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Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2015 May 1;24(7):1031–1039. doi: 10.1002/pro.2684

Advanced protein formulations

Wei Wang 1,2,*
PMCID: PMC4500304  PMID: 25858529

Abstract

It is well recognized that protein product development is far more challenging than that for small-molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long-term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site-specific mutagenesis, and preparation of nontraditional types of dosage forms—semiaqueous solutions, nonfreeze-dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution.

Keywords: stability, viscosity, stabilizers, conjugation, suspension

Introduction

One of the key steps in the development of a successful protein drug product is to formulate the drug candidate into a s dosage form to achieve a minimum of 18-month shelf life. Due to the high molecular sensitivity of proteins to different process stresses, temperature, and other environmental factors, formulation development for proteins can be a complex, and often challenging process.1

Over the past two decades, a variety of formulation approaches have been tried or being developed to stabilize a protein candidate. A commonly accepted process for formulation development is to conduct stability studies upon alteration of a variety of formulation variables, such as pH, ionic strength, type/concentration of buffering agents, and type/concentration of other stabilizing agents, such as surface-active agents, and tonicity-adjustment agents.2 However, these traditional formulation approaches are generally not adequate to make a protein stable enough to commercialize a liquid protein product for long-term storage at room temperature. Many proteins are not even stable enough at 2–8 °C in solution and have to be made into a solid form for clinical evaluation or commercialization. Commercially, only few plasma products, such as Hizentra, approved in the United States in 2010, a 20% immune globulin solution for subcutaneous injection (IGSC), can be stored at room temperature (up to 25 °C) for up to 30 months (US Package Insert).

In recent years, monoclonal antibodies have become a major therapeutic class.3,4 In association with their practical applications for subcutaneous administration, high concentrations are frequently needed to accommodate the low administration volume. Unfortunately, the physical behavior of such a product may change dramatically with increasing protein concentrations.5,6 These properties may include significant enhancement of solution opalescence,7,9 viscosity,10–12 and protein aggregation/immunogenicity.13–15 These altered properties challenge the drug product manufacturing processes, product administration, and marketability.

Several nontraditional or novel concepts and technologies have been or are being developed to address the above issues. This short review summarizes these concepts and technologies as advanced formulations for stabilization of proteins (i.e., improvement in any protein stability indicators, such as Tm, aggregation tendency, etc.) and/or development of high-protein concentration products (>100 mg/mL).

Use of Complex or Uncommon Stabilizers

Proteins generally require a formulation excipient(s) as a protein stabilizer in a liquid state. Protein stabilization by a stabilizer(s) can be achieved through the traditional preferential interaction mechanism16 and/or other proposed mechanisms such as nonspecific interaction with surface hydrophobic pockets17 or charged amino acids,18 specific ligand binding,19 and enhancement of solution viscosity.20 To enhance the stability of proteins, simultaneous use of multiple stabilizers has been tested in expectation of addressing different stability issues via different mechanisms and/or possible synergistic effect. For example, a mixture of three amino acids—l-arginine (positively charged), l-glutamic acid (negatively charged), and l-isoleucine (nonpolar) can stabilize recombinant factor VIII (FVIII) during lyophilization and storage to the same degree as achieved by using albumin as a stabilizing excipient.21 Similarly, protein solubility, shown to correlate closely with protein aggregation tendency,22 can also be significantly enhanced by combination of multiple excipients. Use of both l-arginine hydrochloride and l-glutamic acid together showed synergistic effect on enhancement of the solubility of proteins due to a reduction in protein–protein interactions and additional hydrogen bonding interactions between the excipients on the surface of the protein.23 In reality, most mAbs seem to have high solubility potentials (>100 mg/mL)24 and do not need solubilization. Most of above stabilizing excipients are charged amino acids, suggesting that excipient charges play a significant role in protein stabilization, supporting some of the above proposed stabilization mechanisms. Use of multiple charged excipients for protein stabilization/solubility enhancement is likely similar to combination of multiple buffering agents for protein stabilization.25 On the other hand, use of multiple excipients certainly add additional burden during protein formulation characterization and stability studies.

