding environment. Still,

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the average value is close to the canonical value for the individual amino acids. The large

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number of ionizable groups and their dispersity across the pH range emphasizes the

ability of proteins to function as buffers themselves. For example, the buffering capacity

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of such polyprotic systems has been discussed in detail34, 36, 37.

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The degree of ionization (i.e., the pKa value of a buffer) will also be affected by the
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presence of salts38, 39, as predicted by well-known schemes such as DLVO theory. The

addition of various salts was shown to reduce the pH of a sodium phosphate buffer40,
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decreasing the pH from 7.0 to less than 6.5 by the time 1 M salt was added. Likewise, the
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addition of sodium salts alter the pH of various buffer systems, usually following
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Hofmeister behavior39, 41. Thus, one needs to be aware that salts or electrolytes can and

will alter the pH profile of a buffer system.

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Other excipients, besides salts, can also impact buffer behavior and degree of ionization.
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For example, the presence of PVP was found to affect the viscosity behavior of the
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buffers, MES, MOPS, and MOPSO, leading to variations in the calculated Jones-Dole

viscosity B-coefficients of these compounds42.

Stabilization by Buffers: General Considerations

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Importance of pH. There is no doubt that pH itself impacts protein stability more than any

single factor, whether one considers chemical instability or physical instability.

Therefore, controlling the pH of a formulation is critical. This has been recognized in

review articles on protein stabilization for some time3-5, 43, 44. Consequently, buffers are

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critical components solely from this perspective.

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In this context, buffering capacity is then a consideration when choosing a buffer.

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However, while buffer capacity is a useful metric for guiding the selection of a buffer for

a given formulation, one can find examples of buffers being used in commercial

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pharmaceutical formulations where the pH is well outside the nominal buffering capacity
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range. In those cases, the buffer is likely influencing stability by some mechanism other

than simply controlling the pH. Those stabilization mechanisms include the ability to
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increase conformational stability, to alter colloidal stability, and to impact the behavior at
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interfaces. For example, it is well known that ionic compounds can affect interfacial
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behavior as well, especially interfaces, like the air-water interface, where charged species

can accumulate45, 46. Thus, while buffer capacity is an important consideration, this is not
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the only criterion for selecting a buffer species for a therapeutic protein formulation.
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General Considerations. Buffers have a remarkable ability to stabilize or destabilize

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proteins beyond the direct effect of pH on chemical and physical stability47. It should be

noted that many reports do not ascribe a particular mechanism for the stabilization that is

observed. In those cases, the observation of buffer-induced stabilization is shared, with

only limited discussion on the actual basis for the increased stability.

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Sometimes, the effects of buffers on stability are clear, but in other cases, one must look

at more subtle differences, as when considering the effect of buffers on the rate of

aggregation. In one such study of an IgG2 MAb at low pH, it was found the total amount

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of conjugate acid appears to govern the aggregation rate, when considering citrate and

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acetate buffers48. While the mechanistic basis for this observation may not be readily

apparent, it illustrates how sensitive proteins can be to the presence of certain buffer

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species. So, whether one considers effects on storage stability or on the conformational

stability of a protein, there are numerous reports of buffers having a favorable impact.

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Even in the absence of a mechanistic description, these various case studies and general
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trends are worth careful consideration for the formulation scientist. For example, it was

discovered during buffer screening that, in general, succinate and acetate provided better
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stability of those monoclonal antibodies (MAbs) than citrate, histidine, phosphate, or

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imidazole49. The exact rationale for such observations was not discussed. Moreover, in
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all of those cases, the addition of NaCl greatly reduced storage stability at 37° C49,

presumably by lowering colloidal stability. According to this study, the general trend was
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improved stability at lower pH for a given buffer species, similar to what was seen in the

work by Hari et al.48. Similarly, in another screening study, the stability of a monoclonal
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antibody was found to vary significantly in the presence of different buffers50, with citrate
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and succinate leading to greater chemical instability that was not seen with histidine and

acetate.

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Such studies illustrate not only the universal effects of buffers on protein stability, but

also how the presence of other excipients may modulate this behavior. For example, a

recent screening study using differential scanning fluorimetry (DSF) examined 252

proteins in 28 different formulations, all containing some amount of NaCl. Three buffers

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were identified as providing enhanced stability, as indicated by increases in Tm. They

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were citrate (pH 6), bis-tris (pH 6.5), and ADA (pH 6.5)51. Note that each of these

‘preferred’ formulations also contained 200 mM NaCl. The authors note that the latter

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two buffers are used less commonly than tris, possibly due to the cost, as they are 11- and

36-fold more expensive than tris, respectively51. The findings do illustrate that different

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classes of proteins may have a preference for different types of buffers.
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Mechanisms of Stabilization. The physical stability of any protein has three aspects,
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conformational, colloidal, and interfacial. These three facets of protein stabilization have
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become more and more widely appreciated and understood. Moreover, these three are
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interrelated and formulation components (i.e., excipients) often have an effect on more

than one aspect of protein stability. This is true for buffers as well. The effect of buffers
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on conformational stability is the most widely described aspect of protein stabilization.

This is usually reflected by an increase in the apparent Tm value of the protein in the
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presence of a particular buffer species. In addition, some studies have described improved
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stability during long-term storage, likely as the result of increase thermodynamic

stabilization of the globular structure.

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Conformational Stabilization by Buffers: Ligand Binding

Ligand binding is the dominant mechanism of conformational stabilization of proteins. In

the case of buffers, it is likely the most common one for increasing the thermodynamic

stability of a protein. Many studies do not discuss the exact mechanism of stabilization,

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but only report observations. Yet, the effects are profound and worth noting, even in the

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absence of a mechanistic explanation. In general, increasing the conformational stability

will reduce the proportion of aggregation-competent species in solution, thereby slowing

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aggregation rates. The impact of ligand binding follows the Wyman linkage function52. It

assumes that the buffer (ligand) interacts or binds preferentially to the native state,

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resulting in a net stabilization of the native state, while having essentially no impact on
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the denatured or unfolded state (Figure 1).
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The interaction of buffers with proteins has been discussed in general terms in various
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review articles in the context of protein-excipient interactions53-55. These studies, and

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many others, provide a framework for understanding the ability of buffers and other

ligands56 to increase the conformational stability of proteins. Note that ligand binding can
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also impact the activity of the protein, especially enzymes. For example, Tris buffer was

observed to inhibit starch synthase57. However, effects on the activity of a protein are
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relatively rare.
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Conformational Stabilization: Phosphate. Both citrate and phosphate were found to

protect green fluorescent protein (GFP) against inactivation at elevated temperatures,

presumably by increasing conformational stability58. As more of each of these buffers

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was added, the activation energy for inactivation increased substantially. Similarly, these

same two buffers have been reported to produce a 5° to 7° C increase in Tm for

ovotransferrin59.

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The ability of phosphate buffer to stabilize proteins has been widely reported. For

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example, it was found that phosphate yielded a higher Tm value for cFMS over a number

of other buffers, such as PIPES, HEPES, or MOPS60. Phosphate also increases the

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thermal stability of β-lactoglobulin in NaCl-containing solutions, while imidazole and
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sulfate did not, possibly by direct binding of phosphate ion to the protein . In fact,

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ligand binding seems to be the most likely mechanistic explanation for stabilization in
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many of these cases. For example, phosphate was found to increase the Tm of Protein L in

a concentration-dependent manner62, consistent with phosphate binding to the protein.

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In the case of F-actin, denaturation in the presence of phosphate was slower than when
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using MES as the buffer63. Similar stabilization was found for the vnd/NK-2

homeodomain, where phosphate, as well as NaCl, increased the Tm significantly64.

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Phosphate was also found to protect LDH against damage caused by the addition of a

polyelectrolyte65. The use of phosphate buffer has been claimed to stabilize natalizumab
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in a sodium chloride-based formulation during long-term storage66, 67.

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Phosphate was also found to improve enzymatic activity for lyophilized enzymes,

consistent with its kosmotropic effects in the Hofmeister series68. For another lyophilized

enzyme placed into organic media, subtilisin BPN’, the choice of buffer has an effect on

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optimal activity in solvents like DMF69. Citrate was found to impact the activity of

HRP41. Still, reports on buffer effects on activity are relatively uncommon.

Conformational Stabilization: Citrate. In general, it may be that ligand binding may be

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the primary explanation why many citrate formulations appear to be quite stable, even

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when used at pH values where citrate has limited buffering capacity. For example, a

patent by Lu et al. claims stable formulations of an anti-CD40 antibody in the presence of

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citrate buffer across the pH range of 5.0 to 7.0 when arginine hydrochloride is included70.

Stabilization by citrate has been reported for an antibody71 and for malate

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dehydrogenase72, both of which are likely due to ligand binding of the buffer species to
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the native state of the protein, resulting in net stabilization. Citrate was reported to

provide greater stabilization than phosphate at pH 6.0 for L-asparginase, but the effect
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was not deemed to be statistically significant73. It has also been shown that sodium citrate
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inhibits the unfolding and aggregation of the lens protein, γD-crystalline74. More urea is
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required to unfold photoactive yellow protein (PYP) in the presence of citrate compared

to acetate buffer at pH 575.

