Besides pH, buffer components also play a role in stabilizing mAbs. Sodium phosphate and histidine are the most commonly used buffer components in commerical formulations. While sodium phosphate is more common for low concentraiton mAb formulations, histidine is the primary buffer of choice for higher mAb concentration. Goswami (Antibodies 2031, 2, 452-500)

Carpenter (WO2008/039761) discloses stable liquid antibody formulations of antibodies comprising non-zwitterionic buffers such as phosphate (e.g., Na3PO4), tris, citrate, succiante and acetate buffers. 

Ionic strenght, pH and Temperature Considerations: 

The first step in formulation development is pH optimziation. The most common approach is to identify the pI of the mAb form the primary sequence using common available software such as ExPASy or Sednterp, followed by formulation of mAb at pH away form the pI. Various pathways of chemical degrdation are pH sensitive. For example, deamidation and isomerization are favored at neutral and basic pH. Thus it is preferably to formulate biopharmaceuticals at slightly acidic pH. In fact, out of 28 commercially approved mAb type biopharmaceuticals, 20 are formulated at pH less than or equal to 6.5. Goswami (“Developments and challenges for mAb-based therapeutics” Antibodies 2013, 2, 452-500)

US 4,3596,608 and 4,499,073 disclose that in order to obtain an immunogloublin composition which is substantially free of an aggregate of immunoglobulin and has a monomer content of immune serum globulin exceeding about 90%, a solution of the immune serum globulin is adjusted to have an ionic strenght less than about 0.001 and a pH of 3.5 to 5.0.

JP-A-7-238036 discloses that for the improvement of stability, the use of acid treatment or storgage at room temperature.

disclose that conformational stability of IgG preparations decreases with decreasing pH, while the resistance against aggregation improves. The optimum pH range for storage was 5-6, as a compromise between conformational stability and the tendency for oligomerization. Biologicals 34 (2006) pp. 5-14). 

Importance of isoelectric point: (see also engineering Abs to alter pI)

One important characteristic of mAbs is their isoelectrin point (pI) which essentailly is the pH at whcih teh antibody has no net electrical charge and its value depedns on the charged amino acids the antibody contains. If the pH of the surrounding environment is below the antibody’s pI, then the molecule carries a net postive charge wehreas the antibody will carry a net negative charge when the pH is above the pI. It is generally observed that increases in net positive charge of antibodies result in increased blood clearnace and increased tissue retention with shorter half-life, whereas antibodies with lower pI generally have decreased tissue uptake and longer half-life, although observations can be conflicting regarding correlation between mAb learance and pI. Even subtle manipulation such as molecular surface remodeling to disrupt positive path regions can influence pK properties. (Schneider “Important of isoelectric point (pI) of antibodies, 2017. 

There is interest in identifying antibody candiddates with highly repulsive seelf-interactions in stadnard formualtions (e.g., low ionic strengh buffers at pH 5-6) fo high solubility and low visosity. Likewise, there is interest in identifying antibody candidates with low levels of non-specific interactions in physiological solution condition (PBS, pH 7.4) to promote favorable phamacokinetic properteis. (Gupta “Antibodies with weakly basic isoelectric points minimize trade-offs between formulation and physiological colloidal proeprties” Mol. Pharmaceutics 2022, 19, 775-787). 

Gupta “Antibodies with weakly basic isoelectric points minimize trade-offs between formulation and physiological colloidal proeprties” Mol. Pharmaceutics 2022, 19, 775-787) discloses that IgG1 wieth weakly basic isoelectric points between 8 and 8.5 and variable fragments (Fvs) isoelectirc points between 7.5 and 9 typically display the best comibnations of strong repulsive self-interactions and weak non-specific interactions. 

An inherent protein property of interest is its isoelectric point (IEP). The IEP is defined as the pH at which net charge becoems azero. It is a rule of thumb to define the formulation’s pH not too close to IEP as this increases the risk of preciptiation. The IEP of prtoeins can be theorecctically derived from amino acid sequence or determined experimentally using different methods including isoelectric focusing (IEF) of IEF capillary electrophoresis (iCE). (Stefan fischer “Assessmetn of net charge and prtoien-protein interactions of different monoclaonl antibodies” J. Pharma Sciences, 100(7) 2011).

Addition of Amino Acids:

Most of the liquid IgG which is formulated with amino acids on the market must matinain an acid pH to avoid aggregation, preferably between a pH of 4-50 at at a temperature of 2-8C if they are stabilised with 0.2M or 0.25 M glycine such as Gamunex®, Kiovig® or Gammard® lquiud (both from Bater) or up to 25C if stablized with 0.25M proline such as Privigen® (CSL Behring, Germany).  However, an excessively acid pH over an extended period of time favours fragmentation of IgG. Ristol Debart (US13/838424). 

