US 9,932,443 B2  10  9  In further embodiments, the subject invention provides  the use of energy-dispersive X-ray spectroscopy to measure  the presence of counterions in peptide fibers, wherein the  rate of annealing by irradiation is measured as a function of  counterions leaching out of cross-linked fibers.  This invention focuses on peptides for making materials.  Characterizing peptide-based materials and determining  relationships between polymer structure and solution and  material properties matter most here. The scalability of  polymer synthesis and materials manufacture; convenience;  cost of synthesis and purification; and limitations on poly› mer structure are important.  Linear homopolypeptides and heteropolypeptides, uni› form and non-uniform sequence, respectively, can be pre› pared by solid-phase or solution-phase methods (chemical  approaches)  or  recombinant  methods  (biological  approaches) (see FIG.  3).  These synthesis approaches have  advantages and disadvantages for different purposes. For  example, chemical synthesis is advantageous for small› peptide biologics production. Solution-phase approaches are  usually favored for therapeutic peptides shorter than 15  amino acid residues and quantities over 100 kg, whereas  complex or longer sequences are usually made by solid› phase synthesis, and peptides longer than 50 residues are  made by recombinant methods (Thayer, 2011 ). All industrial  enzymes are made by biological methods.  Ring-opening polymerization of a-amino acid N-car› boxyanhydrides, the main approach to synthesizing peptides  in solution, is utilized herein. The method yields statistical,  linear polypeptides. Growth occurs only by the probabilistic  addition of monomers to active chain ends. Monomers are  present throughout the process, but their concentration falls  with time. Polymer mass and yield are functions of mecha› nism and other reaction particulars. The product will nor› mally have a Poisson distribution of chain lengths, (Ne/N)  Nexp(-N), where N is degree of polymerization (DP) and N  is number-average DP (Strobl, 2007). N can range from  2 to >2,000. Short-range order is possible; long-range order,  improbable. Synthesis from a heterogeneous monomer  population is expected to yield chains of virtually identical  amino acid composition. The approach is advantageous if  monodisperse polymers and sequence  specificity  are  optional. Polydisperse co-poly(L-glutamic acid4, L-tyrosine  1) (PLEY), a random copolymer, has been studied (Khadka  et al., 2011; Haynie et al., 2012; Haynie et al., 2013).  Solution-phase production of a monodisperse population  of polypeptides of identical sequence is not only possible but  actually done in industry.  It is very expensive, however, if  the DP is greater than about 15 residues. Solid-phase syn› thesis of sequence-specific polypeptides is even more expen› sive per unit mass. DP is practically limited to about 50  residues, as yield decreases with chain length. Double  coupling can improve the efficiency of monomer addition at  each step and the yield of desired product, albeit at a cost.  Consequently, peptides made by chemical methods cannot  be competitive in the marketplace unless consumers are  willing to pay a premium. In general terms, sequence  specificity by chemical production is too expensive except  for limited purposes in medicine, basic research and defense  (see Bray, 2003).  The functional properties of proteins are "microscopic" in  that they typically depend on amino acid sequence. For  instance, enzyme activity requires appropriate positioning of  chemical groups. The physical properties of polymer aggre› gates, by contrast, are "macroscopic." Such properties could  depend on amino acid sequence as much as composition.  One approach to peptide synthesis and processing could  be more advantageous for some amino acid compositions  than others. Recombinant synthesis may be best for protein› like sequences, that is, ones for which the composition  resembles the average composition of proteins. Whether a  solution-phase approach or a recombinant approach for  random peptide production is best for large-scale production  depends on the cost of polymer production, characterization  and processing, and the desirability of the physical, chemical  or biological properties displayed by materials that can  actually be made with a polypeptide of a given sequence or  composition.  Recombinant production will in any case have unique and  complementary advantages to solution-phase synthesis for  materials research and possible technology commercializa› tion.  If control over amino acid composition, sequence and  polydispersity are required at any scale of production, if the  ability to encode manufacturing instructions into polymer  structure requires sequence specificity, and if there is no  need to guarantee freedom from small amounts of bacterial  contaminant, then recombinant production could be the least  expensive approach to synthesis. Peptide purity of >95% is  readily and reliably achieved, and polymers of substantially  greater purity can be obtained without great difficulty. Sys› tematic variation of amino acid composition is more difficult  than for solution-phase synthesis, as at least one gene is  needed per polymer structure, but it can be done by estab› lished and cost-effective methods. A small number of rep› resentative random sequences of a given amino acid com› position and chain length could be made by recombinant  methods and compared experimentally. A blend of different  recombinant random sequences can prove useful for mate› rial properties. Biological peptide production is also poten› tially significant for sustainable manufacturing. Production  in photosynthetic bacteria can be especially advantageous  for the purpose; high yields of recombinant proteins have  been obtained in photosynthetic bacteria.  Chemical synthesis, it should be acknowledged, presents  both advantages and disadvantages. The plus side features  ease of polymer preparation and latitude with regard to  composition. A potential advantage for materials is the  incorporation of unusual amino acids, which proceeds in  essentially the same way as for a usual amino acid. The main  limitations are the prohibitive cost of controlling sequence.  For this subject invention, however, specific sequences are  not needed. Polydispersity can be controlled by reaction  conditions and fractionation. Side chain protecting groups  can influence polymerization, but the final amino acid com› position of products can be measured. Incomplete removal  of protecting groups after synthesis can influence down› stream processing, but the abundance of such groups can be  quantified. How material properties depend on a lack of  sequence uniformity or polydispersity can be tested by  recombinant production of monodisperse peptides of iden- tical composition.  Perhaps the most positive way of regarding solution-phase  synthesis of peptides in a materials context is to focus on  cases in which it is certain that sequence will be less relevant  than a) amino acid composition, b) predictability of water  solubility, c) measurability of number-average mass and  weight-average molecular mass and therefore polydispersity  index and d) reproducibility of polymer production and  materials processing. Chemical composition, average DP  and, to a lesser extent, polydispersity index may be treated  as essentially continuous and independent variables, and the  systematic investigation of relationships between polymer  structure and materials will be possible.  Peptide Synthesis for Materials Manufacturing 