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Citrate, having three distinct pKa values, has been widely investigated as a buffer for a
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variety of proteins, but mostly in the acidic pH range. Citrate has been shown to increase
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the Tm for thermal denaturation value by 8° to 10° C for RNase76. In this same study,

other carboxylate buffers (tartrate, succinate, malonate, gluconate) were also found to

have some ability to improve conformational stability in other model proteins. This

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suggests a common binding site for species with carboxylate moieties. Citrate was found

to bind to a Fab fragment77, which should result in greater stability.

Pentraxin was found to be quite stable across a wide pH range from 6.0 to 8.5. Citrate

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was found to be an effective stabilizer of pentraxin, as were glutamate and aspartate

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buffers78. On the other hand, buffers, such as tris and imidazole, improved the

reversibility of denaturation of MGDF at pH 7.0 compared to citrate and phosphate,

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while having minimal effects on the Tm values79.

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Yet, citrate is not the only carboxylate buffer known to stabilize proteins, as mentioned
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above. For example, succinate is also fairly widely used, being found in a number of

marketed products. Succinate was reported to stabilize antibodies raised against

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botulinum neurotoxins in the pH range between pH 5 and about 6.580.

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Conformational Stabilization: N-Based Buffers. In the case of interferon-tau, histidine

(His) was found to reduce the aggregation rate substantially at 50° C compared to tris and
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phosphate, despite having the same pH, the same ionic strength, and the same buffer

concentration81. It was discovered that His binds to the native state of interferon-tau with
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a binding constant in the millimolar range, which is enough to provide about 1.0 kcal/mol
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in net conformational stabilization. Similar degrees of stabilization have been noted for

chloride binding to RNase82.

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Tris was found to provide greater stabilization than phosphate for the yeast protein,

Ure2p83. On the other hand, the protein, RecA, was stabilized by phosphate, MES and

HEPES to a greater degree than with tris when measuring denaturation temperature

(Tm)84. One study found that tris interacted more favorably with the peptide backbone

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than other N-based buffers, like TABS and TES85, which may be the basis for preferential

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stabilization with this buffer. Tris was also found to increase the Tm value of etanercept

across the pH range of 6.6 to 8.686. Often, these increases in Tm values are determined

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using differential scanning fluorimetry (DSF). A DSF study on buffer effects found that

buffers can affect the formation of protein-protein complexes, as evidenced by shifts in

the Tm values87.
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Of the N-based buffers, the sodium salt of MOPS was found to be most effective at
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inhibiting the onset of thermally induced aggregation of BSA, followed by HEPES-Na,

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and HEPES. All of these buffers were better stabilizers than EPPS88. In a separate study,
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the same group determined that TAPSO was superior to TES and TAPS for the

conformational stabilization of BSA89.

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The buffer, TEMED, was found to stabilize interferon-beta against thermal stress90.
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Further studies on this system found MES was able to inhibit aggregation across a fairly
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broad pH range (pH 4 up to 8)91. MES, along with phosphate, was also found to increase

the Tm values of cystathionine γ-lyase relative to those with CAPS, tris, or HEPES

buffers92.

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Conformational Stabilization: Acetate. Recombinant phytase was found to maintain

higher levels of activity upon heating in the presence of acetate buffer93. Acetate was

found to increase Tm values for apoflavodoxin, consistent with its position in the

Hofmeister series where it is classified as a kosmotrope94.

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Conformational Stabilization: Excluded Solute Effects

Two general mechanisms can be envisioned for increasing the conformational stability of

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a globular protein: ligand binding and preferential exclusion. The latter is rarely seen for

buffers, possible because buffers are rarely added at the higher concentrations needed to

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obtain a significant effect via this mechanism. One notable exception is citrate. In the
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case of citrate, an increase in conformational stability could be the result of preferential

exclusion.
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Conformational stabilization due to preferential exclusion has been known for decades95,
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. Typically, one thinks of polyols and polymers and their ability to increase

conformational stability by this mechanism. However, any solute that is excluded from
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the surface of the protein could lead to an increase in the conformational stability of the

protein. This negative binding destabilizes the native state of the protein, but the
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destabilization associated with negative binding is even greater for the unfolded state due
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to its greater surface area. The result is a net stabilization, as the energy gap between the

two states has increased in the presence of the excluded solute (Figure 2).

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Of all of the widely used buffers, citrate is known to function as an excluded solute,

especially at high concentrations76, 97, 98. In one study, the addition of large amounts of

citrate provided increased retention of bioactivity of recombinant KGF99, similar to what

was reported for α1-antitrypsin100. These high levels of citrate also reduce aggregation of

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KGF as well101. Glycine also stabilizes proteins by this mechanism, but mostly under pH

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conditions where it lacks any appreciable buffering capacity. Not only can buffers be

excluded solutes themselves, but they have the potential to modulate excluded solute

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effects by changing the surface properties of a protein102. However, little is known about

these secondary types of effects.

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By comparison, Bottomley and Tew found that 0.5 M citrate raised the conformational

stability of α1-antitrypsin by 2.2 kcal/mol 100

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. The authors attributed this phenomenon to

ligand binding. This may be the case, but the high concentration employed here suggests
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that preferential exclusion may also be the basis for the improvement in Tm.
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Recently, it was reported that acetate produced a more compact structure for a
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monoclonal antibody than in the presence of citrate, reducing the hydrodynamic radius by

almost 6 nm. This, in turn, led to better viral filtration properties103. Presumably, this
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behavior is due to some type of acetate-protein interaction, but the exact nature is not
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known. Still, the outcome is a reduction in size that is usually associated with the addition

of an excluded solute.

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Moreover, in another study, acetate seems to be a better choice for stabilization of a

monoclonal antibody at low buffer concentrations relative to citrate, which is likely due

to effects on colloidal stability104. In fact, at pH 4, using 10 mM acetate buffer,

aggregation is largely eliminated for this antibody105. By comparison, the use of acetate

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led to significant precipitation/aggregation of a mouse IgG3 antibody106. It has been

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claimed that TACI-immunoglobulin fusion proteins are stable in acetate buffer between

pH 4.9 and 5.1 in the presence of trehalose107. The use of acetate from pH 4 to 6 can

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reduce reversible self-association of antibodies as well108.

Colloidal Stabilization by Buffers

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Another essential aspect of protein stabilization is colloidal stability. Colloidal stability

refers to protein-protein interactions (PPIs), where macromolecules like proteins can

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behave as classical colloids having a net attractive or repulsive force between individual
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molecules. While conformational stability can affect the propensity of proteins to

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aggregate, so can colloidal stability109-112, although this has only become widely

appreciated in the last few years.

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In general, for proteins in aqueous solution at pH values far from the isoelectric point (pI
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value), pH is the factor that dominates colloidal stability due to electrostatic repulsion.
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However, it has been observed that specific excipients, including buffers can have an

impact on protein-protein interactions above and beyond that of pH. In fact, there are a

number of observations of buffer effects, as found in this review, that appear to be related

to changes in colloidal stability. Certain buffers, such as citrate, become multiply charged

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as the pH increases, resulting in more effective charge screening that reduces electrostatic

repulsion, which can lead to poorer colloidal stability.

A recent study examined the binding of citrate to proteins and its effect on colloidal

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stability. As citrate species can carry a significant negative charge, they were found to

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cause charge inversion at the binding site, thereby altering the colloidal stability profile of

the protein113. This preferential accumulation of citrate at the protein surface appears to

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lead to greater charge shielding and poorer colloidal stability, resulting in increased

aggregation104. The colloidal stability of an IgG1 monoclonal antibody was lower in the

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presence of high levels of citrate buffer, which seems to lead to a slightly increased
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aggregation rate at elevated temperature114. Thus, it appears that citrate is accumulating

on the surface of a protein, leading to decreased colloidal stability. This may be the
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mechanism for disaggregation or disassociation of virus particles in the RotaTeq®

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vaccine115. However, the use of citrate does not affect the safety or efficacy of the
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vaccine.
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Other examples are worth noting here. Buffers affect colloidal stability, as measured by

SAXS116, where neglect of the buffer as a cosolute can lead to inaccuracies in estimating
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the osmotic second virial coefficient. The self-association of a monoclonal antibody

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differs between a histidine buffer and the use of a citrate-phosphate system, which also

infers that the colloidal stability has been modulated by the choice of the buffer117.

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The ability of buffers to modulate colloidal stability can impact the sensitivity to

interfacial damage. In one study, histidine was more effective at preventing damage to a

monoclonal antibody caused by agitation than citrate, which was more protective than

succinate or phosphate118. The ordering of stabilization effects correlates with the

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colloidal stability of the protein119, 120
. Recently, it was found that HEPES slowed

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fibrillation of human islet amyloid polypeptide (11-20) compared to phosphate121. It is

thought that the phosphate ions neutralized the charge and reduced the colloidal barrier to

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aggregation.

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In a study of liquid-liquid phase separation (LLPS), phosphate formulations exhibited
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greater opalescence than a histidine-buffered solution, which seems to correlate to the

colloidal stability of the system. In either case, there was still some propensity for LLPS
M

to occur in either buffer system if stored at 4° C122. Adsorption behavior can be affected
D

by the choice of buffer, as in the case of binding of various proteins to calcium silicate123.
TE

Another illustration of the ability of buffers to modulate colloidal stability is their effect
EP

on the viscosity of highly concentrated protein solutions. It was reported that acetate can

significantly reduce the viscosity of His-buffered concentrated MAb solutions, as can the
C

addition of more histidine124. Colloidal behavior also affects solubility. Low

AC

concentrations of either phosphate buffer or McIlvaine’s buffer (citrate-phosphate)125 was

found to improve the solubility of IgG antibodies126.