Arginine: Liu (US 2007/0053900) discloses that arginine, specifically arginine-HCl is particularly suited for highly concentrated liquid protein or antibody formulations.

Glycine and/or histidine/ serine: 

Kakuta (US 2003/01901316) discloses a stabilized preparation containing an antibody in a glycine and/or a histidine buffer.

Yang (WO2004/001007) discloses a composition of antibodies in aqueous solution of histidine or acetate buffer at a concentration in the range of 2-48 mM. 

Zhou (US2014/0056888) teaches stable aqueous solutions having a high concentraiton of anti-C5 having a buffering agent which is an amino acid such as histidine, erine and glycine. 

Histidine and Glutamate (His + Glu):

Ignatius (J. Pharma Sciences, 108 (2019) 11391147 (2019) discloses systematic buffer screening for an aggregation prone bispecific antibody and finding that equimolar combiantion of histidine and glutamate at pH 5 bufer showed enhanced colloidal stability as measured by dyanmic light scatering interaction parameter (Kd). This implies a role of net protein-protein interaction in mediating aggregation propensity of the protein. The His + Glu interaction with the protien is predominantly electrostatic in nature as observed by the switch in the net protein-protein interactions from repulsive to attractive interactions in the presence of 20 mM NaCl. 

Histidine and NaCL

The presence of 50 mM sodium chloride in 10 mM histidine pH 5.6 was found to increase diffusion interaction parameter (KD) or reduce self-interaction, improve relative solubility and reduce turbidity (OD350) of an anti-LAG3 antibody. The stability of anti-LAG3 antibody in 10 mM histidine pH5.6 was investigated in the presence of 40 mM L-arginine hydrochloride using diffusion interaction paraemter (KD), tubidity and relative solubility. The self-interaction and turbidity was found to be dramatically reduced. 

 Methionine/Tryptophan: 

Junyan (US13/536584) discloses a method of stabilizing and preventing oxidation of aromatic amino acid residues within a susceptible antibody by adding methionine in combination with one or more vitamins.

Methionine is an antioxidant that prevetns antibody oxidation durin gprocessing and storage (Ma US 8,613,919)

Ramachandra (US2011/0097340A1) teaches anti-VEGF antiboides which are formulated with antioxidants such as methionine.

Sato (EP1260230A1) teaches stabilization of antiboides and proteins such as GCSF by adding methionine and tryptophan. The composition can also include surfactants and mannitol.

Sloey (US2010/0297117A1) discloses stable formulations of antibodies having one or more antioxidants including methionine, aromatic amino acids such as tryptopyhan and vitamins such as water soluble vitamin B6. Sloey further teaches that the formulations may optionally include surfactants and mannitol.

Warne (US2006/0193850) teaches stabilization of antibody with methionine as an antioxidant in an amount sufficient to inhibit the formation of undesired by-products.

Phenylalanine, proline, leucine and isoleucine: Bruegger (CN1157572 (abstract) disclsoes liquid preparations of IgG for intravenous infusion which are stabilixed against dimer formation by the addition of one or more amphiphilic stabizers. Preferred amphiphilic stabilizers are nicoteinic acid derivatives, particularly nicotinamide and naturally occuring alpha amino acids having a lipophilic side chain such as phenylalamine, proline, leucine and isoleucine.

IgPro10 is a liquid intravenous immunoglobulin (IVIG) product that is formulated with 250 mM L-proline at pH 4.8 (Vox Sanguinis (2009) 96, 219-225. 

Addition of Carbohydrate/Polyols or Poly-alcohols

Formulation of IgG with polyols or poly-alchohols, for example, with maltose and sorbitol prevents aggregation. IgG solutions that are stable up to 25C (with 10% maltose, trade name Octagam®) and up to 30C with 5% sorbitol (trade name Flebogamma®) have been formulated in a slightly acid pH range between 5-6. However, the presence of some sugars in IgG formulations has been questioned. Some cases of kidney failure have been associated with preparations containing saccharose.  Ristol Debart (US13/838424).

Lam (US6,171,586) describes a stable aqueous pharmaceutical formulation having a therapeutically effective amount of an antibody not subjected to prior lyophilization, a buffer maintaining the pH in the range from about 4.5-6, a surfactant and a polyol. 

Addition of Sorbital and Mannitol: 

JP-A-63-192724 discloses a liquid immunoglobulin composition for intravenous injection having low conductivity and pH of 5.5 ±0.2 and containing sorbital as a stabilizer.

