US 9,394,339 B2  8  7  equivalently) is detrimental to asparaginyl stability is a very  important discovery in this work, and is shown dramatically  in Example 1 below.  "Pharmaceutically-acceptable": In the context of this  invention, "pharmaceutically-acceptable" designates com› pounds or compositions in which each excipient is approved  by the Food and Drug Administration, or a similar body in  another country, for use in a pharmaceutical or vaccine for› mulation, or belongs to a succinct class of compounds for  which a Drug Master File is on file with a govermnent regu› latory agency, usually the FDA, or, less preferably, is known  from extensive toxicity studies to be safe for the intended  route of administration (which in the context of this invention  is typically, though not always, parenteral). This also includes  compounds that are major components of approved excipi› ents, which are known to be oflow toxicity taken internally. A  listing of approved excipients, each with the various routes of  administration for which they are approved, was published by  the Division of Drug Information Resources of the FDA in  January, 1996 and entitled "Inactive Ingredient Guide". The  existence of a Drug Master File at the FDA is additional  evidence that a given excipient is acceptable for pharmaceu› tical use, at least for certain routes of administration. For  injectable products, a listing of approved excipients was pub› lished in 1997. See Nema, Washkuhn and Brendel (1997)  PDA  J. of Pharm. Sci.  &  Technol.  51(4):166. It should be  added that there are certain compounds, such as vitamins and  amino acids, which are in injectable products (typically for  parenteral nutrition) as "actives", and are thus known to be  safe upon injection, and such compounds are considered  herein as pharmaceutically-acceptable as excipients as well,  for injection. All of the embodiments of this invention are  understood to be pharmaceutically-acceptable for at least  some route of administration in humans, meaning that every  excipient in each embodiment is approved for use in humans,  in at least one route of administration. This is crucial not only  for product safety but also for utility of the invention, as a key  feature of the invention is that it uses, preferentially, only  FDA-approved excipients, most of which are in fact approved  for injection. Examples of compounds that are not pharma› ceutically-acceptable are taurocholic acid and its salts (not  acceptable for any route of administration), dichloroacetic  acid, sodium p-chlorobenzenesulfonate, and other N-acety› lated amino acids besides N-acety ltryptophan. The surprising  recognition that deamidation-stabilizing formulations can be  found and produced within the narrow range of pharmaceu› tically-acceptable formulations is a key aspect of this inven› tion, particularly as it recognizes the destructive effects of  anions that do not fit the preferred range of pKa as described  herein.  "Saccharin": To avoid any confusion, it is pointed out that  in spite of the name-saccharin, which sounds like it should  be related to saccharides-and the sweet taste, saccharin is  not a pol yo 1, nor any kind of sugar. The name reflects only the  sweetness, not the chemical structure. The chemical structure 55  is  in  fact  2,3-dihydro-3-oxobenzisosulfonazole  (C$_{7}$H$_{5}$NO$_{3}$S).  Description  Surprisingly, in the course of experimental work by the  inventor supported by theoretical considerations, and exem- plified by the Examples below, it was found that asparaginyl  stability of peptide ( or protein) solutions containing organic  anions drops off sharply above an anion pKa of about 3.5, and  that for pKa values equal to or greater than about 3.5 the  organic anion can actually destabilize the drug. A dramatic  cutoff between stabilization and destabilization at the pKa  value of3.5 was reported by the inventor in connection with  Organic zwitterions behave, not surprisingly, very differ› ently from the organic anions within the context of the current  invention, and the effect of an acidic group with a pKa within  the preferred ranges of the invention is largely nullified by the  cationic moiety in the zwitterion. Thus, while the acidic group  on a typical amino acid, for example, is between 2.0 and 2.5,  and so well within the preferred range, the zwitterionic amino  acids are not effective as stabilizers and are not to be deemed  as organic anions of the invention; this is not necessarily true  for acidic amino acids such as aspartic acid and glutamic acid.  Without wishing to be bound by theory, one explanation for  stabilizer effectiveness when the pKa of the stabilizer is in the  preferred range of 0.5-3.5, and particularly in the most pre› ferred range between 1.5 and 3.5, is that the conjugate base  may form a temporary covalent bond, or other type of com› plex, with the carbonyl carbon of the labile group on the  peptide or protein, at least a fraction of the time, limiting  access of the carbonyl to other nucleophilic attack. This is  driven by the susceptibility of the carbon atom on the carbo› nyl group to nucleophilic attack, and in nucleophilic attack on  carbonyl groups  generally,  reactivity correlates  fairly  strongly with basicity. If the pKa of the acid is too low, then  the conjugate base is extremely weak, and will less frequently  attack the carbonyl group. Thus, a stabilizer such as saccharin  Organic anions that have two acidic/anionic groups may  need to be treated in the same way as mixtures of anions. In  particular, one destabilizing acidic group, with a pKa greater  than about 3.5, might nullify the stabilizing effect of an acidic  group on the same molecule with a pKa less than or equal to  3.5. Thus, for example, tartaric acid, with a pKa$_{1 }$ of2.93 and  a pKa$_{2 }$ of 4.23, will not afford effective stabilization in the  context of this invention. This is apparently related to the fact  that the weaker acidic group (higher pKa) forms a stronger  conjugate base than does the stronger acidic group. For a  similar reason, phosphoric acid is a destabilizing anion in the  context of this invention. In this evaluation of organic anions,  any titratable acidic group with a pKa that is too high to be  relevant should be removed from consideration, in particular  if the pKa is more than about 1 pH point above the pH of the  formulation.  a small-molecule de-esterification hydrolysis reaction, in  U.S. patent application Ser. No. 11/404,109. Since at the  solution pH value of preferred embodiments of the invention,  at least 90% of the organic anion is deprotonated, and since  the amount protonated (i.e., in acid form) decreases in the  more effective (lower pKa) cases, this trend is almost cer› tainly not strongly dependent on the pH of the solution (pro› vided it is not too strongly acidic), but rather, is largely inde› pendent of pH and thus "universal" in some sense. Hence,  quite broadly, a pKa equal to or less than about 3.5 is strongly  preferred in the practice of this invention. A less dramatic,  though significant, drop-off in stabilizer effectiveness is also  observed as the pKa drops down to negative values. Thus,  saccharin, benzenesulfonic acid, N-acetyltryptophan and  gentisic acid are strongly preferred in this invention, and  much preferred over compounds above pKa 3.5 such as or  acetic or citric acid, which are actually destabilizing. The  destabilizing effect of acetate ion, for example, with its pKa  of 4.5, is demonstrated in Example 1 below. Without wishing  to be bound by theory, when a destabilizing anion (with pKa  greater than 3.5) is present in molar concentrations about  equal to or greater than that of the preferred anion (with pKa  less than or equal to 3.5), it may compete with the preferred  anion for binding to the labile group (viz., at the carbonyl  carbon of the labile group), and effectively displace it because  it is a poorer leaving group, thereby rendering the preferred  anion far less effective, if not substantially ineffective. 