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Conformational Stabilization: General Observations and Case Studies

As stated above, there are a number of reports of buffer-specific stabilization of proteins,

many of which do not address specific mechanisms of stabilization. Still, these case

studies can be informative and are worth noting.

PT
RI
Stabilization by Phosphate and Citrate. In the case of G-CSF (filgrastim), phosphate

protected against aggregation over a broad pH range (pH 3.5 to 7.5) to a much greater

SC
extent than acetate or citrate127. On the other hand, citrate protected filgrastim against

hydrogen peroxide-induced oxidation of Met residues, presumably due to its ability to act

U
as a chelator128. At higher concentrations, citrate actually caused more rapid oxidation to
AN
occur129. More recently, it was discovered that islet amyloid polypeptide (IAPP) from

pufferfish formed fibrils more rapidly in tris buffer than in phosphate-buffered saline at
M

pH 7.4130. Interestingly, the choice of buffer also impacted the binding of Thioflavin T to
D

the fibrils in this study, presenting difficulties in using this dye to assess the degree of
TE

fibrillation.
EP

Stabilization by Acetate. There are a number of systems where acetate appears to be

advantageous. For example, acetate at pH 3.8 to 4.2 was found to be the best buffer for
C

stabilization of interferon-beta over citrate, phosphate, and histidine131.

AC

Acetate, along with glutamate, was found to provide the best stability for G-CSF

compared to citrate and succinate across the pH range of 4.0 to 5.0132. Similarly, it was

found that acetate provided the greatest increases in the unfolding temperature by

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differential scanning fluorimetry (DSF) compared to other buffers, with CHES yielding

the lowest Tm values for endogluconase133. CHES also reduces the stability of

organophosphorus hydrolase, which is partially regained by washing with phosphate-

buffered saline, indicating that the sulfonate ion was more chaotropic than phosphate134.

PT
RI
The use of acetate salts of other ionic species has also been reported to be beneficial for

stability. For example, arginine acetate buffer was found to stabilize an IgG1 antibody

SC
against deamidation and aggregation at pH 4.5 to 6.0135. In another patent, the

stabilization of G-CSF (i.e., filgrastim) by acetate between pH 4.15 and 4.3 was

U
claimed136. In fact, this is the buffer system used in the marketed product.
AN
Stabilization by N-Based Buffers. Examples of increased stability with N-based buffers,
M

such as tris and histidine, have been reported as well. It was discovered that EPO was
D

more stable at pH 7 in tris or His buffer than in citrate or phosphate buffer137. Another N-
TE

based buffer, MES, was most effective at reducing aggregation rates among all of the

buffers examined in the aggregation of an IgG antibody, while phosphate and citrate
EP

produced the fastest rates138. This was found to be true despite little, if any, change in Tm

values.
C
AC

Histidine is reported to be the preferred buffer for long-term stability of antibodies

against PD-1139. In the case of palivizumab, solely having histidine as the buffer at

concentrations up to 100 mM is claimed to be sufficient to stabilize this antibody140. For

anti-TFPI antibodies, histidine is claimed to be the preferred buffer for formulations from

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pH 4.0 to 6.0141. Formulations of antibodies to IL-beta are claimed to be most stable

using histidine buffer from pH 5.5 to 7.0142. Similarly, histidine was listed as stabilizing

anti-PRLR antibodies between pH 5.0 and 6.5143. Histidine was also reported to reduce

viscosity of high concentration formulations (> 80 mg/ml) of MAbs144. All of these

PT
stabilization properties may be related to the fact that histidine acts as a kosmotrope145. If

RI
one considers all of the monoclonal antibody pharmaceutical formulations on the market

in 2011, the average pH was 6.0 ± 0.4, making histidine the logical choice for initial

SC
assessment of new MAb formulations146.

U
Another recent patent claims that anti-IgE antibodies can be stabilized in the presence of
AN
histidine buffer at pH 6.0, provided 200 mM arginine is added along with a

polysorbate147. Histidine, in the presence of arginine, was determined to be the preferred

M

buffer for stabilization of a mouse IgG3 antibody, with 50 mM providing optimal

D

stability106. This is similar to the concentration of His (60 mM) reported as being
TE

effective at stabilizing an IgG1 monoclonal antibody148. Tris, along with arginine buffer,

provided better long-term stability of LysB28ProB29-insulin at pH 7.4149. The effect of

EP

buffers and pH on the chemical stability of IL-1β was evaluated using both IEX and RP-

HPLC150. More basic pH conditions resulted in faster degradation at 60º C. Interestingly,

C

Tris was found to provide some stabilization over acetate at pH 5.2, despite being far
AC

from its ideal buffering capacity. The storage stability of cystatins was optimal when tris

or carbonate buffers were employed151.

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In the case of hemoglobin, autooxidation was slower in the presence of HEPES than with

either Tris or phosphate buffers152. Histidine was found to inhibit aggregation of a fully

human MAb, even protecting against freezing damage148. Addition of phosphate or Tris

reduced the activity of HRP in a pH-dependent fashion, but the inactivation was more

PT
pronounced with phosphate than it was with Tris153.

RI
Sometimes, the effects of buffers are not only reflected in changes in Tm values or other

SC
measures of conformational stability, but in other measures. For example, imidazole and

tris were found to increase the reversibility of thermal denaturation significantly

U
compared to phosphate and citrate, which yielded completely irreversible unfolding of
AN
MGDF79. A similar effect was seen for interferon-γ where lower buffer concentrations
M

allowed the thermal denaturation to be more reversible than at higher buffer

concentrations154. In addition, the enthalpies of unfolding also decreased as the buffer

D

concentration was increased, leading to greater degrees of aggregation for acetate and
TE

phosphate buffered samples. In the case of glucose oxidase, both stability and activity

were affected by the choice of buffer species155. The activity of α-chymotrypsin was
EP

found to be enhanced by the choice of buffer in the order tris > TES > TAPS >

TAPSO156. Both MES and HEPES were found to form ordered arrays at interfaces, like
C

mica and lipid membranes, whereas phosphate formed thick amorphous meshes at the
AC

surface157.

Other Buffers. In some systems, the effects of the buffer are seen in the extent to which

they alter enzyme activity, with some buffers increasing the measured activity. For

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example, buffers appear to have a profound effect on the activity of deoxynucleotidyl

transferase158, while the activity of hydroxynitrile lyase can vary by more than 20-fold

depending on the buffer159. In the latter case, glutamate buffer was superior to phosphate

and citrate, but both of these were better than acetate. Similarly, glycine buffer was found

PT
to provide greater stability of L-amino acid oxidase than phosphate or TEA buffers at pH

RI
8.6160. Furthermore, glycine was found to be superior to borate in the stabilization of L-

asparaginase at pH 9.073. Glycine was also found to protect the activity of a thermophilic

SC
acid phosphatase better than acetate near pH 4161.

U
Other lesser known buffers, such as gluconate, may be of value. For example, gluconate
AN
has been claimed to stabilize an anti-TNF antibody across the pH range of 4.0 to 8.0162.

Malonate buffer has been found to be remarkably effective at allowing crystallization of

M

proteins to occur163, 164. While assessing this behavior, malonate was also found to be a
D

cryoprotectant as well163.
TE

Destabilization of Proteins by Buffers

EP

Clearly, there are many examples where buffers are beneficial in enhancing the stability

of proteins in aqueous formulations. However, at the same time, there have been reports
C

of buffers destabilizing proteins as well, both chemically and physically. A number of

AC

excipient screening studies have found buffers to have a profound effect on stability, both

favorably and disfavorably, at least under accelerated stress conditions. For example,

buffers have been discovered to have a significant impact in monoclonal antibody

stability165, a fusion protein166, and epidermal growth factor (EGF)167, as determined by

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screening various excipients. The mechanistic bases for some of these effects are not

known, but a great deal has been reported regarding mode of destabilization by buffers of

various proteins.

PT
Physical Destabilization. It has been reported that phosphate destabilizes lysozyme, as

RI
evidenced by a lower Tm value168. In addition, it has been shown that sodium phosphate

lowers the Tm of horseradish peroxidase (HRP), possibly by binding to the denatured

SC
state of the protein169. Another report indicated that phosphate destabilized HRP at

elevated temperature170. The use of His buffer affected unfolding of an antibody, with

U
thermal unfolding being less cooperative in the presence of His compared to
AN
phosphate171.
M

In the case of aggregation of tau protein, which has been implicated in neurodegenerative
D

diseases, acetate yielded a greater extent of fibrillation than a comparable concentration

TE

of phosphate and both were more destabilizing than water itself172. However, the

aggregates formed in the presence of acetate were more reproducible than with
EP

phosphate.
C

There has been a report of tris causing fibrillation of rat amylin; this same behavior does
AC

not occur in the presence of a phosphate buffer173. The impact of a buffer on stability can

be highly pH-dependent. For example, His was shown to cause greater degradation rates

of EGF than succinate or citrate at pH 6, but was found to be stabilizing at pH 8174. In the

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case of a N-acetyltransferase, HEPES was found to bind in the active site and alter the

conformation of the enzyme175.