Use of a large polymeric excipient/substance has been found to be effective in stabilization of proteins. Neutral polymers, as crowding agents, can stabilize a protein due to the excluded volume effect (steric repulsion).26 Several neutral polymers were found to stabilize various proteins, such as PVP,27 Ficoll-70000,28 and hydroxyethyl (heta) starch, or PEG 4000.29 Recently, new polymers have been synthesized and have demonstrated utility for protein stabilization. Examples include functionalized dextrans30,31 and glycopolymers containing trehalose as the side chain units for enhanced process and storage stability.32,33 Glycopolymers made from modified trehalose monomers were also shown to achieve stabilization for several model proteins.34 Polyanions/polycations have long been recognized as possible stabilizers due to their delicate formation of protein–polyion interactions, such as heparin35–39 and dextran sulfate36,37,40 pentosan polysulfate, polyphosphoric acid, poly-l-glutamic acid, poly(acrylic acid), poly(methacrylic acid).36,37 Interaction between polycationic chitosan and negatively charged lactate dehydrogenase (LDH) leads to significant stabilization during air-jet nebulization.41,42 On the other hand, interaction of negatively heparin and keratinocyte growth factor 2 (KGF-2) actually facilitated protein aggregation during agitation.43 It is apparent that strong interactions may lead to protein destabilization, as illustrated by the preferential interaction mechanism.16

Similarly, amphoteric polymers or proteins can be used as effective protein stabilizers. Successful examples include albumin, caseins,44 heat shock proteins (HSPs),45–48 valosin-containing protein (VCP),49 α-crystallin,50–52 and gelatin.29,53 Among these examples, use of α-crytallin at 0.2 mg/mL greatly improved resistance of insulin to fibrillation, better than the effect of HSA at 2.5 mg/mL.54 The chaperone-like effect is proposed due to interaction between crystallin and non-native protein species involved in the fibrillation process. A special case is the use of recombinant hyaluronidase enzyme in a protein formulation for facilitating rapid tissue distribution and thus, administration of a larger-than-normal injection volume.55 Again, the use of polymers or proteins in a protein formulation increases the complexity of the formulation and complicates protein formulation characterization and stability studies.

The limited degree of protein stabilization achieved by commonly-used excipients prompted evaluation of uncommon excipients/substances in improving protein/peptide stability. These include resveratrol, a natural phenol,56 hydroxybutyrate,57 polyamines,44 octanoic acid,58 and quinone-tryptophan derivatives.59 Among these, hydrophobic salts, such as salt of pentaine-1,5-dimaine and camphor-10-solfonic acid, have been shown to reduce the viscosity of mAb solutions by 10 folds.60 However, uncommon excipients may need safety evaluations, which may include significant in vivo studies.

Chemical Conjugation/Protein Fusion

Chemical conjugation or protein fusion was initially designed as a strategy to increase the in vivo half-life for longer duration of action. An added potential benefit upon chemical conjugation or protein fusion is the enhanced protein stability due to steric hindrance and/or changes in surface properties. Conjugation moieties include PEG,61 glycans,62 and other hydrophilic substances.63 PEGs are arguably the most widely used conjugation agents and have been found to stabilize proteins against different stresses, such as thermal stability,64,65 pH-induced or protease-induced degradation,66 and oligomerization.67 Similarly, glycosylation of proteins has been found to inhibit protein aggregation, using maltodextrins (with a dextrose equivalent of 18),68 differing lengths of lactose and dextran (10 kDa),69 and modified trehalose polymer.32 Conjugation of ovalbumin simply with more hydrophilic substituents increased its thermal stability by altering the intermolecular interactions.70

There are many examples of enhanced stability of proteins upon fusion with a more stable partner. Protein fusion with albumin, a relatively stable protein, has been found to reduce the aggregation rate of GCSF71 and to limit the self-association of human interferon-α2b into reversible dimers/trimers, while interferon-α2b forms stable dimers, and other multimers.67 Similarly, fusion of maltose-binding protein (MBP) with protein MpAFP698 (MBP-MpAFP698) made the protein 10 °C more thermally stable than MBP protein.72

Both chemical conjugation and protein fusion require additional processing steps during drug substance manufacturing, and the larger size of the final drug candidate would be more challenging for characterization and stability monitoring.