Barrera (60 Ann. Rheum. Dis. 660-69 (2001) discloses s single does of antibody to study short term effects in rheumatoid arthritis patients, using a preparation of 25 mg/ml mAb in 1.2% mannitol, 0.12% citric acid, 0.02% sodium citrate in an intravenous infusion. 

Hirao (EP0278422B1) teaches an intravenously injectable solution of gamma-globulin in solution containing 5 to less than 10% by wweight per volume of sorbitol as a stabilizer and having an electrical conductivity of not higher than 1 mmho and pH of 5.5.

Zhou (US2014/0056888) teaches highly concentration antibody formulations having at least one carbohydrate excipient such as sorbitol and mannitol. 

 Addition of Polymers:

Polyelectrolytes:

Gibson (WO95/10605) discloses stabilisation of proteins such as antibodies and enzymes which includes a polyelectrolytes. The polyelectrolyte can be cationic, anionic or amphoteric. 

–Anionic Polyelectrolytes:

Gibson (WO95/10605) disclsoes stablisation of rptoeins such as antibodies with anionic polyelectrolytes which are preferably a polymer with anionic groups distributed along the molecular chain. The anionic groups, which may include carboxylate, sulphonate, sulphate and other negatively charged ionisable groupings may be disposed upon groups pendant from the chain or bonded directly to teh polymer backbone. 

–cationic polyeletrolytes:

Gibson (WO95/10605) disclsoes stabilisation of proteins such as antibodies which include cationic electrolytes. The cationic groups, which are preferably quaternary ammonium derived functions may be disposed in side groups pendant form the chain or may be incorproated in it.  Examples of caitonic polyelectrolytes include coplymers of vinyl pyrollidone and quaternary methyl methacrylate. 

—–Polyamino compounds (e.g., spermine and spermidine):

Spermidine is an aliphatic polyamine. Spermidine synthase catalyzes its formation from putrescine. It is a precursor to other polyamine, such as spermine and its structural isomer thermospermine (Wikipedia). 

Bowen (US 2013/0058958) discloses using certain charged amiono acids for reducing the viscocity of aqueous protein contianing formulations. In oueous formulation is spermidine or spermine. ne embodiment the protein is an antibody and the the compound that is capable of reducign the viscosity of the aq

Gelder (WO 2006/094974) disclsoes a pharmaceutical composition that incldues a stabiliser composition with a biological molecule such as an antibody. In a preferred embodiemtn, the stabiliser composition includes at least one polyamine (compound with two or more amino groups) such as spermidine and psermine. The composition can also include a sugar such as glucose, lactose, sucrose, maltose osorbitol or mannitol and an amino acid such as glutamate (gluamic acid) or glycine. The composition is particularly useful for stablising vaccines. 

Gibson (WO95/10605) discloses stabilisation of proteins such as antibodies which includes low molecular weight polyamino compounds such as spermine and spermidine. 

Polyethyleneglycol (PEG): JP-W-59-501546 discloses ultrafiltration treatment of an immunoglobulin prepration at pH 5 to 5.6 in the presence of 0.05 to 2 w/v % polyethyleneglycol (PEG).

US 5,132,406 discloses a method for the production of immunoglobulin preprations for intravenous injection, which comprises a combination of a heat treatment step, a supernantant fraction recvoering step by a fractionation treatment with 4 to 10% PEG and a preciptaition fraction recovering step by a 10 to 15% PEG fractionation treatment.

Hirao (US 6,159,471) discloses an immunoglobulin prepration having good storeage stability in the form of a solution by fractionating with an aqueous solution with 4 to 10 w/v % of PEG haivng a MW of from 1k to 10k at a pH of from 4.5 to 6.5 at an ionic strenght of from 0.0001 to 0.1 M and a temperature of from 0 to 4 degrees C.

Addition of Surfactants

Surfactants are also included in typical mAb formualtions. Surfactants are surface active agents that are amphipathic in nautre (polar head and hydrophobic tail). Surfactants tend to accumulate at various interfaces, resulting in reduced interfacial tension. Goswami (Antibodies 2031, 2, 452-500)

Addition of Non-Ionic surfactants

WO95-3826 discloses the immunogloublin preparation comprising 0.1 g/L or less of non-ionic surfactant as stabilizer for maintaining solution state, and being substantially free of albumin.

–polysorbates: 

Lam (US6,171,586) discloses a stable aqueous formulation of an antibody with a buffer maintaining the pH in the range of about 4.5-6 and a surfactant such as polysorbants (polysorbant 20, 80, etc) which reduce aggregation.

Zhou (US2014/0056888) teaches hingly concnetrated antibody formulations having a surfactant such as polysorbate 20 or polysorbate 80.