Citrate has often been found to destabilize proteins with respect to aggregation, likely due

PT
to lowering the colloidal stability. For example, at low pH (pH 4), monoclonal antibodies

RI
were found to undergo aggregation during longer hold times after Protein A purification.

The rate of aggregation was significantly increased in the presence of citrate relative to

SC
acetate or glycine. Similar effects were seen upon the addition of salt. Therefore, it was

concluded that the effect was due to reduced colloidal stability in the presence of citrate

U
buffer176. Similarly, accumulation of citrate at the protein surface was the rationale for
AN
lower stability of a monoclonal antibody upon heating105.
M

There has been a report that borate buffer at pH 8.0 causes a marked decrease in the
D

enzymatic activity of β-galactosidase, compared to solutions buffered by citrate-

TE

phosphate or phosphate alone, in the presence of organic solvents177. The inactivation

appears to arise from direct interaction of borate ion with the carbohydrates of the
EP

enzyme. Interestingly, if one is to add a polyhydroxy compound to this mixture, the

borate appears to interact with the polyol instead and the activity is partially restored.
C

This type of interaction between buffers and sugars is discussed in more detail below in
AC

the context of lyophilized products.

Another physical instability, at least indirectly, can be decomposition of the container.

For example, phosphate has been reported to promote delamination of glass vials, leading

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to particulate formation178. It also appears that histidine can promote leaching of iron

from stainless steel148.

Chemical Instability. There have been a number of reports of histidine (His) buffer

PT
impacting the chemical stability of proteins. For example, the rate of hinge region

RI
hydrolysis was reported to be lower when using His as the buffer compared to phosphate

at identical pH and ionic strengths171. Possibly, this is because His is a better chelating

SC
agent than phosphate, as this reaction has been reported to be metal catalyzed179-184.

There are reports of His binding different metals. The ability of His to bind copper and

U
cause hydrolytic cleavage of DNA has been reported185.
AN
The chelation ability of histidine was reported to affect the stability a MAb formulation
M

containing 60 mM His, where this higher buffer concentration may also promote leaching
D

of iron from stainless steel148. An increase in soluble iron species would likely lead to an
TE

increase in metal-catalyzed oxidation (MCO) of the protein. In addition, His has been

shown to promote formation of hydroxyl radicals, which can then lead to increased rates
EP

of hinge region degradation186, 187, although the exact mechanism of degradation was not

described. Histidine has been found to participate in other oxidative processes as well.
C

For example, histidine has been found to be a photosensitizer188, which, in turn, leads to
AC

increased photooxidation of the protein.

This ability of certain buffers to bind to metals can result in an increase in the reactivity

of the metal rather than diminish it, as occurs with most chelating agents. For example,

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the complex of phosphate and ferrous ion is more reactive than free Fe2+ alone189.

Ascorbate, the conjugate base of ascorbic acid, is another buffer known to complex

metals and make them more effective at carrying out MCO190-192. At the same time,

ascorbate can be effective at protecting against certain types of oxidative damage193.

PT
RI
Still, redox-active metals can cause damage to proteins, even at fairly low levels. In the

case of copper, as little as 75 ppb is sufficient to cause oxidative damage194. Residual

SC
metals in tris and phosphate buffers were found to contribute to oxidation of methionine

residues195. These effects can be magnified by certain buffers. Phosphate buffer was

U
shown to accelerate MCO damage of a peptide in an ascorbate/Fe3+ system compared to
AN
Tris, HEPES, or MOPS buffers196. Citrate complexes of iron have been shown to cause

oxidative damage of proteins197. The ability of other carboxylate buffers (tartrate, malate,
M

succinate) to bind iron has been demonstrated as well198. However, even Tris has been
D

shown to function as a chelating agent199, as can ACES, which is a Good’s-type buffer

TE

(see below)200.
EP

Citrate is known to be a chelator of metal ions128, 129, 201, 202. If a protein is stabilized by

metal binding203 or if the metal is essential to activity, then such buffers could be
C

physically destabilizing, as in the case of proteases and amylases found in laundry

AC

detergent. These metal-binding proteins can be destabilized by the addition of chelators,

including citrate, which remove the calcium needed for conformational stability204. On

the other hand, complexes of divalent metal ions with citrate appear to stabilize oxytocin,

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both in solution205 and in spray-dried solids206. The same stabilization strategy works for

metal complexes of aspartate buffer as well207.

The ability of zwitterionic N-substituted aminosulfonic acids, also known as Good’s

PT
buffers, to bind metals has been reviewed208. Good’s buffers have been known for

RI
decades17, 209
, although they are not widely employed in biopharmaceutical products.

They have the ability to act as free radical scavengers as well as chelating agents, making

SC
them useful examples of how buffers can affect stability by various mechanisms. The

effect of metal binding on the behavior of Good’s buffers in biochemical and biological

U
systems was discussed. In addition, when the anionic species of Good’s buffers are
AN
combined with cholinium chloride to form ionic liquids, they display an ability to

stabilize proteins, such as BSA210.

M
D

Buffer Catalysis of Hydrolytic Reactions. It has been known for some time that buffers
TE

can promote the hydrolysis of amide groups, whether of the peptide backbone

(proteolysis) or of an Asn or Gln side chain (deamidation). Deamidation is known to be

EP

primarily driven by pH, where the optimal pH for slowing deamidation appears to be

between pH 3 and 63, 211, 212

. This is particularly illustrated by the sensitivity of
C

deamidated peptides to undergo racemization as well via deprotonation of the tetrahedral

AC

intermediate213. In addition, other hydrolytic reactions, such as proteolysis and

isomerization are acutely sensitive to pH184, 214, 215.

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Beyond the direct effect of pH, these are reactions that can be catalyzed by buffer

species. Buffer catalysis has been demonstrated for buffers as diverse as borate,

phosphate, and glycine. In general, the reaction appears to involve specific rather than

general buffer catalysis216. The effect is known to be pH-dependent211, buffer

PT
concentration-dependent, and even temperature-dependent217. As a result, one will

RI
usually want to use a minimal amount of buffer if deamidation is problematic for a given

protein.

SC
There have been a number of reports of buffers having a differential effect on the extent

U
of deamidation in a peptide or protein. Phosphate was found to cause more rapid
AN
deamidation of equine α-lactalbumin than tris buffer218. Likewise, phosphate caused

more deamidation than tris and bicarbonate of a tripeptide219, although all three buffers
M

showed some degree of buffer catalysis. Phosphate also catalyzed deamidation in other
D

short peptides220. Similarly, various bicarbonate buffers were found to accelerate

TE

deamidation relative to tris, even at pH 6221. Both glycine and phosphate buffers were

found to catalyze deamidation of Asn-His-containing peptides in a pH- and

EP

concentration-dependent manner222. Even larger proteins, like lysozyme, exhibit more

rapid deamidation when the phosphate concentration increases223. Four different buffers
C

(citrate, phosphate, tartrate, succinate) were evaluated for their effect on deamidation of
AC

an IgG1 MAb and it was determined that citrate yielded the slowest deamidation rates 224.

Various buffers were evaluated for their effect on deamidation of human epidermal

growth factor as well225. Of note, tris does not seem to promote deamidation226.

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Buffer effects on Asp isomerization have also been reported. In the case of a MAb,

succinate was found to exhibit the least amount of buffer catalysis compared to phosphate

and citrate227. The same group found buffers impact the rate of formation of

pyroglutamate at the N-terminus of polypeptide chains, with tris exhibiting slower

PT
reactions rates than when phosphate was used228.

RI
With proteolysis of Asp-Pro sequences, acetate was reported to yield slower rates than

SC
with succinate buffer229. Hydrolytic scission of the hinge region was found to be buffer-

dependent as well171. The distribution of cleavage sites is pH-dependent, emphasizing the

U
importance of both buffer composition and pH184. Likewise, diketopiperazine formation
AN
was found to be affected by the nature of the buffer230. Both glycine and phosphate were

found to accelerate DKP formation in a pH- and concentration-dependent fashion231. In

M

general, it appears that most hydrolytic reactions that can occur in proteins and peptides
D

can be buffer-catalyzed, so buffer selection and concentration is critical for long-term

TE

stability.
EP

Buffers as Scavengers

Over fifty years ago, there was a publication that described some buffers as having the
C

ability to scavenge free radicals209. Over time, these buffers became to be known as
AC

Good’s buffers after the first author of this article. These buffers tend to be N-based

buffers. There are four groups or classes of Good’s buffers (Table 5). In general, these

types of buffers (e.g., HEPES, PIPES, EPPS) can take part in modulating free radical

processes232. In addition, various zwitterionic butanesulfonic acids were described as

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having similar properties to Good’s buffers233. Among these, HEPES may be the most

widely investigated. For example, HEPES was found to protect apoferritin by functioning

as a free radical scavenger234.

PT
Although Good’s buffers are not necessarily found in marketed protein pharmaceutical

RI
products, they illustrate the fact that buffers can potentiate oxidative processes by acting

as free radical scavengers. In addition to Good’s buffers, histidine buffer was shown to be

SC
as effective as the well known free radical scavenger, ascorbate, in protecting fibrinogen

from sterilizing doses of gamma irradiation235. Ascorbate serves not only as a buffer, but

U
also as a radiolytic stabilizer for metalloradio-pharmaceuticals236.
AN
It has been reported that, in the process of chemical modification of Tyr residues using
M

click chemistry, a side reaction can occur to form an isocyanate byproduct. This
D

unwanted side-product can be effectively scavenged by tris, where the amine

TE

functionality readily reacts with the isocyanate. The Tyr modification reaction can be

performed in the presence of a variety of buffers, but only the addition of tris was found
EP

to be an effective method to mitigating the side reaction237.