Site-Specific Mutagenesis of Stability-Controlling Residues

Site-specific mutagenesis can be very effective for protein stabilization. For examples, a lipase mutant increased the Tm of the wild-type protein by as much as ∼22 °C (from 78 °C) and the optimum activity temperature by as much as 30 °C, devoid of heat-induced aggregation.73 The melting temperature of a mutated RNase Sa was 28 °C higher than the wild-type enzyme.74 Mutation of even one amino acid at a key position may alter the stability of a protein.75,76 However, accurate selection of the right location and amino acid(s) for mutation can be challenging. Comparison of 11 different stability prediction methods proves that such methods are at best moderately accurate (about 60%).77 Therefore, protein stabilization through mutation is frequently based on trial and error. Nevertheless, some rough rules of mutation for protein stabilization were proposed—consideration of mutation in an α-sheet or β-sheet instead of those in loops, and exposed instead of buried residues, and nonconserved sites instead of conserved sites.76 Other investigators have found that rigidification (anchoring) of the mobile regions of a protein such as loops and helix termini is effective to attain higher thermostability and reduction/perturbation of the large hydrophobic patches for enhanced resistance to aggregation.73 In addition, two general mutation strategies can be used—improvement in electrostatic interactions and reduction in the conformational entropy of the denatured state by adding proline residuals in β-turns or other locations in proteins.74

It is obvious that there are specific sites (flexibility hotspots) in proteins that are important for both binding and stability.78 Therefore, any mutations in these sites may lead to irreversible, partial loss of protein activity.

Nontraditional Dosage Forms

The nontraditional or novel concepts and technologies in protein formulation include (1) use of polar organic solvents, (2) suspensions, (3) nonfreeze-dried solid formulations, and (4) other emerging concepts.

Use of Polar Organic Solvents

Generally, addition of a miscible organic solvent into an aqueous protein solution makes the protein unstable due to promotion of protein unfolding. On the other hand, changing the solvent property could lead to a reduction in protein solution viscosity. Polar organic solvents have been evaluated for this purpose. The viscosity of an IgG1 solution was reduced significantly in the presence of a small amount of dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).79 The effect is roughly equivalent to addition of 200 mM arginine chloride. However, the three melting temperatures of the protein dropped linearly with increasing concentrations of these two solvents. Therefore, storage stability of the protein could be compromised in this case. Similarly, reconstitution of a solid product with nonaqueous diluents could enable a significant reduction in injection volume for ease of drug administration (www.xerispharma.com/html/).

Suspensions

Suspensions have not been widely considered for proteins as a commercial dosage form. A successful example is insulin suspension based on its good crystallization tendency under certain conditions. The main purpose of designing such an insulin formulation is to increase the duration of action for less frequent injections.

A protein could be prepared hypothetically either as a crystalline or amorphous particulate suspension for protein stabilization and/or accommodation to high-concentration formulations. A decade ago, a group of scientists from Altus demonstrated feasibility of preparing crystal forms of three mAb's—rituximab, trastuzumab, and infliximab.80 Such crystalline suspensions were prepared with no detectable structural alteration to these proteins and with full retention of their biological activities in vitro. At 150 mg/mL, the viscosity of infliximab was at 275 cP but the equivalent crystalline suspension has a viscosity of less than 40 cP. In addition, the T1/2 of the crystalline infliximab was increased twice after SC injection in rats. A similar crystalline form, spherulite, was also successfully prepared for insulin80 and interferon.81 The respective T1/2 for the soluble and spherulite interferon was 2.5 and 13.1 h in rabbits. Crystallization appears to be a feasible approach for SC delivery of a small volume of a highly concentrated form of mAbs. On the other hand, there are several potential crystallization-related issues for consideration. A solution condition optimal for crystallization may not be suitable for long-term protein storage. Any additives used for promoting protein crystallization need to be safe for injections. Filling a crystalline suspension drug product is generally considered to be a manufacturing challenge due to the constant settling of crystals.