C

Buffer Effects on Chromatography/Separation Techniques

AC

Buffers can play a critical role in the efficiency of separation techniques, whether for

analytical purposes or purification. The effect of temperature on buffer effects during

separation has been reviewed238. Buffering capacity and buffer concentration appears to

be critical for adequate separation of proteins on hydroxyapatite columns239.

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Specific buffer effects have been reported for various separation techniques. A two-buffer

system replaces polyampholyte for cation exchange chromatofocusing of MAbs240.

Buffers have also been found to be influential in mitigating reversible self-association

PT
that is seen during cation-exchange chromatography241. In the anion exchange

RI
chromatographic purification of α-lactalbumin from enriched whey protein concentrate,

acetate was the preferred buffer and could be used over a wide pH range (up to pH 7)242.

SC
In a comparison of buffers for purification of virus particles by anion exchange

chromatography, phosphate was found to yield greater capacity than tris buffer243.

U
Surface-mediated denaturation has been reported for an aglycosylated MAb during
AN
cation-exchange chromatography. The extent of unfolding was reduced by using citrate

or glycine, both of which can function as excluded solutes97.

M
D

A multi-component buffer system capable of maintaining pH over a range of 7.5 pH units

TE

was developed for in pH-gradient ion exchange chromatography244. The use of mixed

electrolytes, including buffer species such as citrate, acetate, and glycine, in hydrophobic
EP

interaction chromatography was examined245. A citrate-phosphate buffer system was

reported to be preferable for sample preparation for CE-SDS compared to the buffer
C

supplied by the vendor246. The ability of acetate to control protein unfolding during
AC

reversed phase chromatography was evaluated, relative to other salt species247. Histidine

was found to be the best choice for Protein A purification of MAbs248. Polishing of

antibodies using convective flow devices worked best at pH 7.5 to 8.0 using phosphate

buffer, which was found to work better than HEPES or Tris buffers249.

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After purification, proteins are typically held for further processing for some time.

Holding them in buffers commonly used for ion-exchange chromatography (tris, acetate,

phosphate) helps reduce the extent of aggregation176.

PT
RI
Citrate was found to improve the resolution of two forms of β-galactosidase when using

capillary electrophoresis250. In the separation of peptides using electrochromatographic

SC
techniques, the role of the buffer species was found to be critical in achieving

selectivity251. For determination of molecular weights by electrophoresis, it was found

U
that tris provided superior resolution without the need for other additives252.
AN
M

Degradation of Buffers

Buffer themselves can degrade by a variety of processes. This is especially true for
D

organic buffers, like histidine. For convenience, the degradation of buffers discussed in
TE

this section is divided between decomposition occurring upon light exposure and other

mechanisms. This includes adduct formation with peptides or proteins.

EP

Photodegradation. Photodegradation has been reported for aromatic buffers like His253,
C

254
. It has been reported that histidine can degrade during an accelerated stress study,
AC

being converted to trans-urocanic acid255. Addition of alanine or cysteine can effectively

inhibit this process. The same group demonstrated that the degradant can be isolated and

characterized. It appears that the light from a diode array detector alone can contribute to

the production of trans-urocanic acid256. In one study, the photodegradation of citrate has

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been reported, with the decomposition of citrate resulting in an increase in protein

acetonation257.

Chemical Degradation. Thermal degradation of His buffer has been reported as well.

PT
Heating histidine buffer can lead to production of formaldehyde products, which can

RI
result in chemical damage to proteins258. Free histidine, like His residues in proteins, can

be damaged by metal-catalyzed oxidation (MCO)259, usually producing 2-oxohistidine259-

SC
261
. Often, chemical degradation of His buffer results in the appearance of a yellow color.

U
Free radical degradation of certain buffer species has been known for some time71, 262.
AN
Since buffers can degrade, one must be vigilant regarding the purity of the buffer, as

excipient purity can adversely impact protein stability263. Certain Good’s buffers were
M

found to be oxidized by hydrogen peroxide, where the morpholine ring of MOPS and
D

MES and the piperazine ring of PIPES, HEPES, and EPPS were oxidized to the
TE

corresponding N-oxide forms264. Peroxynitrite can react with HEPES to form hydrogen

peroxide, suggesting that HEPES and similar Good’s buffers should not be used with
EP

proteins that are sensitive to oxidation265. In a similar fashion, hydroxyl radical are

known to react with Tris buffer, leading to formaldehyde products266.

C
AC

Buffer-Polypeptide Reactions. There have been reports about the direct chemical reaction

of certain buffers with peptides and proteins. For example, citrate adducts have been

described as occurring in oxytocin formulations267 and in monoclonal antibody

preparations268, although the potential for such reactions with citrate and succinate was

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mentioned much earlier269. In the case of antibodies and also with antibody-drug

conjugates (ADCs), formation of covalent adducts with citrate at the hydroxyl groups of

Ser and Thr residues has been observed270. The same study found evidence of adducts

formed with Tris and His as well as citrate.

PT
RI
Physical Instability of Buffer Species. Of all of the buffers, phosphate is the best known

for physical instability in aqueous solution. In large part, this is due to formation of

SC
complexes with certain metal ions that are highly insoluble. These include calcium and

zinc ions. As mentioned above, these incompatibilities are well known271.

U
AN
Buffer Free Formulations

There have been reports of stable protein formulations that do not contain any added
M

buffer as an excipient. These systems, which are comprised of protein at high

D

concentrations, are self-buffering. In other words, the protein itself is providing the
TE

majority of the buffering capacity. The notion of proteins being able to act as buffers has

been known for some time34, 36, 37

. Now, the concept has been applied to developing
EP

stable pharmaceutical formulations. For example, in monoclonal antibody (MAb)

formulations at 80 mg/ml, the buffering capacity was determined to be similar to that of

C

20 mM acetate buffer30. By comparison, another study found that MAb solutions at 50

AC

mg/ml had buffering capacities equivalent to 6 mM citrate or 14 mM His32. A more

recent study examined buffer-free formulations of MAbs at high concentrations and

determined their buffering capacities over different pH ranges31. The buffer-free

formulations exhibited less aggregation upon shaking than those containing a buffering

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agent. This significant buffering capacity comes from the sheer number of ionizable

groups in a protein. For example, if a typical monoclonal antibody, there are

approximately ~25 His residues, 50-60 Asp residues, and 55-75 Glu residues32, not to

mention other functional groups. The average pKa values of these groups in folded

PT
proteins has been tabulated by Grimsley et al. and these can be found in Table 433.

RI
A detailed study was undertaken comparing buffer-free lyophilized formulations of a

SC
MAb at high concentrations. It was determined that the buffer-free formulations were as

stable as those containing a conventional buffer system, provided there was a

U
lyoprotectant present. In addition, no shift in pH was observed with the buffer-free
AN
formulations272. It is likely that commercial, high concentration antibody products will

emerge that eschew the use of buffer and rely upon the buffering capacity of the protein.
M
D

Multiple Buffer Systems

TE

Occasionally, the use of more than one buffer species is reported for a protein

formulation. In the case of the marketed MAb, Humira (generic name: adalimumab), both
EP

citrate and phosphate are present in a formulation having a pH of 5.2. This composition

for adalimumab has been described in various patents273, although the same inventors
C

claim that phosphate works just as well274 across the pH range of 4.0 to 8.0. As phosphate
AC

has little or no buffering capacity at this pH, it is not clear about the origin of the actual

benefits of phosphate in this system and the patents do not discuss this. Although the

mechanism of stabilization may not always be clear in the case of these mixed buffer

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systems, the compositions are claimed in multiple patents held by AbbVie regarding

adalimumab.

Other antibodies appear to be stable in this citrate/phosphate buffer system, provided that

PT
the pH is between approximately 5 and 6275. On the other hand, citrate alone appears to

RI
provide better stability to interferon-α2b with respect to aggregation compared to citrate-

phosphate or phosphate itself276. The citrate-phosphate buffer system has been claimed to

SC
be beneficial for preparing high concentration formulations of MAbs by ultrafiltration if

the pH is 5.2277. It also has been reported to stabilize G-CSF127.

U
AN
Together, the combination of citrate and phosphate is known as McIlvaine’s buffer125, 278,
279
M

and has been known for almost a century. The primary advantage is that it can be used

for screening protein stability across a larger pH range than either buffer component
D

alone. However, with the exception of the examples listed above, this composition is not
TE

widely used as a buffer system in a commercial therapeutic protein product. It was

recently reported to be used during purification of a scFV antibody fragment280. Another

EP

interesting aspect of the citrate-phosphate buffer is that MAb self-association increases

117
with increasing pH in this buffer system, but not when a His buffer is employed .
C

Presumably, this is related to differences in colloidal stability. Also, McIlvaine’s buffer

AC

was found to perform similarly to citrate and succinate in the thermostabilization of G-

CSF, all of which are outperformed by acetate and glutamate buffers in terms of

stabilization132. McIlvaine’s buffer has also been reported to be effective at reducing

aggregation of IgG antibodies126.

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The use of histidine and phosphate together was found to be effective in the stabilization

of a hepatitis B vaccine at pH 5.2281, 282

. This particular combination is the basis for

various intellectual property claimed by Arecor. A recent patent listed the combination of

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acetate and histidine at pH 5.6 to 6.2 as being favorable for the stabilization of antibodies

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against interleukin-4 receptor283, 284
. Similarly, the histidine-acetate buffer system was

described in a patent on stabilization of antibodies against HER2 in the pH range of 5.5 to

SC
6.5285. In fact, the commercial product, Perjeta, uses the histidine acetate buffer system

(Table 1). The temperature dependence of the histidine acetate system has been described

U
and compared to the chloride salt of His22. While it is not clear if the acetate has a
AN
significant impact on histidine ionization, it may be beneficial to use histidine acetate22 in

systems where chloride may be contraindicated148. The stability of a fusion protein was
M

evaluated in histidine acetate and compared to the stability in citrate buffer166.

D
TE

Buffers in Frozen Formulations

Frequently, protein solutions are frozen, especially for storage of the bulk drug substance.
EP

It is also a common way to store drug product when it is to be used in early stage clinical

trials. Moreover, protein samples can also be frozen to facilitate analysis at a convenient
C

time. While freezing a solution can be convenient, there can be stability issues associated
AC

with the freezing and thawing of protein solutions. For example, it is well known that

sodium phosphate buffer at pH 7, comprised of both monobasic and dibasic phosphate

species, can undergo selective crystallization of the dibasic species upon freezing, leading

to acidification of the sample by as much as 3 pH units286, 287. It has been demonstrated

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that this effect is dependent on the concentration of the buffer, with higher phosphate

concentrations leading to a greater degree of acidification. Interestingly, potassium

phosphate displays just the opposite behavior, with the monobasic species crystallizing

during freezing, leading to a rise in pH288. Upon freezing, Tris has also been reported to

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exhibit abrupt decreases in pH289. It has been reported that if one mixes HEPES with

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potassium phosphate, the resulting temperature independent buffer system will hold a

relatively constant pH all the way down to -180° C26. In all of these cases, the addition of

SC
other excipients or increasing the protein concentration will hinder the crystallization

process, thereby diminishing the extent of the pH shift288, 289.

U
AN
Not surprisingly, this potential shift in pH can dramatically alter the stability of a protein.

The selective crystallization of sodium phosphate has been widely studied in terms of its
M

relationship to the stability of proteins290-294, whether for frozen dosage forms or in the
D

context of freeze-drying. Similarly, the phase change and associated pH shift in an

TE

acetate buffer appears to contribute to multimer formation in atrial natriuretic peptide

during the freeze-drying process295. Consequently, other buffers besides sodium

EP

phosphate are used when dealing with frozen protein solutions. For example, use of Tris

buffer in place of sodium phosphate buffer leads to lower amounts of aggregation of

C

LDH upon freezing and thawing at pilot scale296. Using HEPES instead of phosphate
AC

reduced damage to haemagglutinin by limiting the change in pH297. The authors classify

HEPES buffer as cryoprotectants, although it was used at 2 mM, which may account for

the limited shift in pH upon freezing. Others have seen larger pH changes with HEPES

upon cooling and freezing298. Still, HEPES-buffered solutions were more stable to freeze-

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thaw stresses than PBS-based formulations, where the pH shift could be as much as 3 pH

units, unless sugars were present.

As noted above, the selective crystallization of the dibasic form of sodium phosphate

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results in acidification. By comparison, the monobasic form selectively crystallizes upon

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freezing when using potassium phosphate, resulting in basification. The differences in pH

when using both phosphate salts have been observed in the turbidity of bovine IgG293. In

SC
both cases, the crystallization is inhibited by the presence of other solutes, which means

that in most formulations, little change in pH is actually observed288.

U
AN
Other buffers have been shown to undergo crystallization upon freezing as well. Freezing

effects on the succinate buffer system, which can have three components (succinic acid,
M

monosodium succinate, and disodium succinate), has been studied in detail299. If the
D

initial pH is 4.0, the pH of the freeze-concentrated state could rise to as much as 8.0
TE

initially, followed by a decrease all the way to 2.2. A similar swing, but in the opposite

direction, is seen if the initial pH was 6.0299. This behavior is due to sequential
EP

crystallization of the various deprotonated components300. Addition of glycine or

mannitol or sucrose inhibited the crystallization of succinate buffer species to varying

C

degrees301.
AC

Various types of carboxylate buffers were studied for their behavior upon freezing.

Tartrate and citrate display a rise in pH upon freezing, if the initial pH is less than 2302,
303
. Interestingly, malate showed no selective crystallization upon cooling and freezing.

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Knowledge of the relative solubility of each component allows one to predict accurately

the crystallization behavior303. The initial pH also impacts which buffer species

crystallizes upon freezing for His and Gly buffers302. Glycine, when it crystallizes, can

form a variety of polymorphs. The exact polymorphic distribution obtained upon freezing

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depends on the initial pH and solute concentration304.

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Some buffers remain amorphous in the frozen state as well as in the dried state. In doing

SC
so, they can provide some degree of stabilization to a protein. The cryoprotective ability

of carboxylate buffers on hen egg white lysozyme appears to increases as the number of

U
carboxylate groups increases305. It has been reported that citrate is a better stabilizer than
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acetate for azurin in ice, consistent with its higher position on the Hofmeister series306.

By remaining amorphous, the buffers contribute to the glass transition temperature of the
M

maximally freeze concentrated state (Tg’). In general, the Tg’ values of buffers tend to be
D

quite low (Table 6). For example, Tg’ for glycine is between -70° C and -85° C307. Chang
TE

and Randall tabulated Tg’ values for various buffer species, many of which are commonly

used in therapeutic protein formulations. They also measured the eutectic melt
EP

temperatures (Te) for many buffers that crystallize in frozen solution as well308.
C

While some buffers remain amorphous upon freezing, others crystallize easily. This
AC

behavior becomes even more complex in the presence of sugars, which are effective

stabilizers of proteins, both in the frozen state and in dried solids. Yet, sugars and buffers

are frequently observed to interact, altering the physical properties of each component.

For example, it has been reported that when sugars (glucose or maltose) are frozen with

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various phosphate species, Tg’ (once again, the glass transition temperature of the

maximally freeze-concentrated state) is altered. The Tg’ of glucose increases in the

presence of sodium phosphates as does the Tg’ for maltose, but to a lesser degree. This

was attributed to direct phosphate-sugar interactions309. Tetrasodium tripolyphosphate

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was found to increase Tg’ values for sucrose by 4.2° C, for trehalose by 2.1° C and for

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sorbitol by 14.9° C310.

SC
More often, buffer salts reduce the Tg’ values of sugars and polymers. Various types of

chloride salts were reported to lower the Tg’ of sucrose, trehalose,311 and PVP312. In like

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fashion, phosphate salts lower the Tg’ of PVP, although not nearly as much as NaCl
AN
does313. Phosphate buffer salts also can affect the crystallization of PEG, especially at

higher concentrations314. Buffers can also cause phase separation with polymeric
M

additives315. Allowing stabilizing excipients to remain amorphous is critical for long-term

D

stability in the frozen state, especially at -20° C. This appears to be more critical than pH
TE

shifts caused by buffer crystallization or temperature dependence22. Excipients that

crystallize can no longer stabilize the protein in the frozen state316, 317
. Buffers like
EP

HEPES and histidine were also reported to lower Tg’ values for sugars318.
C

Freeze concentration can occur during freezing, especially depending on the cooling rate.
AC

It was found that the concentration distribution of phosphate and tris were comparable

when freezing HRP in bottles153.

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Buffers in Dried Formulations

Control of pH is also important in freeze-dried (i.e., lyophilized) formulations. Keep in

mind that as water is removed, the traditional Bronsted-Lowry definition of pH no longer

applies. It is the ionization state of each group that matters, a phenomenon termed

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microenvironmental pH319. It appears that, for dried solids, whether in a powder or

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suspended in a nonaqueous solvent, the ionization state of the protein does impact its

activity and stability. The ionization state can be established either by the addition of a

SC
buffer at the appropriate pH or by the addition of a counter ion that can provide the

optimal pH of the salt form of the drug319. In this context, buffers and the initial pH play

U
an important role in controlling the stability of lyophilized proteins. As a result, outside
AN
of the crystallization events that could occur during the freezing step, buffers have been

shown to have a profound impact on the properties of freeze-dried protein formulations.

M

Glycocholate buffer, for example, provides greater stability to lyophilized interferon-γ

D

than succinate320. Histidine was found to be a superior buffer for stabilization of

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lyophilized LDH compared to phosphate and citrate321. Sodium citrate buffer was found

to provide better stability than sodium phosphate for freeze-dried IL-1 receptor antagonist
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(IL-1ra) at pH 6.5322. The pH appears to be critical here, as the stability of IL-1ra

decreased significantly when the pH was lowered, even just to pH 6.0 or 5.5. In other
C

cases, the selection of buffer has minimal effect323. In the case of lignin peroxidase, the
AC

buffer dimethyl succinate, was found to be a lyoprotectant324.