Use of a neat organic solvent could significantly change the interaction of proteins with the solvent. This change in solvent interaction may enable preparation of a nonaqueous suspension to reduce the viscosity of protein preparations. Feasibility has been demonstrated in a few studies. Several years ago, it was reported that milled lysozyme microparticles can be suspended at nearly 400 mg/mL in benzyl benzoate or benzyl benzoate mixtures with safflower oils and injected through a syringe with a 25–27G needle at room temperature.82 The suspensions were resuspendable after a year, with essentially constant particle size after two months as measured by static light scattering. Another group of scientists have demonstrated the utility of several solvents in the preparation of protein suspensions, including Miglyol 840, benzyl benzoate, and ethyl lactate.83 All of these solvents can be used to prepare suspensions having a viscosity of less than 10 cP. The aqueous solution of γ-globulin at 300 mg/mL showed a viscosity of 370 cP at 25 °C but its suspensions in a number of organic solvents (ethanol, methanol, isopropanol, 1,4-butanediol, propylene glycol, benzyl benzoate, PEG200, ethyl acetate, toluene, acetonitrile, etc.) exhibited viscosities up to 38 times lower than those of the corresponding aqueous solutions84 (Alexander Klibanov from MIT). Monoclonal antibodies should follow the same general trend with the magnitude of viscosity reduction depending on the property of the solvent, particularly its hydrogen-bonding properties. A major concern of using organic solvents is their toxicity and tolerability for human use.

Nonfreeze-Dried Solid Formulations

Traditionally, solid protein products were prepared to accommodate unstable proteins in solutions. Freeze-drying has been the method of choice for producing solid formulations of biotechnology products due to its minimal process stress and elegance of appearance of the final products.85,86 Practically, a protein could be freeze-dried to tolerate long-term room-temperature storage, and/or to accommodate the need for achieving high protein concentrations (freeze-drying a protein solution at a low protein concentration and reconstituting the product to a lower volume). In reality, use of a freeze-drying process to achieve room-temperature stability for proteins is not ideal due to the added manufacturing cost (long freeze-drying process) and the extra reconstitution step needed for drug administration.

Several alternative drying methods have been tried, including spray-drying, spray freeze-drying, supercritical fluid drying, and foam drying.87 Spray-drying is now emerging as a competitive drying technology for production of solid formulations, due to its high efficiency, low cost, and scalability. It has long been explored as a potential alternative to prepare stable bulk drug substance for easier storage and shipping, and to generate protein particulates of certain size and shapes for pulmonary drug delivery. Protein examples include insulin,88–91 human growth hormones,92,93 trypsin,94 and monoclonal antibodies.95–98 It is anticipated that spray drying would be more stressful to proteins than the freeze-drying process, due to the high drying temperature and high air–water interfaces generated during the process, as demonstrated for Met-hGH.93

An alternative to spray-drying is spray freeze-drying. This method eliminates the use of elevated temperature during spray drying but air–water interfaces are still created and remain a potential cause for protein aggregation, such as interferon-γ,99 lysozyme,100 Darbepoetin α,101,102 and BSA.103 Also, the freezing process is still a potential stress for proteins. Therefore, this method may or may not be advantageous to spray-drying.104–106 In fact, it is demonstrated that spray-freeze drying of Darbepoetin α by ultrasonic atomization at 120 kHz and 25 kHz generated 9% insoluble aggregates while spray drying at bench-top and pilot scales did not lead to detectable formation of insoluble or high MW soluble aggregates, using a two-fluid nozzle.101,102

Supercritical fluid (SCF) drying is another drying method that has been tested. Limited success has been achieved with model proteins.107 Protein aggregation seemed to be a key issue for this process.107 In addition, stringent requirement of instrumentation, process-induced excipient crystallization, and difficult protein reconstitution could be barriers for implementing this drying method.108

In comparison, vacuum foam drying shows promise as an alternative drying method.109–111 Some advantages may include process efficiency, reduced tendency for excipient crystallization, and better stability relative to the freeze-drying process. On the other hand, stringent control of process conditions may be required to maintain a high drying efficiency without accidental spill-over.

Among these alternative drying methods, no clear winner stands out. Comparative studies may be needed for a protein candidate and the final choice of a drying method is likely dictated by the process efficiency and protein's process and storage stabilities.

Other Emerging Concepts

One of the latest emerging concepts to tackle protein stability and high viscosity issues is the creation of “nanoclusters”—a densely packed protein molecules formed in the presence of a crowder(s) such as trehalose. It was demonstrated that protein molecules can be crowded into colloidally stable dispersions of distinct nanoclusters (35–80 nm) that exhibit equilibrium hydrodynamic diameters without gelation at very high concentrations (up to 320 mg/mL).112 The nanoclusters of an IgG protein are in equilibrium with monomers, which can be less than 2%. One nanocluster dispersion at 220 mg/mL in the presence of 70 mg/trehalose exhibited a viscosity of 36 cP, which is syringeable through a 25G needle. Subcutaneous injection of these nanocluseter preparations resulted in indistinguishable pharmacokinetics versus a standard antibody solution in mice.113

It is conceivable that the distances between protein molecules in nanoclusters are smaller than those in the bulk solution. The shorter distance could enhance protein–protein interactions and potentially lead to stability issues.114 The nanocluster concept is relatively new and needs further evaluation.

Summary

Many nontraditional concepts and technologies have been or are being tested to address protein stability and/or high-protein concentration issues. These concepts/technologies and their limitations/uncertainties are summarized in Table1. It is obvious that no one technology appears to be mature, ideal, and/or adequate to address the above issues.

Table 1.

Advanced Protein Formulations and Their Limitations/Uncertainties

Concepts/technologies Examples Limitations/uncertainties Selective references
Use of multiple or uncommon stabilizers Use of multiple stabilizers Complication of formulation composition 21
Formation of a complex with hydrophobic salts Safety of hydrophobic salts, and possible effect on protein aggregation 115, 60
Use of newly-designed polymers Limited degree of stabilization and complication of formulation composition 32–33.
Chemical conjugation or fusion Conjugation with PEGs Cost of PEG, extra conjugation step, yield drop, and characterization burden 64
Conjugation with newly designed glycopolymers Cost of polymer, extra conjugation step, yield drop, and characterization burden 32–33
Fusion with human serum albumin Additional design, complication of manufacturing and characterization 71
Single or multiple mutations Mutation of a lipase Design and evaluations, and potential activity change 73
Nontraditional formulations Use of a mixable organic solvent Safety evaluation and long-term stability 79
Solidification of proteins by alternative drying processes Process-induced instability, equipment design, and process control 98
Suspensions with protein crystals Strict crystallization conditions and filling challenges 80
Suspensions with organic solvents Safety evaluation and effect on protein stability 82, 84
Formation of nanoclusters Limited effect and potential stability issues 112–113
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Among these different concepts and technologies, solidification of proteins by one of the efficient drying methods represents the first choice and is a practical approach for protein stabilization. Use of multiple or uncommon protein stabilizers can potentially address both protein stability and high-protein concentration issues. The choice of these protein stabilizers certainly requires better understanding of the protein's tertiary structure, surface properties, and protein–protein interactions. Additionally, protein–stabilizer interactions should be better defined. Although the use of uncommon protein stabilizers, including organic solvents, may require significant safety evaluation, it is a worthy approach, especially when such stabilizers may offer potential platform applications.

Overall, a significant gap still exists in addressing the room-temperature protein instability and high-concentration issues. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution.

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