One factor to consider is that buffers can interact directly with carbohydrates (sugars,

starches, and polyols). In doing so, they can alter the properties of the matrix surrounding

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a protein in a freeze-dried solid. For example, phosphate ion was shown to increase the

Tg (glass transition temperature of the dried solid) values of both sucrose and trehalose in

a pH-dependent manner325. The effect is sizable, with increases of approximately 20º C

being observed. In a similar fashion, borate increases Tg values of glucose and maltose,

PT
which actually leads to greater structural distortion of the protein in the dried state326. On

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the other hand, tris and other buffers were found to lower Tg and Tg’ values for trehalose
327
. However, more recently, it was found that the interaction of trehalose and phosphate

SC
buffer is more subtle than previously appreciated. While monobasic phosphate is a

plasticizer of trehalose, dibasic phosphate was found to increase the Tg of trehalose328.

U
Mixtures of choline dihydrogen phosphate and trehalose display Tg behavior that can be
AN
described by the Kwei equation329. Citrate increases the Tg of sucrose-based

formulations330. Combinations of phosphate species and sugars were found to be more

M

protective for lyophilized liposomes than the sugars themselves331.

D
TE

While buffer salts can alter the Tg of sucrose-based formulations, one must also recognize

that the potential crystallization of sodium phosphate and the associated acidification of
EP

the solution can lead to sucrose hydrolysis, but this is only likely to happen at quite high

buffer concentrations332. The same study also found that buffers can impact the amount of
C

residual moisture left in the freeze-dried cake.

AC

Another aspect of buffers in freeze-dried formulations is the ability of the buffer to

impact crystallization of bulking agents. Phosphate buffer was found to impact the

crystallization of both mannitol and glycine333. Potassium phosphate was also reported to

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inhibit crystallization of mannitol. The remaining amorphous mannitol provides sufficient

stability to the protein334. Sodium phosphate and sodium citrate inhibit crystallization of

mannitol as well335.

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The ability of buffers to interact with amino acids has led to alternatives to sugars for

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suitable glassy matrices for protein stabilization. In one study, the interaction of buffers

having multiple carboxylate groups (e.g., citrate, tartrate) with arginine was shown to

SC
produce amorphous glasses capable of stabilizing proteins336, 337
. In another study, the

combination of glycine and HEPES buffer was found to be an effective stabilizer of

Factor VIII and Factor V338.

U
AN
Some buffers can actually be lyoprotectants themselves, forming an amorphous matrix in
M

which to stabilize the protein. Histidine, along with arginine, was found to be the
D

preferred stabilizer for monoclonal antibodies in lyophilized formulations over other

TE

amino acids339, 340.

EP

The Tg values of buffer species have been reported. For example, the Tg of dried citric

acid was found to be 10º C341, 342

. The Tg’ values for various other buffers have been
C

described as well. The Tg’ values for tris are quite low (-51º C for the base and -65º C for
AC

the conjugate acid). One study found the Tg’ value of citrate buffer to be -40º C, while

sodium phosphate buffer (a 1:1 mixture of monobasic and dibasic forms) was reported as

having Tg’ at -80º C 343.

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Some buffer systems are known to be volatile, meaning that they have an appreciable

vapor pressure under vacuum. This can be especially problematic during the sublimation

phase of freeze-drying, where the amount of buffer present in the final product may vary

due to its volatility. Volatile buffers have been reviewed12. Such buffer systems include

PT
acetate (acetic acid-sodium acetate), trimethylammonium formate, trimethylammonium

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acetate, and ammonium carbonate. The removal of volatile buffers may be advantageous

in subsequent procedures, such as chromatography and electrophoresis12.

SC
Toxicological/Safety Issues with Buffers; Pain upon Injection Issues

U
Another consideration for selecting a buffer for use in a therapeutic protein product is
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related to its safety profile. Even though one tends to think of excipients as inactive, this

is not always the case344, 345. For example, pamoic acid buffer system actually is a potent
M

activator of the G-coupled protein receptor, GPR35346.

D
TE

As for safety, many buffers were found to exhibit no cellular toxicity, even at

concentration up to and above 50 mM16. So while systemic toxicity of buffers has been
EP

rarely noted347, there is a greater concern regarding local irritation or toxicity, especially

for products given by subcutaneous (sc) injection. Of all of the buffers commonly used in
C

protein formulations, citrate is usually considered as the one having the greatest risk of
AC

causing pain upon injection, especially subcutaneous (sc) injection. To whatever extent

this may be true, it is likely related to the ability of citrate to act as a chelating agent for

calcium, which is known to play a role in pain sensation348, 349. Contrary to what one may

believe, there is actually relatively little literature on this topic. The limited number of

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clinical studies has been discussed repeatedly in medical journals, so it is worthwhile to

consider the specific findings from the original studies. Most of this literature focuses on

pain perception. Certainly, other adverse events (irritation, tissue damage, etc.) can occur

as well, but the there is little mention of this as it relates to buffers or any other excipient,

PT
for that matter.

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The issue of pain upon injection was reviewed in 1998349. Even at that point in time, a

SC
number of studies had been published on the possible role of buffers causing irritation or

pain upon injection, especially SC injection. Most of these studies focused on

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erythropoietin (EPO). During the early 1990s, EPO was beginning to be administered by
AN
SC administration compared to an intravenous (iv) injection, which was the standard

treatment. It was reported that Eprex (epoetin alfa), one of the EPO products on the
M

market at the time, caused pain upon injection350-352. The conclusion of this series of
D

studies was that this was likely due to the presence of citrate buffer. Another EPO study
TE

suggested the same fact353, although they raised the possibility that other excipients, such

as human serum albumin (HSA) and polysorbate, could alter the perception of pain. For
EP

example, a multi-dose formulation of epoetin alfa was found to be less painful than the

original formulation, suggesting that preservatives may modulate pain perception354.

C

Similar results were reported when comparing two different EPO products355-357. Another
AC

study compared citrate-buffered EPO with one buffered by histidine and found the

citrate-containing product caused greater discomfort358. This finding was supported by a

subsequent study by Laursen et al.359 . On the other hand, a study reported by Boyce et al.

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(1995b) found no difference in pain upon injection with epoetin alfa (buffered with

citrate) and epoetin beta, which was buffered with phosphate360.

Therefore, these studies clearly suggest that citrate could be problematic in terms of

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potentially causing pain upon injection, which could, in turn, compromise patient

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compliance361. However, one must be careful in such a judgment. Certainly, there are

products on the market using citrate as the buffer that are administered by SC

SC
administration. Other factors are known to play a role in pain perception as well. For

example, a study using phosphate-buffered formulation of IGF-1 found that a 50 mM

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phosphate formulation at pH 6 caused increased pain upon injection362. Yet, there is
AN
rarely a concern over using phosphate as the buffer for a SC product. The same study

found that a phosphate concentration of 50 mM could be tolerated if the pH was 7 rather

M

than at 6. It also reported that a pH 6 formulation containing only 10 mM phosphate also

D

did not exhibit pain issues upon SC injection. Thus, one must conclude that using the
TE

minimal amount of buffer necessary is one strategy for mitigating pain upon injection

concerns. In the case of iduronate-2-sulfatase treatment by intrathecal administration, a

EP

patent was issued where the phosphate buffer concentration was limited to no more than

5 mM363. Also, having a formulation that is closer to the pH of the body (pH 7.4), will
C

also be helpful in reducing discomfort. This latter point is reinforced by the observation
AC

that both alkaline and acidic pH conditions can lead to perception of increased pain upon

injection364.

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Having the appropriate buffering capacity, no matter what pH is employed, may be

helpful in reducing pain perception365. This study also found that warming the solution

prior to injection also decreased pain. So, the temperature difference between the protein

solution and SC space (34º C)366 could be an important consideration. Finally, injection

PT
volume was found to impact pain perception for a citrate-buffered EPO, where 0.4 mL

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injection volumes appear to be better tolerated than 1.0 mL SC injections367. This point

regarding injection volume has been reiterated in many publications, where the

SC
recommended maximal volume for SC injection is reported to be 1 ml or less368-370. In

addition, the injection time and the injection site may also play a role in pain perception

U
with SC administration371. Thus, a variety of factors (needle gauge, frequency of
AN
administration, etc.) need to be considered when assessing SC pain upon injection, not

simply the buffer composition.

M
D

Interestingly, the nature of the protein may also be influential in the perception of pain.
TE

Injections of CERA, an EPO analog, were more painful than for darbepoietin, another

EPO-like compound372. By comparison, darbepoetin alfa was found to be more painful

EP

than epoetin beta when administered subcutaneously, despite having a phosphate

buffer373. Finally, glutamate has also been reported to cause pain upon injection at pH
C

7.2374, but little is known about how varying the pH or buffer concentration may impact
AC

this effect. It may be related to calcium binding by the carboxylate groups of glutamate.

In addition to issues related to SC administration, it has been reported that buffers can

exhibit myotoxicity as well when given by intramuscular (im) injection. For example,

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succinate, as well as acetate, was found to display low myotoxicity, while citrate was

found to show higher values of myotoxcity375. It has been reported that IM injections of

an influenza vaccine causes more pain and discomfort in children than the corresponding

product given subcutaneously376.

PT
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Another potential cause for toxicity would be precipitation of the buffer in the presence

of metal ions, such as calcium. These incompatibility issues are well known in the

SC
pharmaceutical sciences271. For example calcium phosphate is notoriously insoluble,

which could lead to precipitation377, 378 and obstruction of capillaries, in a process known

U
as crystalluria379 and also cause acute phosphate nephropathy due to calcium phosphate
AN
precipitation in distal tubules380.
M

Other Considerations
D

Buffers can also impact the analysis of proteins. For example, it has been known for
TE

some time that Tris buffer can interfere with protein content determination using the

Lowry method381. Other buffers besides Tris, such as HEPES, EPPS, and bicine, can also
EP

interfere with the Lowry assay12, 382. Both tris and glycine buffers are reported to interfere

with the Bradford assay12. Tris is also known to interfere with electrospray ionization-
C

mass spectrometry383, 384, although a fused droplet approach to ESI-MS appears to allow
AC

one to use very high concentrations of tris without any issues385. It has also been reported

that different buffers work better than others as extraction media for assessing chemical

and physical changes in proteins undergoing stress386. Buffers can even impact the

measurement of the pI value. For example, the isoelectric points of wild-type aspartate

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transcarbamoylase and two mutants were found to be similar in MES buffer, but differed

in the presence of 10 mM potassium phosphate387.

SUMMARY

PT
The solution behavior of buffers has been examined for decades. Their ability to

RI
modulate pH has been known for almost a century and many excellent review articles are

available on buffer properties, purification, and preparation. Beyond their effect on pH, as

SC
seen in this review, buffers can have profound effects on protein stability. In some cases,

this arises from increased conformational stability due to ligand binding. In other cases,

U
the buffer is able to improve colloidal stability. In any case, one must assess protein
AN
stability in the presence of different buffers at a constant pH, and, if possible, constant

ionic strength. Likely, one buffer will emerge as preferential in terms of its ability to
M

stabilize under those conditions. At the same time, formulations are being developed
D

where the protein is the primary buffering species, meaning that conventional buffers
TE

may not be necessary for the maintenance of the pH. This has become the focus of many

high concentration MAb formulations.

EP

Not all of the effects of buffers on protein stability are favorable. Buffers can catalyze
C

hydrolytic reactions, such as deamidation. They can bind to metals needed for stability
AC

and/or activity. In addition, they can even promote aggregation by binding to partially

unfolded states of the protein and accelerating the formation of aggregates. These

observations reinforce the need to screen a variety of buffers for their effects on stability

during storage and under stress conditions.

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Finally, there is some information available on the safety of buffers, both systemically

and locally. There can still be issues with local irritation or pain upon injection, which

may be one reason why most recombinant proteins, including MAbs, are still

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administered by IV infusion.

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FIGURE LEGENDS

SC
Scheme 1. Chemical structures of some common buffers

Figure1. Conformational stabilization of a protein by the ligand binding mechanism

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AN
Figure 2. Conformational stabilization of a protein by the excluded solute
mechanism
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D
TE
C EP
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AC

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AC

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356. Veys N, Dhondt A, Lameire N. Pain at the injection site of subcutaneously

administered erythropoietin: phosphate-buffered epoetin alpha compared to
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371. Pourghaznein T, Azimi AV, Jafarabadi MA. The effect of injection duration and
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382. Vankley H, Bartholo D. Interference by tris and other buffers in Lowry assay for
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388. LLC S-AC. Buffer reference center 2016 [cited 2016 4 June 2016]. Available
from: www.sigmaaldrich.com/life-science/core-nioreagents/biological-
buffers/learning-center/.

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Table 1. Summary of buffer usage and pH for select MAb products

Product* [MAb] pH Buffer Lyophilized
Ilaris 150 mg/ml 6.2-6.8 Histidine Yes
Praluent 150 mg/ml 6.0 Histidine No
Xolair 125 mg/ml 5.5-6.5 Histidine Yes
Raptiva 100 mg/ml 6.2 Histidine Yes
Synagis 100 mg/ml 6.0 Histidine Yes

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Simponi 100 mg/ml 5.5 Histidine No
Anthim 100 mg/ml 5.5 Histidine No
Stelara 90 mg/ml 5.7-6.3 Histidine No

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Gazyva 25 mg/ml 6.0 Histidine No
Herceptin 21 mg/ml 6.0 Histidine Yes
Keytruda 25 mg/ml 5.5 Histidine Yes

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Cyramza 10 mg/ml 6.0 Histidine No
Nucala 100 mg/ml 7.0 Phosphate Yes
Campath 30 mg/ml 6.8 Phosphate No

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Avastin 25 mg/ml 6.2 Phosphate No
Remicade 10 mg/ml 7.2 Phosphate Yes
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Repatha 140 mg/ml 5.0 Acetate No
Pralia 60 mg/ml 5.0-5.5 Acetate No
Tecentriq 60 mg/ml 5.8 Acetate No
Amjevita 50 mg/ml 5.2 Acetate No
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Arzerra 20 mg/ml 5.3-5.7 Acetate No

Vectibix 20 mg/ml 5.6-6.0 Acetate No
Cinqair 10 mg/ml 5.5 Acetate No
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Humira 50 mg/ml 5.2 Citrate-Phosphate No

Perjeta 30 mg/ml 6.0 Histidine-Acetate No
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Taltz 80 mg/ml 5.3-6.1 Citrate No

Portrazza 16 mg/ml 6.0 Citrate No
Rituxan 10 mg/ml 6.5 Citrate No
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Opdivo 10 mg/ml 6.0 Citrate No

Yervoy 5 mg/ml 7 Tris No
* Brand names that are registered trademarks of originator companies
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Table 2. Summary of buffer usage and pH for select non-MAb biopharmaceutical products
Product* pH Buffer Lyophilized
Aldurazyme 5.5 Phosphate No
Aranesp 6.2 Phosphate No
Fabrazyme 7.0 Phosphate Yes
Alferon 7.4 Phosphate No
Neumega 7.0 Phosphate Yes

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Kineret 6.5 Citrate No
Erelzi 6.3 Citrate No
Kanuma 5.9 Citrate No

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Trulicity Citrate No
Leukine 6.7-7.7 Tris Yes
Neupogen 4.0 Acetate No

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* Brand names that are registered trademarks of originator companies

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Table 3. Summary of pKa and d(pKa)/dT values for various buffers

Buffer pKa dpKa/dT Reference Buffer pKa dpKa/dT Reference
13 13 233 388 51
Phosphate 2.15 0.0044 MOPS 7.20 0.015 , , ,
13 17
7.2 -0.0028 7.15
13 14
12.33 -0.026 7.02 -0.015
13 18
Succinate 4.21 -0.0018 7.18

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17 13 233 388
4.25 MES 6.10 -0.011 , ,
18 51
4.21 6.20
14 14
5.28 6.02 -0.011

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13 18 17
5.64 0 , 6.15
17 18
5.60 6.27
193 193
Lactate 4.14 Aspartate 1.99

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13 18 193
Acetate 4.76 0.0002 , 3.90
193 193
4.75 10.00
30 193
4.73 Ascorbate 4.17

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51 193
4.80 11.57
14 18
4.64 0.0002 Glycine 2.35
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193 18 13 18
Citrate 3.13 , 9.78 -0.025 ,
13 193 18 17
4.76 -0.0016 , , 9.90
14 13
5.80 Carbonate 6.35 -0.0055
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13 193 18 13
6.40 0 , , 10.33 -0.009
13 17
Tris 8.06 -0.028 10.25
51 18
8.10 ADA 1.59
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14 18
8.00 -0.031 2.48
17 18
8.30 6.84
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18 13
8.07 Borate 9.23 -0.008
13 18
Bis-Tris 6.80 9.24
388 51
6.50 ,
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14
6.32 -0.017
18
6.48
18
Histidine 1.50
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18
6.07
22
-0.022
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18
9.34
13
HEPES 7.48 -0.014
233
7.50
17
7.55
388
7.48
51
7.60
14
7.39 -0.014
18
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Table 4. Average pKa values in proteins from Grimsley33

Group n pKa (avg ± s.d.)
Asp 139 3.5 ± 1.2
Glu 153 4.2 ±0.9
His 131 6.6 ± 1.0
Cys 25 6.8 ± 2.7

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Tyr 20 10.3 ± 1.2
Lys 35 10.5 ± 1.1
C-terminus 22 3.3 ± 0.8

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N-terminus 16 7.7 ±0.5

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Table 5. Four groups or families of Good’s buffers and their respective pKa values233

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Family Buffer pKa Useful Range
Morpholine MES 6.1 5.5-6.7
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MOPS 7.2 6.5-7.9
MOBS 7.6 7.0-8.3
Trizma TES 7.4 6.8-8.2
TAPS 8.4 7.7-9.1
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TABS 8.9 8.2-9.5

HEP HEPES 7.5 6.8-8.2
EPPS 8.0 7.3-8.7
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HEPBS 8.3 7.6-9.0

CHA CHES 9.3 8.6-10.0
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CAPS 10.4 9.7-11.1

CABS 10.7 10.0-11.4
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Table 6. Summary of glass transition temperatures Tg’ values measured by two different
methods and eutectic temperatures (Te) for buffers in frozen systems308
Buffer Tg’ (DSC) Tg’ (TMA) Te
KH2PO4 -55 -49
Tris base -51 -6
Tris-HCl -65 -13
Tris-acetate -54

PT
Citric acid -54
Sodium citrate -41
Potassium citrate -62

RI
Sodium acetate -64 -60
Na2HPO4 -45
Na2HPO4 • 12H2O -1

SC
Na2CO3 -3
Imidazole -8
NaHCO3 -52

U
HEPES -63 -9
TAPS -49
AN
Glycine -4
Glutamic Acid -17
Histidine -33
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC